Research Article Yupingfeng Pulvis Regulates the Balance of T Cell Subsets in Asthma Mice Zhigang Wang, 1,2 Xueting Cai, 1,2 Zhonghua Pang, 1,2 Dawei Wang, 1,2 Juan Ye, 1,2 Kelei Su, 1,2 Xiaoyan Sun, 1,2 Jing Li, 1,2 Peng Cao, 1,2 and Chunping Hu 1,2 1 Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, China 2 Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, Jiangsu 210028, China Correspondence should be addressed to Chunping Hu; [email protected] Received 10 January 2016; Accepted 22 March 2016 Academic Editor: Vincenzo De Feo Copyright © 2016 Zhigang Wang et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Yupingfeng Pulvis (HFBP) had played an active role in many diseases, especially respiratory tract infections. Exploring the possible prevention mechanism of HFBP may provide new ideas in clinical applications for this well-known herbal formula. Purpose. To study the possible mechanisms of therapy effect of HFBP on asthma mice via regulating the balance of Tregs and 17 cells. Method. e female BALB/c mice were divided into five groups: control group, model group, prednisone (5.5 mg/kg) group, and 22 g/kg HFBP and 44 g/kg HFBP groups. Ovalbumin was used to make the asthma model of mice; the drug was ig administered daily aſter atomization for consecutive 15 d. e mice were killed aſter the last administration. e paraffin-embedded tissue sections of the lungs were stained by H&E. Tregs and 17 cells in bronchoalveolar lavage fluid were detected by flow cytometry. IL-4, TGF- , and TNF- in the serum were detected by ELISA assay. Results. HFBP could alleviate the inflammation in the lung tissue of mice, decrease the proportion of 17 cells, and increase the proportion of Treg cells in bronchoalveolar lavage fluid. HFBP could decrease IL-4 and TNF- level and increase TGF- level in blood. Conclusion. HFBP could treat the asthma through impacting the balance of 17 cells and Treg cells as well as the levels of related inflammatory cytokines in asthma mice. 1. Introduction Asthma is a common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, reversible airflow obstruction, and bronchospasm. Common symptoms include wheezing, coughing, chest tightness, and shortness of breath. Over the past few decades the world asthma prevalence and mortality have been a rising trend year by year, having impact on social economy and health. e disease is affecting more than 300 million persons all over the world, with approximately 250,000 annual deaths [1], and it is expected that the number of the patients will increase by more than 100 million by 2025 [2]. e pathogenesis of asthma is not yet clear and may be related to genetic, immune, environment, spirit, sex, and other related factors, in which the immunological patho- genesis of asthma has become a current research hot spot. Asthma is classically recognized as the typical 2 disease, with increased IgE levels and eosinophilic inflammation in the airway. Emerging 2 cytokines modulate the airway inflammation, which induces airway remodeling [3]. Biolog- ical agents, which have specific molecular targets for these 2 cytokines, are available and clinical trials for asthma are ongoing. However, the relatively simple paradigm has been doubted because of the realization that strategies designed to suppress 2 function are not effective enough for all patients in the clinical trials. In the future, it is required to understand more details for phenotypes of asthma. Nowadays, it is known that 17 cells and Treg cells also modulate asthma. 17 cells produce IL-17A, IL-17F, and IL-22. ese cytokines induce airway inflammation and IL-17A enhances smooth muscle contractility. IL-17 can promote fibroblast cells, epithelial cells, endothelial cells, macrophages, and smooth muscle cells activation, make Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2016, Article ID 6916353, 7 pages http://dx.doi.org/10.1155/2016/6916353
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Research ArticleYupingfeng Pulvis Regulates the Balance ofT Cell Subsets in Asthma Mice
Zhigang Wang12 Xueting Cai12 Zhonghua Pang12 Dawei Wang12 Juan Ye12 Kelei Su12
Xiaoyan Sun12 Jing Li12 Peng Cao12 and Chunping Hu12
1Affiliated Hospital of Integrated Traditional Chinese and Western Medicine Nanjing University of Chinese Medicine NanjingJiangsu 210028 China2Laboratory of Cellular and Molecular Biology Jiangsu Province Academy of Traditional Chinese Medicine NanjingJiangsu 210028 China
Correspondence should be addressed to Chunping Hu njhcp66126com
Received 10 January 2016 Accepted 22 March 2016
Academic Editor Vincenzo De Feo
Copyright copy 2016 Zhigang Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Background Yupingfeng Pulvis (HFBP) had played an active role inmany diseases especially respiratory tract infections Exploringthe possible prevention mechanism of HFBP may provide new ideas in clinical applications for this well-known herbal formulaPurpose To study the possible mechanisms of therapy effect of HFBP on asthma mice via regulating the balance of Tregs andTh17cellsMethod The female BALBc mice were divided into five groups control group model group prednisone (55mgkg) groupand 22 gkgHFBP and 44 gkgHFBP groups Ovalbuminwas used tomake the asthmamodel ofmice the drug was ig administereddaily after atomization for consecutive 15 dThemicewere killed after the last administrationTheparaffin-embedded tissue sectionsof the lungs were stained by HampE Tregs andTh17 cells in bronchoalveolar lavage fluid were detected by flow cytometry IL-4 TGF-120573 and TNF-120572 in the serum were detected by ELISA assay Results HFBP could alleviate the inflammation in the lung tissue ofmice decrease the proportion of Th17 cells and increase the proportion of Treg cells in bronchoalveolar lavage fluid HFBP coulddecrease IL-4 and TNF-120572 level and increase TGF-120573 level in blood Conclusion HFBP could treat the asthma through impacting thebalance of Th17 cells and Treg cells as well as the levels of related inflammatory cytokines in asthma mice
1 Introduction
Asthma is a common chronic inflammatory disease of theairways characterized by variable and recurring symptomsreversible airflow obstruction and bronchospasm Commonsymptoms include wheezing coughing chest tightness andshortness of breath Over the past few decades the worldasthmaprevalence andmortality have been a rising trend yearby year having impact on social economy and health Thedisease is affectingmore than 300million persons all over theworld with approximately 250000 annual deaths [1] and it isexpected that the number of the patientswill increase bymorethan 100 million by 2025 [2]
The pathogenesis of asthma is not yet clear and maybe related to genetic immune environment spirit sex andother related factors in which the immunological patho-genesis of asthma has become a current research hot spot
Asthma is classically recognized as the typical Th2 diseasewith increased IgE levels and eosinophilic inflammation inthe airway Emerging Th2 cytokines modulate the airwayinflammation which induces airway remodeling [3] Biolog-ical agents which have specific molecular targets for theseTh2 cytokines are available and clinical trials for asthma areongoing However the relatively simple paradigm has beendoubted because of the realization that strategies designed tosuppressTh2 function are not effective enough for all patientsin the clinical trials In the future it is required to understandmore details for phenotypes of asthma
Nowadays it is known that Th17 cells and Treg cellsalso modulate asthma Th17 cells produce IL-17A IL-17Fand IL-22 These cytokines induce airway inflammationand IL-17A enhances smooth muscle contractility IL-17 canpromote fibroblast cells epithelial cells endothelial cellsmacrophages and smooth muscle cells activation make
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 6916353 7 pageshttpdxdoiorg10115520166916353
2 Evidence-Based Complementary and Alternative Medicine
Table 1 The composition of HFBP
Species Chinese name Plant part Origin Grams g Saposhnikovia divaricata (Trucz) Schischk Fangfeng Root Hebei China 30 250Astragalus membranaceus (Fisch) Bunge Huangqi Root Shanxi China 30 250Atractylodes macrocephala Koidz Baizhu Rhizoma Jiangsu China 60 500Total amount 120 1000
these cells highly express a variety of proinflammatoryfactor such as IL-6 and IL-8 and release granulocyte colonystimulating factor IL-17 also recruits dendritic cells T cellsand neutrophils to inflammation section increasing airwayinflammation [4 5] CD4+CD25+Foxp3+ regulatory T cells(regulatory T cells Tregs) are a group which has the functionof immune suppression of T cell subgroup Treg cells produceinhibitory cytokines (TGF-120573 and IL-10) and express mem-brane molecules (CTLA-4 GITR etc) and Foxp3 Studieshave found that in patients with asthma the specific tran-scription factor Foxp3 expression reduced and the inhibitionfunction and the proportion of CD25hi regulatory T cells alsoreduced [6 7] Therefore studying the differentiation andregulation mechanism of Th17 cell will help us to deepenunderstanding of the relationship betweenTh17 cells and thepathogenesis of asthma as well as the development of newimmunosuppressive drugs
Yupingfeng Pulvis (HFBP) is recorded in Effective Pre-scriptions Handed Down for Generations written by Yi-linWei in Yuan Dynasty It consists of three commonly usedherbs including Saposhnikovia divaricata (Trucz) SchischkAstragalus membranaceus (Fisch) Bunge and Atractylodesmacrocephala Koidz Currently clinical reports about HFBPare increasing It had played an active role in many diseasesespecially treatment of respiratory tract infections To studythe possible mechanisms of therapy effect of HFBP onasthma asthmamodel mice were established and the balanceof Tregs andTh17 cells was evaluated Exploring the possibleprevention mechanism of HFBP may provide new ideas inclinical applications for this well-known herbal formula
2 Materials and Methods
21 Animals Female BALBc mice were purchased fromShanghai SLRC Laboratory Animal Co Ltd (ShanghaiChina) Mice were housed under specific pathogen-freeconditions and provided with a standard rodent laboratorydiet from Shanghai SLRC Laboratory Animal Co Ltd
22 HFBP Preparation HFBP is composed of three medici-nal components Saposhnikovia divaricata (Trucz) SchischkAstragalus membranaceus (Fisch) Bunge and Atractylodesmacrocephala Koidz (Table 1) All medicinal plants usedto prepare formulas were provided by Jiangsu ProvinceHospital on Integration of Chinese and Western Medicine(Nanjing Jiangsu China) The plant name and part usedwere shown in Table 1 (the plant name has been checkedwith httpwwwtheplantlistorg) All the herbal drugs wereauthenticated by Professor Song-Lin Li (Jiangsu Province
Academy of Traditional Chinese Medicine Nanjing China)according to the monographs documented in the ChinesePharmacopeia (Part I 2010 Version) Voucher specimens ofcrude drugs were deposited at the Laboratory of Cellularand Molecular Biology at Jiangsu Province Academy of Tra-ditional Chinese Medicine (Nanjing China) HFBP extractwas prepared according to the following procedure singlecrude herb was homogenized with a Waring blender Thepowders of three medicinal herbs were mixed in proportion(Table 1) and refluxed with ten volumes of water for 2 h aftermaceration for 24 hThe filtrates obtained from 2 cycles of theextraction procedure were combined and dried by a vacuum-drier at 60∘Cand groundTheyield of dried extracts forHFBPwas 22 (ww) of the weight of original herbs
23 HPLC Analysis of HFBP After the centrifugation at3000 rpm for 5min the HFBP supernatant was filtered bymembrane filter (045 120583m) and subjected for HPLC analysisThe experiment was conducted by an Agilent 1200 HPLCinstrument (Agilent USA) equipped with a XTerra MSC18 (250mm times 46mm 5 120583m) column The mobile phaseconsisted of 01 (vv) formic acid (A) and acetonitrile (B)at a flow rate of 10mLmin A gradient program was used asfollows 0min 5 B 10min 15 B 20min 20 B 30min28 B 40min 40 B 45ndash50min 60 B 60min 70 B65ndash70min 95 B The diode array detector scanned from200 nm to 400 nm and the monitor wavelength was set at250 nm
24The Establishment ofMouseModels of Asthma Weused aprotocol slightly modified from that described by McMillanand Lloyd [8] Briefly female BALBc mice were sensitizedintraperitoneally with 20120583g ovalbumin (OVA) and 2mgAl(OH)
3on days 1 and 14 (OVA groups 119899 = 40) The
blank control group (119899 = 10) was injected with salineat the respective time points On day 28 OVA groupsrsquomice were random divided into four groups (model group22 gkg HFBP group 44 gkg HFBP group and 55mgkgprednisone group) (119899 = 10) and inhaled aerosol 1 OVAsolution for 30min for five daysThe blank control group wasgiven the respective vehicle aerosol inhalation HFBP groupswith different doses were administrated intragastrically withHFBP every day 55mgkg prednisone was administratedintragastrically every day to the positive control group Theblank control group and model group were administratedwith equal volume of saline since aerosol inhalation Allgroups were administrated for 15 days Blood was drawnat 24 h after the last intragastric administration to detectcytokines IL-4 TGF-120573 and TNF-120572 Bronchoalveolar lavage
Evidence-Based Complementary and Alternative Medicine 3
fluid (BALF) was collected to count the number of Th17cells and Treg cells Left upper lung lobes were collected forpathologic histology
25 ELISA The concentrations of cytokines IL-4 TGF-120573and TNF-120572 in the blood from all groups were measuredusing a commercially available ELISA kit (Nanjing JianchengBioengineering Institute China) ELISA assaywas performedaccording to the manufacturerrsquos instructions of the ELISAkits
26 Flow Cytometric Analysis For detecting the percentageofTh17 cells cells in BALFwere collected and stimulatedwith10 ngmL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich USA) and 10 ngmL brefeldin A (BFA) (Sigma-Aldrich USA) in RPMI-1640 medium (Invitrogen USA) in96-well plates After being stimulated for 5 h (37∘C 5 CO
2)
the cells were collected and washed once with PBS The cellswere then incubated with CD4-FITC antibody at 4∘C for30 minutes Next the cells were fixed and permeabilizedand stained with anti-human IL-17-PE antibody at 37∘Cfor 30 minutes For detecting the percentage of Treg cellsthe cells were washed in PBS Then the cells were stainedwith CD4-FITC and CD25-APC antibodies at 4∘C for 30minutes Then the cells were incubated with Foxp3-PEantibody after fixation and permeabilization according to themanufacturerrsquos instruction All stained cells were analyzedby flow cytometer (Guava 6HT Merck-Millipore USA) Thedata were analyzed using the software Guava 25
27 Immunohistochemistry Assay Lung tissue samples ofeach group were cut into sections of approximately 05 cm2sizes and fixed in 10 formalin for at least 48 hours Thefixed samples were placed in plastic cassettes and dehydratedusing an automated tissue processor The processed tissueswere embedded in paraffin wax (Leica Germany) and theblocks trimmed and sectioned to about 5 times 5 times 4 120583m sizeusing amicrotomeThe tissue sectionsweremounted on glassslides using a hot plate and subsequently treated in order with100 90 and 70 ethanol for two minutes each Finally thesections were rinsed with tap water and stained with Harrisrsquoshaematoxylin and eosin for light microscopy
28 Statistical Evaluation All results shown represent theMean plusmn SD from triplicate experiments performed in a par-allel manner unless otherwise indicated Statistical analyseswere performed using an unpaired two-tailed Studentrsquos 119905-test
3 Results
31 HPLC Fingerprint of HFBP Extracts HPLC analysiswas conducted to confirm the biological composition ofHFBP extract resulting in the identification of 9 chemicalcomponents with reference standards (Figure 1) Since HFBPis a polyherbal formulation with complicated compositiontwo batches of HFBP extracts were thus prepared underthe same condition and analyzed by HPLC The resultsshowed that all the main chromatographic peaks detected
in batch I coincided with batch II demonstrating a goodreproducibility of HFBP chemical composition Figure 1shows the HPLC fingerprint of two batches of HFBP extractsand 3D-HPLC chromatogram of batch I HFBP extracts Inorder to evaluate the quality of HFBP extracts an externalstandard method was applied to quantitatively analyze fivemajor compounds (prim-O-glucosylcimifugin calycosin-7-O-120573-D-glucoside macrotin 5-O-methylvisammioside andononin) in the HFBP samplesThe external standard methodwas validated in terms of linearity precision accuracy andstability The quantitative results are presented in Table 2
32 HFBP Impact on Serum Inflammatory Factors in AsthmaMice Experiments were made to determine the seruminflammatory cytokines IL-4 TGF-120573 and TNF-120572 as shownin Figure 2 IL-4 and TNF-120572 increased in model grouptwo HFBP groups could significantly reduce serum IL-4and TNF-120572 levels so did prednisone group TGF-120573 as asuppression of inflammation factor reduced in the modelgroup prednisone can raise TGF-120573 level by contrast HFBPgroups could increase the level of the serum content of TGF-120573more than prednisone group
33 The Percentages of Th17 and Treg Cells in BALF in FiveGroups Flow cytometry results showed that the percentageofTh17 cells inmodel group significantly enhanced comparedwith blank control group and the proportion of Treg cellsdecreased obviously Positive medicine prednisone couldreduce Th17 cells and increase Treg cells Both HFBP groupscould reduce the percentage of Th17 cells and increase theproportion of Treg cells and the effect of HFBP was betterthan prednisone (Figures 3(a) and 3(b))
34 Effect of HFBP on Lung Tissue Pathology in AsthmaMice HampE staining of lung tissue showed that there wereno obvious pathological changes of lung tissue observed inthe blank control group mice In the model group micea large number of inflammatory cells infiltrated bronchialwall of lung tissue The prednisone group could improvethe lung tissue of the inflammatory cells invasion both highand low dose HFBP groups could improve the bronchialinflammation situation lung damage had a greater degree ofrecovery after HFBP treatment showing that HFBP had agood treatment effect as shown in Figure 4
4 Discussion
Asthma treatment goal is to reduce attack frequency improverespiratory function and life quality control acute onsetprevent deterioration and avoid death At present the treat-ment of asthma is mainly western medicine traditional Chi-nese medicine (TCM) is used as complementary therapiesWestern medicine treatment of asthma is mainly with anti-inflammatory drugs complementarywith spasmolytic agentsand apophlegmatisant Western medicine has achieved verygood curative effect in the control of asthma symptomsbut still cannot reduce the recurrence rate of asthma andhas the side effects due to long-term use of these drugsTraditional Chinese medicine has a long history in treating
4 Evidence-Based Complementary and Alternative Medicine
Table2Th
econ
tentso
ffive
major
compo
unds
inHFB
Pby
HPL
Canalysis
Peak
number119905 119877
(min)
Com
poun
dname
Linearity
Sensitivity(120583gmL)
Precision
(RSD
119899=6)
Stability(RSD
119899=3)
Con
tent
()
Equatio
n1198772
LOD
LOQ
Intra-day
Inter-day
11637
Prim
-O-glucosylcim
ifugin
119910=40565119909+14955
09998
003
005
024
138
040
0202
21923
Calycosin
-7-O
-120573-D
-glucosid
e119910=73499119909+64019
09997
0027
0055
026
158
051
0043
32133
Macrotin
119910=60171119909+50145
09997
0047
0023
016
159
056
0035
42403
41015840-O
-120573-D
-Glucosyl-5
-O-m
ethylvisa
mminol119910=45658119909+17985
09996
0031
0051
017
133
018
0221
52969
Ono
nin
119910=76593119909+60616
09997
020
140
048
0021
Evidence-Based Complementary and Alternative Medicine 5
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(a)
8 9
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(min)
(mAU
)
50
40
30
20
10
0
30 35 40 45 50 55
(b)
1 23 4 5
6 7
8
9
(mAU
)
10 20 30 40 50 60
(min)
1200
1000
800
600
400
200
0
(c)12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
200250300350400 100
200
300
400
(d)
Figure 1 HPLC fingerprint of HFBP extracts and reference compounds (a) HFB extract (batch I) (b) HFB extract (batch II) (c) referencecompounds (d) 3D-HPLC fingerprint of HFBP extract (batch I) 1 prim-O-glucosylcimifugin 2 calycosin-7-O-120573-D-glucoside 3 macrotin4 5-O-methylvisammioside 5 ononin 6 calycosin 7 sec-O-glucosylhamaudol 8 formononetin 9 atractylenolide I
000
10000
20000
30000
40000
50000
IL-4
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowast
(a)
000
20000
40000
60000
80000
100000
120000
TGF-120573
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowastlowast
lowastlowast
(b)
000
20000
40000
60000
80000
TNF-120572
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
(c)
Figure 2 Detection of levels of IL-4 (a) TGF-120573 (b) and TNF-120572 (c) in serumofmice by ELISA (Meanplusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt 001versus model group
6 Evidence-Based Complementary and Alternative Medicine
104
104
103
103
102
102
101
101100
100
IL-17
-PE
(YEL
-HLo
g)IL
-17
-PE
CD4-FITC (GRN-HLog)CD4-FITC
Plot P03 gated on P01R1
097
Control104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
513
Model104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
353
Prednisone(55mgkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
113
HFBP(22 gkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
127
HFBP(44gkg)
(a)
104
103
102
101
100
104103102101100
213Plot P03 gated on P01R1R2
Foxp
3-P
E (Y
EL-H
Log)
Foxp
3-P
E
CD25-APC (RED2-HLog)CD25-APC CD25-APC CD25-APC CD25-APC CD25-APC
Control
104103102101100
052Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Model
104103102101100
071Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Prednisone(55mgkg)
104103102101100
077Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(22 gkg)
104103102101100
102Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(44gkg)
(b)
Figure 3 Detection for proportion of Th17 cells (a) and Treg cells (b) in mice by flow cytometry (Mean plusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt001 versus model group
Control Model Prednisone (55mgkg) HFBP (22 gkg) HFBP (44gkg)
Figure 4 HampE staining of lung of asthma mice of each group (optical microscopy times200)
asthma and has rich pharmaceutical with low cost andsmall side effects Traditional Chinese medicine blocked achain reaction of inflammation in particular to improve themicroenvironment to eliminate airway chronic inflammationin the airway prevent the occurrence of airway remodelingand regulate the immune system by changing the immunegene expression reduce airway hyperresponsiveness stabi-lize environment within the body and enhance the adaptiveadjustment ability TCM gives full play to the advantages ofthe overall treatment
The ingredients of HFBP are fewer but better only threecomponents radix astragali radix saposhnikoviae and rhi-zoma atractylodis macrocephalae Radix astragali is especiallysuitable for the treatment of deficiency and night sweatpeople which is the main drug of the formulas Rhizomaatractylodis macrocephalae is the adjuvant drug of the for-mulas Radix saposhnikoviae called ldquoPinfengrdquo can releaseexterior and dispel wind In recent years more and more
pharmacological effects of HFBP have been revealed for itsldquoreplenishing qi and consolidating exteriorrdquo pharmacody-namics basis Previous studies have shown that HFBP hasgood immunity effect on allergic diseases such as asthmaallergic rhinitis and allergic conjunctivitis [9ndash11] Its phar-macological mechanismmay be through promotingTh1 cellsand the expression of IFN-120574 inhibition of Th2 cells and thesecretion of cytokines thus improving the Th1Th2 ratio andthe state of inflammation of the respiratory tract [9] Th17cells and Treg cells have obvious change in bronchial asthmapatients Higher levels of Th17 cells raise the inflammation oflung tissue lower level of Treg cells reduces the inhibitioneffect of inflammatory cells and inflammatory factor whichcan promote the occurrence of asthma disease [12 13] HFBPhas a good immunity effect on allergic diseases yet there is noevidence about its relationship with the balance of Th17 cellsand Treg cells
Evidence-Based Complementary and Alternative Medicine 7
Therefore we designed the experiment to investigate therelationship between Th17 cells differentiation mechanismand the onset of asthma and at the same time observeTh17 related cytokines expression with HFBP treatment inacute asthma mice The results showed that HFBP couldreduce asthma mice lung tissue bronchioles and perivascularinflammatory cell infiltration improve the state of airwayinflammation and reduce mucus secretion ELISA assay wasused to detect IL-4 TGF-120573 and TNF-120572 level in the blood ofmice According to the results IL-4 and TNF-120572 of asthmamice were increased compared with the blank control group(119875 lt 005) suggesting that IL-4 and TNF-120572 participate in theonset of asthma After treatingwith prednisone orHFBP IL-4and TNF-120572 level significantly reduced (119875 lt 005) suggestingthat HFBP could inhibit IL-4 and TNF-120572 expression in theblood of asthma mice TGF-120573 as a suppression of inflamma-tion factor reduced in the model group after treatment withprednisone or HFBP TGF-120573 level increased obviously Flowcytometry analysis showed that HFBP could decrease theproportion of Th17 cells and increase the proportion of Tregcells in bronchoalveolar lavage fluid which indicated thatHFBP could treat the asthma through impacting the balanceof Th17 cells and Treg cells as well as the levels of relatedinflammatory cytokines in mice
Differentiation of naıve T cells into effector cells isrequired for optimal protection against different classes ofmicrobial pathogen and for the development of immunememory Recent findings have revealed important roles forthe Notch signaling pathway in T cell differentiation intoall known effector subsets including Th1 Th2 and Tregs[14] Inhibiting Notch signaling has been shown to blockTh2 polarization by preventing Notch mediated upregulationof GATA-3 Zhou found that Astragalus injection exertedprotective effects on bleomycin-induced pulmonary fibrosisvia downregulating Jagged1Notch1 in lung [15] Atractyleno-lide I one of the main naturally occurring compounds ofAtractylodes macrocephala Koidz had the effect of reductionof expressions of Notch1 Jagged1 and downstream proteinHes1 Hey1 of Notch pathway [16] Above all we doubtedthat HFBP may impact the balance of Th17 cells and Tregcells through Notch signaling pathway Of course furtherexperiments needed to be done
Abbreviations
HFBP Yupingfeng PulvisTCM Traditional Chinese medicineTregs Regulatory T cellsOVA OvalbuminBALF Bronchoalveolar lavage fluid
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 81503573 81202967 and 81470179)
and Jiangsu Provincersquos Outstanding Leader Program of Tra-ditional Chinese Medicine
References
[1] J Bousquet T J H Clark S Hurd et al ldquoGINA guidelines onasthma and beyondrdquo Allergy vol 62 no 2 pp 102ndash112 2007
[2] M Masoli D Fabian S Holt R Beasley and Global Initiativefor Asthma (GINA) Program ldquoThe global burden of asthmaexecutive summary of the GINA Dissemination Committeereportrdquo Allergy vol 59 no 5 pp 469ndash478 2004
[3] M Kudo Y Ishigatsubo and I Aoki ldquoPathology of asthmardquoFrontiers in Microbiology vol 4 article 263 2013
[4] Y Zhao J Yang Y-D Gao and W Guo ldquoTh17 immunity inpatients with allergic asthmardquo International Archives of Allergyand Immunology vol 151 no 4 pp 297ndash307 2010
[5] L Cosmi F Liotta E Maggi S Romagnani and F AnnunziatoldquoTh17 cells new players in asthma pathogenesisrdquo Allergy vol66 no 8 pp 989ndash998 2011
[6] A Ray A Khare N Krishnamoorthy Z Qi and P RayldquoRegulatory T cells in many flavors control asthmardquo MucosalImmunology vol 3 no 3 pp 216ndash229 2010
[7] N Ohkura Y Kitagawa and S Sakaguchi ldquoDevelopment andmaintenance of regulatory T cellsrdquo Immunity vol 38 no 3 pp414ndash423 2013
[8] S J McMillan and C M Lloyd ldquoProlonged allergen challengein mice leads to persistent airway remodellingrdquo Clinical andExperimental Allergy vol 34 no 3 pp 497ndash507 2004
[9] H-Z Wang M Hong L-L Gui Y-Q Hua and H-Q XuldquoEffect of Yupingfeng San against OVA-induced allergic asthmainmicerdquo hongguo Zhong Yao ZaZhi vol 38 no 7 pp 1052ndash10552013
[10] H-Y Shi Y Zhuang and X-Y Wang ldquoEffect of yupingfengdroppill in treatment of allergic rhinitisrdquo Zhongguo Zhong YaoZa Zhi vol 39 no 12 pp 2364ndash2366 2014
[11] Y Chen ldquoEfficacy of sodium cromoglicate eye drops combinedwith yupingfeng granules in the treatment of allergic conjunc-tivitisrdquo Eye science vol 28 no 4 pp 201ndash203 2013
[12] S Langier K Sade and S Kivity ldquoRegulatory T cells in allergicasthmardquo Israel Medical Association Journal vol 14 no 3 pp180ndash183 2012
[13] H Y Kim R H Dekruyff and D T Umetsu ldquoThe many pathsto asthma phenotype shaped by innate and adaptive immunityrdquoNature Immunology vol 11 no 7 pp 577ndash584 2010
[14] D Amsen C Helbig and R A Backer ldquoNotch in T celldifferentiation all things consideredrdquo Trends in Immunologyvol 36 no 12 pp 802ndash814 2015
[15] Y Zhou S Liao Z Zhang B Wang and L Wan ldquoAstragalusinjection attenuates bleomycin-induced pulmonary fibrosis viadown-regulating Jagged1Notch1 in lungsrdquo Journal of Pharmacyand Pharmacology 2016
[16] LMa RMao K Shen et al ldquoAtractylenolide I-mediatedNotchpathway inhibition attenuates gastric cancer stem cell traitsrdquoBiochemical andBiophysical ResearchCommunications vol 450no 1 pp 353ndash359 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
Table 1 The composition of HFBP
Species Chinese name Plant part Origin Grams g Saposhnikovia divaricata (Trucz) Schischk Fangfeng Root Hebei China 30 250Astragalus membranaceus (Fisch) Bunge Huangqi Root Shanxi China 30 250Atractylodes macrocephala Koidz Baizhu Rhizoma Jiangsu China 60 500Total amount 120 1000
these cells highly express a variety of proinflammatoryfactor such as IL-6 and IL-8 and release granulocyte colonystimulating factor IL-17 also recruits dendritic cells T cellsand neutrophils to inflammation section increasing airwayinflammation [4 5] CD4+CD25+Foxp3+ regulatory T cells(regulatory T cells Tregs) are a group which has the functionof immune suppression of T cell subgroup Treg cells produceinhibitory cytokines (TGF-120573 and IL-10) and express mem-brane molecules (CTLA-4 GITR etc) and Foxp3 Studieshave found that in patients with asthma the specific tran-scription factor Foxp3 expression reduced and the inhibitionfunction and the proportion of CD25hi regulatory T cells alsoreduced [6 7] Therefore studying the differentiation andregulation mechanism of Th17 cell will help us to deepenunderstanding of the relationship betweenTh17 cells and thepathogenesis of asthma as well as the development of newimmunosuppressive drugs
Yupingfeng Pulvis (HFBP) is recorded in Effective Pre-scriptions Handed Down for Generations written by Yi-linWei in Yuan Dynasty It consists of three commonly usedherbs including Saposhnikovia divaricata (Trucz) SchischkAstragalus membranaceus (Fisch) Bunge and Atractylodesmacrocephala Koidz Currently clinical reports about HFBPare increasing It had played an active role in many diseasesespecially treatment of respiratory tract infections To studythe possible mechanisms of therapy effect of HFBP onasthma asthmamodel mice were established and the balanceof Tregs andTh17 cells was evaluated Exploring the possibleprevention mechanism of HFBP may provide new ideas inclinical applications for this well-known herbal formula
2 Materials and Methods
21 Animals Female BALBc mice were purchased fromShanghai SLRC Laboratory Animal Co Ltd (ShanghaiChina) Mice were housed under specific pathogen-freeconditions and provided with a standard rodent laboratorydiet from Shanghai SLRC Laboratory Animal Co Ltd
22 HFBP Preparation HFBP is composed of three medici-nal components Saposhnikovia divaricata (Trucz) SchischkAstragalus membranaceus (Fisch) Bunge and Atractylodesmacrocephala Koidz (Table 1) All medicinal plants usedto prepare formulas were provided by Jiangsu ProvinceHospital on Integration of Chinese and Western Medicine(Nanjing Jiangsu China) The plant name and part usedwere shown in Table 1 (the plant name has been checkedwith httpwwwtheplantlistorg) All the herbal drugs wereauthenticated by Professor Song-Lin Li (Jiangsu Province
Academy of Traditional Chinese Medicine Nanjing China)according to the monographs documented in the ChinesePharmacopeia (Part I 2010 Version) Voucher specimens ofcrude drugs were deposited at the Laboratory of Cellularand Molecular Biology at Jiangsu Province Academy of Tra-ditional Chinese Medicine (Nanjing China) HFBP extractwas prepared according to the following procedure singlecrude herb was homogenized with a Waring blender Thepowders of three medicinal herbs were mixed in proportion(Table 1) and refluxed with ten volumes of water for 2 h aftermaceration for 24 hThe filtrates obtained from 2 cycles of theextraction procedure were combined and dried by a vacuum-drier at 60∘Cand groundTheyield of dried extracts forHFBPwas 22 (ww) of the weight of original herbs
23 HPLC Analysis of HFBP After the centrifugation at3000 rpm for 5min the HFBP supernatant was filtered bymembrane filter (045 120583m) and subjected for HPLC analysisThe experiment was conducted by an Agilent 1200 HPLCinstrument (Agilent USA) equipped with a XTerra MSC18 (250mm times 46mm 5 120583m) column The mobile phaseconsisted of 01 (vv) formic acid (A) and acetonitrile (B)at a flow rate of 10mLmin A gradient program was used asfollows 0min 5 B 10min 15 B 20min 20 B 30min28 B 40min 40 B 45ndash50min 60 B 60min 70 B65ndash70min 95 B The diode array detector scanned from200 nm to 400 nm and the monitor wavelength was set at250 nm
24The Establishment ofMouseModels of Asthma Weused aprotocol slightly modified from that described by McMillanand Lloyd [8] Briefly female BALBc mice were sensitizedintraperitoneally with 20120583g ovalbumin (OVA) and 2mgAl(OH)
3on days 1 and 14 (OVA groups 119899 = 40) The
blank control group (119899 = 10) was injected with salineat the respective time points On day 28 OVA groupsrsquomice were random divided into four groups (model group22 gkg HFBP group 44 gkg HFBP group and 55mgkgprednisone group) (119899 = 10) and inhaled aerosol 1 OVAsolution for 30min for five daysThe blank control group wasgiven the respective vehicle aerosol inhalation HFBP groupswith different doses were administrated intragastrically withHFBP every day 55mgkg prednisone was administratedintragastrically every day to the positive control group Theblank control group and model group were administratedwith equal volume of saline since aerosol inhalation Allgroups were administrated for 15 days Blood was drawnat 24 h after the last intragastric administration to detectcytokines IL-4 TGF-120573 and TNF-120572 Bronchoalveolar lavage
Evidence-Based Complementary and Alternative Medicine 3
fluid (BALF) was collected to count the number of Th17cells and Treg cells Left upper lung lobes were collected forpathologic histology
25 ELISA The concentrations of cytokines IL-4 TGF-120573and TNF-120572 in the blood from all groups were measuredusing a commercially available ELISA kit (Nanjing JianchengBioengineering Institute China) ELISA assaywas performedaccording to the manufacturerrsquos instructions of the ELISAkits
26 Flow Cytometric Analysis For detecting the percentageofTh17 cells cells in BALFwere collected and stimulatedwith10 ngmL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich USA) and 10 ngmL brefeldin A (BFA) (Sigma-Aldrich USA) in RPMI-1640 medium (Invitrogen USA) in96-well plates After being stimulated for 5 h (37∘C 5 CO
2)
the cells were collected and washed once with PBS The cellswere then incubated with CD4-FITC antibody at 4∘C for30 minutes Next the cells were fixed and permeabilizedand stained with anti-human IL-17-PE antibody at 37∘Cfor 30 minutes For detecting the percentage of Treg cellsthe cells were washed in PBS Then the cells were stainedwith CD4-FITC and CD25-APC antibodies at 4∘C for 30minutes Then the cells were incubated with Foxp3-PEantibody after fixation and permeabilization according to themanufacturerrsquos instruction All stained cells were analyzedby flow cytometer (Guava 6HT Merck-Millipore USA) Thedata were analyzed using the software Guava 25
27 Immunohistochemistry Assay Lung tissue samples ofeach group were cut into sections of approximately 05 cm2sizes and fixed in 10 formalin for at least 48 hours Thefixed samples were placed in plastic cassettes and dehydratedusing an automated tissue processor The processed tissueswere embedded in paraffin wax (Leica Germany) and theblocks trimmed and sectioned to about 5 times 5 times 4 120583m sizeusing amicrotomeThe tissue sectionsweremounted on glassslides using a hot plate and subsequently treated in order with100 90 and 70 ethanol for two minutes each Finally thesections were rinsed with tap water and stained with Harrisrsquoshaematoxylin and eosin for light microscopy
28 Statistical Evaluation All results shown represent theMean plusmn SD from triplicate experiments performed in a par-allel manner unless otherwise indicated Statistical analyseswere performed using an unpaired two-tailed Studentrsquos 119905-test
3 Results
31 HPLC Fingerprint of HFBP Extracts HPLC analysiswas conducted to confirm the biological composition ofHFBP extract resulting in the identification of 9 chemicalcomponents with reference standards (Figure 1) Since HFBPis a polyherbal formulation with complicated compositiontwo batches of HFBP extracts were thus prepared underthe same condition and analyzed by HPLC The resultsshowed that all the main chromatographic peaks detected
in batch I coincided with batch II demonstrating a goodreproducibility of HFBP chemical composition Figure 1shows the HPLC fingerprint of two batches of HFBP extractsand 3D-HPLC chromatogram of batch I HFBP extracts Inorder to evaluate the quality of HFBP extracts an externalstandard method was applied to quantitatively analyze fivemajor compounds (prim-O-glucosylcimifugin calycosin-7-O-120573-D-glucoside macrotin 5-O-methylvisammioside andononin) in the HFBP samplesThe external standard methodwas validated in terms of linearity precision accuracy andstability The quantitative results are presented in Table 2
32 HFBP Impact on Serum Inflammatory Factors in AsthmaMice Experiments were made to determine the seruminflammatory cytokines IL-4 TGF-120573 and TNF-120572 as shownin Figure 2 IL-4 and TNF-120572 increased in model grouptwo HFBP groups could significantly reduce serum IL-4and TNF-120572 levels so did prednisone group TGF-120573 as asuppression of inflammation factor reduced in the modelgroup prednisone can raise TGF-120573 level by contrast HFBPgroups could increase the level of the serum content of TGF-120573more than prednisone group
33 The Percentages of Th17 and Treg Cells in BALF in FiveGroups Flow cytometry results showed that the percentageofTh17 cells inmodel group significantly enhanced comparedwith blank control group and the proportion of Treg cellsdecreased obviously Positive medicine prednisone couldreduce Th17 cells and increase Treg cells Both HFBP groupscould reduce the percentage of Th17 cells and increase theproportion of Treg cells and the effect of HFBP was betterthan prednisone (Figures 3(a) and 3(b))
34 Effect of HFBP on Lung Tissue Pathology in AsthmaMice HampE staining of lung tissue showed that there wereno obvious pathological changes of lung tissue observed inthe blank control group mice In the model group micea large number of inflammatory cells infiltrated bronchialwall of lung tissue The prednisone group could improvethe lung tissue of the inflammatory cells invasion both highand low dose HFBP groups could improve the bronchialinflammation situation lung damage had a greater degree ofrecovery after HFBP treatment showing that HFBP had agood treatment effect as shown in Figure 4
4 Discussion
Asthma treatment goal is to reduce attack frequency improverespiratory function and life quality control acute onsetprevent deterioration and avoid death At present the treat-ment of asthma is mainly western medicine traditional Chi-nese medicine (TCM) is used as complementary therapiesWestern medicine treatment of asthma is mainly with anti-inflammatory drugs complementarywith spasmolytic agentsand apophlegmatisant Western medicine has achieved verygood curative effect in the control of asthma symptomsbut still cannot reduce the recurrence rate of asthma andhas the side effects due to long-term use of these drugsTraditional Chinese medicine has a long history in treating
4 Evidence-Based Complementary and Alternative Medicine
Table2Th
econ
tentso
ffive
major
compo
unds
inHFB
Pby
HPL
Canalysis
Peak
number119905 119877
(min)
Com
poun
dname
Linearity
Sensitivity(120583gmL)
Precision
(RSD
119899=6)
Stability(RSD
119899=3)
Con
tent
()
Equatio
n1198772
LOD
LOQ
Intra-day
Inter-day
11637
Prim
-O-glucosylcim
ifugin
119910=40565119909+14955
09998
003
005
024
138
040
0202
21923
Calycosin
-7-O
-120573-D
-glucosid
e119910=73499119909+64019
09997
0027
0055
026
158
051
0043
32133
Macrotin
119910=60171119909+50145
09997
0047
0023
016
159
056
0035
42403
41015840-O
-120573-D
-Glucosyl-5
-O-m
ethylvisa
mminol119910=45658119909+17985
09996
0031
0051
017
133
018
0221
52969
Ono
nin
119910=76593119909+60616
09997
020
140
048
0021
Evidence-Based Complementary and Alternative Medicine 5
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(a)
8 9
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(min)
(mAU
)
50
40
30
20
10
0
30 35 40 45 50 55
(b)
1 23 4 5
6 7
8
9
(mAU
)
10 20 30 40 50 60
(min)
1200
1000
800
600
400
200
0
(c)12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
200250300350400 100
200
300
400
(d)
Figure 1 HPLC fingerprint of HFBP extracts and reference compounds (a) HFB extract (batch I) (b) HFB extract (batch II) (c) referencecompounds (d) 3D-HPLC fingerprint of HFBP extract (batch I) 1 prim-O-glucosylcimifugin 2 calycosin-7-O-120573-D-glucoside 3 macrotin4 5-O-methylvisammioside 5 ononin 6 calycosin 7 sec-O-glucosylhamaudol 8 formononetin 9 atractylenolide I
000
10000
20000
30000
40000
50000
IL-4
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowast
(a)
000
20000
40000
60000
80000
100000
120000
TGF-120573
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowastlowast
lowastlowast
(b)
000
20000
40000
60000
80000
TNF-120572
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
(c)
Figure 2 Detection of levels of IL-4 (a) TGF-120573 (b) and TNF-120572 (c) in serumofmice by ELISA (Meanplusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt 001versus model group
6 Evidence-Based Complementary and Alternative Medicine
104
104
103
103
102
102
101
101100
100
IL-17
-PE
(YEL
-HLo
g)IL
-17
-PE
CD4-FITC (GRN-HLog)CD4-FITC
Plot P03 gated on P01R1
097
Control104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
513
Model104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
353
Prednisone(55mgkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
113
HFBP(22 gkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
127
HFBP(44gkg)
(a)
104
103
102
101
100
104103102101100
213Plot P03 gated on P01R1R2
Foxp
3-P
E (Y
EL-H
Log)
Foxp
3-P
E
CD25-APC (RED2-HLog)CD25-APC CD25-APC CD25-APC CD25-APC CD25-APC
Control
104103102101100
052Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Model
104103102101100
071Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Prednisone(55mgkg)
104103102101100
077Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(22 gkg)
104103102101100
102Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(44gkg)
(b)
Figure 3 Detection for proportion of Th17 cells (a) and Treg cells (b) in mice by flow cytometry (Mean plusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt001 versus model group
Control Model Prednisone (55mgkg) HFBP (22 gkg) HFBP (44gkg)
Figure 4 HampE staining of lung of asthma mice of each group (optical microscopy times200)
asthma and has rich pharmaceutical with low cost andsmall side effects Traditional Chinese medicine blocked achain reaction of inflammation in particular to improve themicroenvironment to eliminate airway chronic inflammationin the airway prevent the occurrence of airway remodelingand regulate the immune system by changing the immunegene expression reduce airway hyperresponsiveness stabi-lize environment within the body and enhance the adaptiveadjustment ability TCM gives full play to the advantages ofthe overall treatment
The ingredients of HFBP are fewer but better only threecomponents radix astragali radix saposhnikoviae and rhi-zoma atractylodis macrocephalae Radix astragali is especiallysuitable for the treatment of deficiency and night sweatpeople which is the main drug of the formulas Rhizomaatractylodis macrocephalae is the adjuvant drug of the for-mulas Radix saposhnikoviae called ldquoPinfengrdquo can releaseexterior and dispel wind In recent years more and more
pharmacological effects of HFBP have been revealed for itsldquoreplenishing qi and consolidating exteriorrdquo pharmacody-namics basis Previous studies have shown that HFBP hasgood immunity effect on allergic diseases such as asthmaallergic rhinitis and allergic conjunctivitis [9ndash11] Its phar-macological mechanismmay be through promotingTh1 cellsand the expression of IFN-120574 inhibition of Th2 cells and thesecretion of cytokines thus improving the Th1Th2 ratio andthe state of inflammation of the respiratory tract [9] Th17cells and Treg cells have obvious change in bronchial asthmapatients Higher levels of Th17 cells raise the inflammation oflung tissue lower level of Treg cells reduces the inhibitioneffect of inflammatory cells and inflammatory factor whichcan promote the occurrence of asthma disease [12 13] HFBPhas a good immunity effect on allergic diseases yet there is noevidence about its relationship with the balance of Th17 cellsand Treg cells
Evidence-Based Complementary and Alternative Medicine 7
Therefore we designed the experiment to investigate therelationship between Th17 cells differentiation mechanismand the onset of asthma and at the same time observeTh17 related cytokines expression with HFBP treatment inacute asthma mice The results showed that HFBP couldreduce asthma mice lung tissue bronchioles and perivascularinflammatory cell infiltration improve the state of airwayinflammation and reduce mucus secretion ELISA assay wasused to detect IL-4 TGF-120573 and TNF-120572 level in the blood ofmice According to the results IL-4 and TNF-120572 of asthmamice were increased compared with the blank control group(119875 lt 005) suggesting that IL-4 and TNF-120572 participate in theonset of asthma After treatingwith prednisone orHFBP IL-4and TNF-120572 level significantly reduced (119875 lt 005) suggestingthat HFBP could inhibit IL-4 and TNF-120572 expression in theblood of asthma mice TGF-120573 as a suppression of inflamma-tion factor reduced in the model group after treatment withprednisone or HFBP TGF-120573 level increased obviously Flowcytometry analysis showed that HFBP could decrease theproportion of Th17 cells and increase the proportion of Tregcells in bronchoalveolar lavage fluid which indicated thatHFBP could treat the asthma through impacting the balanceof Th17 cells and Treg cells as well as the levels of relatedinflammatory cytokines in mice
Differentiation of naıve T cells into effector cells isrequired for optimal protection against different classes ofmicrobial pathogen and for the development of immunememory Recent findings have revealed important roles forthe Notch signaling pathway in T cell differentiation intoall known effector subsets including Th1 Th2 and Tregs[14] Inhibiting Notch signaling has been shown to blockTh2 polarization by preventing Notch mediated upregulationof GATA-3 Zhou found that Astragalus injection exertedprotective effects on bleomycin-induced pulmonary fibrosisvia downregulating Jagged1Notch1 in lung [15] Atractyleno-lide I one of the main naturally occurring compounds ofAtractylodes macrocephala Koidz had the effect of reductionof expressions of Notch1 Jagged1 and downstream proteinHes1 Hey1 of Notch pathway [16] Above all we doubtedthat HFBP may impact the balance of Th17 cells and Tregcells through Notch signaling pathway Of course furtherexperiments needed to be done
Abbreviations
HFBP Yupingfeng PulvisTCM Traditional Chinese medicineTregs Regulatory T cellsOVA OvalbuminBALF Bronchoalveolar lavage fluid
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 81503573 81202967 and 81470179)
and Jiangsu Provincersquos Outstanding Leader Program of Tra-ditional Chinese Medicine
References
[1] J Bousquet T J H Clark S Hurd et al ldquoGINA guidelines onasthma and beyondrdquo Allergy vol 62 no 2 pp 102ndash112 2007
[2] M Masoli D Fabian S Holt R Beasley and Global Initiativefor Asthma (GINA) Program ldquoThe global burden of asthmaexecutive summary of the GINA Dissemination Committeereportrdquo Allergy vol 59 no 5 pp 469ndash478 2004
[3] M Kudo Y Ishigatsubo and I Aoki ldquoPathology of asthmardquoFrontiers in Microbiology vol 4 article 263 2013
[4] Y Zhao J Yang Y-D Gao and W Guo ldquoTh17 immunity inpatients with allergic asthmardquo International Archives of Allergyand Immunology vol 151 no 4 pp 297ndash307 2010
[5] L Cosmi F Liotta E Maggi S Romagnani and F AnnunziatoldquoTh17 cells new players in asthma pathogenesisrdquo Allergy vol66 no 8 pp 989ndash998 2011
[6] A Ray A Khare N Krishnamoorthy Z Qi and P RayldquoRegulatory T cells in many flavors control asthmardquo MucosalImmunology vol 3 no 3 pp 216ndash229 2010
[7] N Ohkura Y Kitagawa and S Sakaguchi ldquoDevelopment andmaintenance of regulatory T cellsrdquo Immunity vol 38 no 3 pp414ndash423 2013
[8] S J McMillan and C M Lloyd ldquoProlonged allergen challengein mice leads to persistent airway remodellingrdquo Clinical andExperimental Allergy vol 34 no 3 pp 497ndash507 2004
[9] H-Z Wang M Hong L-L Gui Y-Q Hua and H-Q XuldquoEffect of Yupingfeng San against OVA-induced allergic asthmainmicerdquo hongguo Zhong Yao ZaZhi vol 38 no 7 pp 1052ndash10552013
[10] H-Y Shi Y Zhuang and X-Y Wang ldquoEffect of yupingfengdroppill in treatment of allergic rhinitisrdquo Zhongguo Zhong YaoZa Zhi vol 39 no 12 pp 2364ndash2366 2014
[11] Y Chen ldquoEfficacy of sodium cromoglicate eye drops combinedwith yupingfeng granules in the treatment of allergic conjunc-tivitisrdquo Eye science vol 28 no 4 pp 201ndash203 2013
[12] S Langier K Sade and S Kivity ldquoRegulatory T cells in allergicasthmardquo Israel Medical Association Journal vol 14 no 3 pp180ndash183 2012
[13] H Y Kim R H Dekruyff and D T Umetsu ldquoThe many pathsto asthma phenotype shaped by innate and adaptive immunityrdquoNature Immunology vol 11 no 7 pp 577ndash584 2010
[14] D Amsen C Helbig and R A Backer ldquoNotch in T celldifferentiation all things consideredrdquo Trends in Immunologyvol 36 no 12 pp 802ndash814 2015
[15] Y Zhou S Liao Z Zhang B Wang and L Wan ldquoAstragalusinjection attenuates bleomycin-induced pulmonary fibrosis viadown-regulating Jagged1Notch1 in lungsrdquo Journal of Pharmacyand Pharmacology 2016
[16] LMa RMao K Shen et al ldquoAtractylenolide I-mediatedNotchpathway inhibition attenuates gastric cancer stem cell traitsrdquoBiochemical andBiophysical ResearchCommunications vol 450no 1 pp 353ndash359 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
fluid (BALF) was collected to count the number of Th17cells and Treg cells Left upper lung lobes were collected forpathologic histology
25 ELISA The concentrations of cytokines IL-4 TGF-120573and TNF-120572 in the blood from all groups were measuredusing a commercially available ELISA kit (Nanjing JianchengBioengineering Institute China) ELISA assaywas performedaccording to the manufacturerrsquos instructions of the ELISAkits
26 Flow Cytometric Analysis For detecting the percentageofTh17 cells cells in BALFwere collected and stimulatedwith10 ngmL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich USA) and 10 ngmL brefeldin A (BFA) (Sigma-Aldrich USA) in RPMI-1640 medium (Invitrogen USA) in96-well plates After being stimulated for 5 h (37∘C 5 CO
2)
the cells were collected and washed once with PBS The cellswere then incubated with CD4-FITC antibody at 4∘C for30 minutes Next the cells were fixed and permeabilizedand stained with anti-human IL-17-PE antibody at 37∘Cfor 30 minutes For detecting the percentage of Treg cellsthe cells were washed in PBS Then the cells were stainedwith CD4-FITC and CD25-APC antibodies at 4∘C for 30minutes Then the cells were incubated with Foxp3-PEantibody after fixation and permeabilization according to themanufacturerrsquos instruction All stained cells were analyzedby flow cytometer (Guava 6HT Merck-Millipore USA) Thedata were analyzed using the software Guava 25
27 Immunohistochemistry Assay Lung tissue samples ofeach group were cut into sections of approximately 05 cm2sizes and fixed in 10 formalin for at least 48 hours Thefixed samples were placed in plastic cassettes and dehydratedusing an automated tissue processor The processed tissueswere embedded in paraffin wax (Leica Germany) and theblocks trimmed and sectioned to about 5 times 5 times 4 120583m sizeusing amicrotomeThe tissue sectionsweremounted on glassslides using a hot plate and subsequently treated in order with100 90 and 70 ethanol for two minutes each Finally thesections were rinsed with tap water and stained with Harrisrsquoshaematoxylin and eosin for light microscopy
28 Statistical Evaluation All results shown represent theMean plusmn SD from triplicate experiments performed in a par-allel manner unless otherwise indicated Statistical analyseswere performed using an unpaired two-tailed Studentrsquos 119905-test
3 Results
31 HPLC Fingerprint of HFBP Extracts HPLC analysiswas conducted to confirm the biological composition ofHFBP extract resulting in the identification of 9 chemicalcomponents with reference standards (Figure 1) Since HFBPis a polyherbal formulation with complicated compositiontwo batches of HFBP extracts were thus prepared underthe same condition and analyzed by HPLC The resultsshowed that all the main chromatographic peaks detected
in batch I coincided with batch II demonstrating a goodreproducibility of HFBP chemical composition Figure 1shows the HPLC fingerprint of two batches of HFBP extractsand 3D-HPLC chromatogram of batch I HFBP extracts Inorder to evaluate the quality of HFBP extracts an externalstandard method was applied to quantitatively analyze fivemajor compounds (prim-O-glucosylcimifugin calycosin-7-O-120573-D-glucoside macrotin 5-O-methylvisammioside andononin) in the HFBP samplesThe external standard methodwas validated in terms of linearity precision accuracy andstability The quantitative results are presented in Table 2
32 HFBP Impact on Serum Inflammatory Factors in AsthmaMice Experiments were made to determine the seruminflammatory cytokines IL-4 TGF-120573 and TNF-120572 as shownin Figure 2 IL-4 and TNF-120572 increased in model grouptwo HFBP groups could significantly reduce serum IL-4and TNF-120572 levels so did prednisone group TGF-120573 as asuppression of inflammation factor reduced in the modelgroup prednisone can raise TGF-120573 level by contrast HFBPgroups could increase the level of the serum content of TGF-120573more than prednisone group
33 The Percentages of Th17 and Treg Cells in BALF in FiveGroups Flow cytometry results showed that the percentageofTh17 cells inmodel group significantly enhanced comparedwith blank control group and the proportion of Treg cellsdecreased obviously Positive medicine prednisone couldreduce Th17 cells and increase Treg cells Both HFBP groupscould reduce the percentage of Th17 cells and increase theproportion of Treg cells and the effect of HFBP was betterthan prednisone (Figures 3(a) and 3(b))
34 Effect of HFBP on Lung Tissue Pathology in AsthmaMice HampE staining of lung tissue showed that there wereno obvious pathological changes of lung tissue observed inthe blank control group mice In the model group micea large number of inflammatory cells infiltrated bronchialwall of lung tissue The prednisone group could improvethe lung tissue of the inflammatory cells invasion both highand low dose HFBP groups could improve the bronchialinflammation situation lung damage had a greater degree ofrecovery after HFBP treatment showing that HFBP had agood treatment effect as shown in Figure 4
4 Discussion
Asthma treatment goal is to reduce attack frequency improverespiratory function and life quality control acute onsetprevent deterioration and avoid death At present the treat-ment of asthma is mainly western medicine traditional Chi-nese medicine (TCM) is used as complementary therapiesWestern medicine treatment of asthma is mainly with anti-inflammatory drugs complementarywith spasmolytic agentsand apophlegmatisant Western medicine has achieved verygood curative effect in the control of asthma symptomsbut still cannot reduce the recurrence rate of asthma andhas the side effects due to long-term use of these drugsTraditional Chinese medicine has a long history in treating
4 Evidence-Based Complementary and Alternative Medicine
Table2Th
econ
tentso
ffive
major
compo
unds
inHFB
Pby
HPL
Canalysis
Peak
number119905 119877
(min)
Com
poun
dname
Linearity
Sensitivity(120583gmL)
Precision
(RSD
119899=6)
Stability(RSD
119899=3)
Con
tent
()
Equatio
n1198772
LOD
LOQ
Intra-day
Inter-day
11637
Prim
-O-glucosylcim
ifugin
119910=40565119909+14955
09998
003
005
024
138
040
0202
21923
Calycosin
-7-O
-120573-D
-glucosid
e119910=73499119909+64019
09997
0027
0055
026
158
051
0043
32133
Macrotin
119910=60171119909+50145
09997
0047
0023
016
159
056
0035
42403
41015840-O
-120573-D
-Glucosyl-5
-O-m
ethylvisa
mminol119910=45658119909+17985
09996
0031
0051
017
133
018
0221
52969
Ono
nin
119910=76593119909+60616
09997
020
140
048
0021
Evidence-Based Complementary and Alternative Medicine 5
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(a)
8 9
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(min)
(mAU
)
50
40
30
20
10
0
30 35 40 45 50 55
(b)
1 23 4 5
6 7
8
9
(mAU
)
10 20 30 40 50 60
(min)
1200
1000
800
600
400
200
0
(c)12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
200250300350400 100
200
300
400
(d)
Figure 1 HPLC fingerprint of HFBP extracts and reference compounds (a) HFB extract (batch I) (b) HFB extract (batch II) (c) referencecompounds (d) 3D-HPLC fingerprint of HFBP extract (batch I) 1 prim-O-glucosylcimifugin 2 calycosin-7-O-120573-D-glucoside 3 macrotin4 5-O-methylvisammioside 5 ononin 6 calycosin 7 sec-O-glucosylhamaudol 8 formononetin 9 atractylenolide I
000
10000
20000
30000
40000
50000
IL-4
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowast
(a)
000
20000
40000
60000
80000
100000
120000
TGF-120573
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowastlowast
lowastlowast
(b)
000
20000
40000
60000
80000
TNF-120572
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
(c)
Figure 2 Detection of levels of IL-4 (a) TGF-120573 (b) and TNF-120572 (c) in serumofmice by ELISA (Meanplusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt 001versus model group
6 Evidence-Based Complementary and Alternative Medicine
104
104
103
103
102
102
101
101100
100
IL-17
-PE
(YEL
-HLo
g)IL
-17
-PE
CD4-FITC (GRN-HLog)CD4-FITC
Plot P03 gated on P01R1
097
Control104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
513
Model104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
353
Prednisone(55mgkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
113
HFBP(22 gkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
127
HFBP(44gkg)
(a)
104
103
102
101
100
104103102101100
213Plot P03 gated on P01R1R2
Foxp
3-P
E (Y
EL-H
Log)
Foxp
3-P
E
CD25-APC (RED2-HLog)CD25-APC CD25-APC CD25-APC CD25-APC CD25-APC
Control
104103102101100
052Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Model
104103102101100
071Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Prednisone(55mgkg)
104103102101100
077Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(22 gkg)
104103102101100
102Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(44gkg)
(b)
Figure 3 Detection for proportion of Th17 cells (a) and Treg cells (b) in mice by flow cytometry (Mean plusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt001 versus model group
Control Model Prednisone (55mgkg) HFBP (22 gkg) HFBP (44gkg)
Figure 4 HampE staining of lung of asthma mice of each group (optical microscopy times200)
asthma and has rich pharmaceutical with low cost andsmall side effects Traditional Chinese medicine blocked achain reaction of inflammation in particular to improve themicroenvironment to eliminate airway chronic inflammationin the airway prevent the occurrence of airway remodelingand regulate the immune system by changing the immunegene expression reduce airway hyperresponsiveness stabi-lize environment within the body and enhance the adaptiveadjustment ability TCM gives full play to the advantages ofthe overall treatment
The ingredients of HFBP are fewer but better only threecomponents radix astragali radix saposhnikoviae and rhi-zoma atractylodis macrocephalae Radix astragali is especiallysuitable for the treatment of deficiency and night sweatpeople which is the main drug of the formulas Rhizomaatractylodis macrocephalae is the adjuvant drug of the for-mulas Radix saposhnikoviae called ldquoPinfengrdquo can releaseexterior and dispel wind In recent years more and more
pharmacological effects of HFBP have been revealed for itsldquoreplenishing qi and consolidating exteriorrdquo pharmacody-namics basis Previous studies have shown that HFBP hasgood immunity effect on allergic diseases such as asthmaallergic rhinitis and allergic conjunctivitis [9ndash11] Its phar-macological mechanismmay be through promotingTh1 cellsand the expression of IFN-120574 inhibition of Th2 cells and thesecretion of cytokines thus improving the Th1Th2 ratio andthe state of inflammation of the respiratory tract [9] Th17cells and Treg cells have obvious change in bronchial asthmapatients Higher levels of Th17 cells raise the inflammation oflung tissue lower level of Treg cells reduces the inhibitioneffect of inflammatory cells and inflammatory factor whichcan promote the occurrence of asthma disease [12 13] HFBPhas a good immunity effect on allergic diseases yet there is noevidence about its relationship with the balance of Th17 cellsand Treg cells
Evidence-Based Complementary and Alternative Medicine 7
Therefore we designed the experiment to investigate therelationship between Th17 cells differentiation mechanismand the onset of asthma and at the same time observeTh17 related cytokines expression with HFBP treatment inacute asthma mice The results showed that HFBP couldreduce asthma mice lung tissue bronchioles and perivascularinflammatory cell infiltration improve the state of airwayinflammation and reduce mucus secretion ELISA assay wasused to detect IL-4 TGF-120573 and TNF-120572 level in the blood ofmice According to the results IL-4 and TNF-120572 of asthmamice were increased compared with the blank control group(119875 lt 005) suggesting that IL-4 and TNF-120572 participate in theonset of asthma After treatingwith prednisone orHFBP IL-4and TNF-120572 level significantly reduced (119875 lt 005) suggestingthat HFBP could inhibit IL-4 and TNF-120572 expression in theblood of asthma mice TGF-120573 as a suppression of inflamma-tion factor reduced in the model group after treatment withprednisone or HFBP TGF-120573 level increased obviously Flowcytometry analysis showed that HFBP could decrease theproportion of Th17 cells and increase the proportion of Tregcells in bronchoalveolar lavage fluid which indicated thatHFBP could treat the asthma through impacting the balanceof Th17 cells and Treg cells as well as the levels of relatedinflammatory cytokines in mice
Differentiation of naıve T cells into effector cells isrequired for optimal protection against different classes ofmicrobial pathogen and for the development of immunememory Recent findings have revealed important roles forthe Notch signaling pathway in T cell differentiation intoall known effector subsets including Th1 Th2 and Tregs[14] Inhibiting Notch signaling has been shown to blockTh2 polarization by preventing Notch mediated upregulationof GATA-3 Zhou found that Astragalus injection exertedprotective effects on bleomycin-induced pulmonary fibrosisvia downregulating Jagged1Notch1 in lung [15] Atractyleno-lide I one of the main naturally occurring compounds ofAtractylodes macrocephala Koidz had the effect of reductionof expressions of Notch1 Jagged1 and downstream proteinHes1 Hey1 of Notch pathway [16] Above all we doubtedthat HFBP may impact the balance of Th17 cells and Tregcells through Notch signaling pathway Of course furtherexperiments needed to be done
Abbreviations
HFBP Yupingfeng PulvisTCM Traditional Chinese medicineTregs Regulatory T cellsOVA OvalbuminBALF Bronchoalveolar lavage fluid
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 81503573 81202967 and 81470179)
and Jiangsu Provincersquos Outstanding Leader Program of Tra-ditional Chinese Medicine
References
[1] J Bousquet T J H Clark S Hurd et al ldquoGINA guidelines onasthma and beyondrdquo Allergy vol 62 no 2 pp 102ndash112 2007
[2] M Masoli D Fabian S Holt R Beasley and Global Initiativefor Asthma (GINA) Program ldquoThe global burden of asthmaexecutive summary of the GINA Dissemination Committeereportrdquo Allergy vol 59 no 5 pp 469ndash478 2004
[3] M Kudo Y Ishigatsubo and I Aoki ldquoPathology of asthmardquoFrontiers in Microbiology vol 4 article 263 2013
[4] Y Zhao J Yang Y-D Gao and W Guo ldquoTh17 immunity inpatients with allergic asthmardquo International Archives of Allergyand Immunology vol 151 no 4 pp 297ndash307 2010
[5] L Cosmi F Liotta E Maggi S Romagnani and F AnnunziatoldquoTh17 cells new players in asthma pathogenesisrdquo Allergy vol66 no 8 pp 989ndash998 2011
[6] A Ray A Khare N Krishnamoorthy Z Qi and P RayldquoRegulatory T cells in many flavors control asthmardquo MucosalImmunology vol 3 no 3 pp 216ndash229 2010
[7] N Ohkura Y Kitagawa and S Sakaguchi ldquoDevelopment andmaintenance of regulatory T cellsrdquo Immunity vol 38 no 3 pp414ndash423 2013
[8] S J McMillan and C M Lloyd ldquoProlonged allergen challengein mice leads to persistent airway remodellingrdquo Clinical andExperimental Allergy vol 34 no 3 pp 497ndash507 2004
[9] H-Z Wang M Hong L-L Gui Y-Q Hua and H-Q XuldquoEffect of Yupingfeng San against OVA-induced allergic asthmainmicerdquo hongguo Zhong Yao ZaZhi vol 38 no 7 pp 1052ndash10552013
[10] H-Y Shi Y Zhuang and X-Y Wang ldquoEffect of yupingfengdroppill in treatment of allergic rhinitisrdquo Zhongguo Zhong YaoZa Zhi vol 39 no 12 pp 2364ndash2366 2014
[11] Y Chen ldquoEfficacy of sodium cromoglicate eye drops combinedwith yupingfeng granules in the treatment of allergic conjunc-tivitisrdquo Eye science vol 28 no 4 pp 201ndash203 2013
[12] S Langier K Sade and S Kivity ldquoRegulatory T cells in allergicasthmardquo Israel Medical Association Journal vol 14 no 3 pp180ndash183 2012
[13] H Y Kim R H Dekruyff and D T Umetsu ldquoThe many pathsto asthma phenotype shaped by innate and adaptive immunityrdquoNature Immunology vol 11 no 7 pp 577ndash584 2010
[14] D Amsen C Helbig and R A Backer ldquoNotch in T celldifferentiation all things consideredrdquo Trends in Immunologyvol 36 no 12 pp 802ndash814 2015
[15] Y Zhou S Liao Z Zhang B Wang and L Wan ldquoAstragalusinjection attenuates bleomycin-induced pulmonary fibrosis viadown-regulating Jagged1Notch1 in lungsrdquo Journal of Pharmacyand Pharmacology 2016
[16] LMa RMao K Shen et al ldquoAtractylenolide I-mediatedNotchpathway inhibition attenuates gastric cancer stem cell traitsrdquoBiochemical andBiophysical ResearchCommunications vol 450no 1 pp 353ndash359 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
Table2Th
econ
tentso
ffive
major
compo
unds
inHFB
Pby
HPL
Canalysis
Peak
number119905 119877
(min)
Com
poun
dname
Linearity
Sensitivity(120583gmL)
Precision
(RSD
119899=6)
Stability(RSD
119899=3)
Con
tent
()
Equatio
n1198772
LOD
LOQ
Intra-day
Inter-day
11637
Prim
-O-glucosylcim
ifugin
119910=40565119909+14955
09998
003
005
024
138
040
0202
21923
Calycosin
-7-O
-120573-D
-glucosid
e119910=73499119909+64019
09997
0027
0055
026
158
051
0043
32133
Macrotin
119910=60171119909+50145
09997
0047
0023
016
159
056
0035
42403
41015840-O
-120573-D
-Glucosyl-5
-O-m
ethylvisa
mminol119910=45658119909+17985
09996
0031
0051
017
133
018
0221
52969
Ono
nin
119910=76593119909+60616
09997
020
140
048
0021
Evidence-Based Complementary and Alternative Medicine 5
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(a)
8 9
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(min)
(mAU
)
50
40
30
20
10
0
30 35 40 45 50 55
(b)
1 23 4 5
6 7
8
9
(mAU
)
10 20 30 40 50 60
(min)
1200
1000
800
600
400
200
0
(c)12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
200250300350400 100
200
300
400
(d)
Figure 1 HPLC fingerprint of HFBP extracts and reference compounds (a) HFB extract (batch I) (b) HFB extract (batch II) (c) referencecompounds (d) 3D-HPLC fingerprint of HFBP extract (batch I) 1 prim-O-glucosylcimifugin 2 calycosin-7-O-120573-D-glucoside 3 macrotin4 5-O-methylvisammioside 5 ononin 6 calycosin 7 sec-O-glucosylhamaudol 8 formononetin 9 atractylenolide I
000
10000
20000
30000
40000
50000
IL-4
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowast
(a)
000
20000
40000
60000
80000
100000
120000
TGF-120573
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowastlowast
lowastlowast
(b)
000
20000
40000
60000
80000
TNF-120572
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
(c)
Figure 2 Detection of levels of IL-4 (a) TGF-120573 (b) and TNF-120572 (c) in serumofmice by ELISA (Meanplusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt 001versus model group
6 Evidence-Based Complementary and Alternative Medicine
104
104
103
103
102
102
101
101100
100
IL-17
-PE
(YEL
-HLo
g)IL
-17
-PE
CD4-FITC (GRN-HLog)CD4-FITC
Plot P03 gated on P01R1
097
Control104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
513
Model104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
353
Prednisone(55mgkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
113
HFBP(22 gkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
127
HFBP(44gkg)
(a)
104
103
102
101
100
104103102101100
213Plot P03 gated on P01R1R2
Foxp
3-P
E (Y
EL-H
Log)
Foxp
3-P
E
CD25-APC (RED2-HLog)CD25-APC CD25-APC CD25-APC CD25-APC CD25-APC
Control
104103102101100
052Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Model
104103102101100
071Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Prednisone(55mgkg)
104103102101100
077Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(22 gkg)
104103102101100
102Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(44gkg)
(b)
Figure 3 Detection for proportion of Th17 cells (a) and Treg cells (b) in mice by flow cytometry (Mean plusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt001 versus model group
Control Model Prednisone (55mgkg) HFBP (22 gkg) HFBP (44gkg)
Figure 4 HampE staining of lung of asthma mice of each group (optical microscopy times200)
asthma and has rich pharmaceutical with low cost andsmall side effects Traditional Chinese medicine blocked achain reaction of inflammation in particular to improve themicroenvironment to eliminate airway chronic inflammationin the airway prevent the occurrence of airway remodelingand regulate the immune system by changing the immunegene expression reduce airway hyperresponsiveness stabi-lize environment within the body and enhance the adaptiveadjustment ability TCM gives full play to the advantages ofthe overall treatment
The ingredients of HFBP are fewer but better only threecomponents radix astragali radix saposhnikoviae and rhi-zoma atractylodis macrocephalae Radix astragali is especiallysuitable for the treatment of deficiency and night sweatpeople which is the main drug of the formulas Rhizomaatractylodis macrocephalae is the adjuvant drug of the for-mulas Radix saposhnikoviae called ldquoPinfengrdquo can releaseexterior and dispel wind In recent years more and more
pharmacological effects of HFBP have been revealed for itsldquoreplenishing qi and consolidating exteriorrdquo pharmacody-namics basis Previous studies have shown that HFBP hasgood immunity effect on allergic diseases such as asthmaallergic rhinitis and allergic conjunctivitis [9ndash11] Its phar-macological mechanismmay be through promotingTh1 cellsand the expression of IFN-120574 inhibition of Th2 cells and thesecretion of cytokines thus improving the Th1Th2 ratio andthe state of inflammation of the respiratory tract [9] Th17cells and Treg cells have obvious change in bronchial asthmapatients Higher levels of Th17 cells raise the inflammation oflung tissue lower level of Treg cells reduces the inhibitioneffect of inflammatory cells and inflammatory factor whichcan promote the occurrence of asthma disease [12 13] HFBPhas a good immunity effect on allergic diseases yet there is noevidence about its relationship with the balance of Th17 cellsand Treg cells
Evidence-Based Complementary and Alternative Medicine 7
Therefore we designed the experiment to investigate therelationship between Th17 cells differentiation mechanismand the onset of asthma and at the same time observeTh17 related cytokines expression with HFBP treatment inacute asthma mice The results showed that HFBP couldreduce asthma mice lung tissue bronchioles and perivascularinflammatory cell infiltration improve the state of airwayinflammation and reduce mucus secretion ELISA assay wasused to detect IL-4 TGF-120573 and TNF-120572 level in the blood ofmice According to the results IL-4 and TNF-120572 of asthmamice were increased compared with the blank control group(119875 lt 005) suggesting that IL-4 and TNF-120572 participate in theonset of asthma After treatingwith prednisone orHFBP IL-4and TNF-120572 level significantly reduced (119875 lt 005) suggestingthat HFBP could inhibit IL-4 and TNF-120572 expression in theblood of asthma mice TGF-120573 as a suppression of inflamma-tion factor reduced in the model group after treatment withprednisone or HFBP TGF-120573 level increased obviously Flowcytometry analysis showed that HFBP could decrease theproportion of Th17 cells and increase the proportion of Tregcells in bronchoalveolar lavage fluid which indicated thatHFBP could treat the asthma through impacting the balanceof Th17 cells and Treg cells as well as the levels of relatedinflammatory cytokines in mice
Differentiation of naıve T cells into effector cells isrequired for optimal protection against different classes ofmicrobial pathogen and for the development of immunememory Recent findings have revealed important roles forthe Notch signaling pathway in T cell differentiation intoall known effector subsets including Th1 Th2 and Tregs[14] Inhibiting Notch signaling has been shown to blockTh2 polarization by preventing Notch mediated upregulationof GATA-3 Zhou found that Astragalus injection exertedprotective effects on bleomycin-induced pulmonary fibrosisvia downregulating Jagged1Notch1 in lung [15] Atractyleno-lide I one of the main naturally occurring compounds ofAtractylodes macrocephala Koidz had the effect of reductionof expressions of Notch1 Jagged1 and downstream proteinHes1 Hey1 of Notch pathway [16] Above all we doubtedthat HFBP may impact the balance of Th17 cells and Tregcells through Notch signaling pathway Of course furtherexperiments needed to be done
Abbreviations
HFBP Yupingfeng PulvisTCM Traditional Chinese medicineTregs Regulatory T cellsOVA OvalbuminBALF Bronchoalveolar lavage fluid
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 81503573 81202967 and 81470179)
and Jiangsu Provincersquos Outstanding Leader Program of Tra-ditional Chinese Medicine
References
[1] J Bousquet T J H Clark S Hurd et al ldquoGINA guidelines onasthma and beyondrdquo Allergy vol 62 no 2 pp 102ndash112 2007
[2] M Masoli D Fabian S Holt R Beasley and Global Initiativefor Asthma (GINA) Program ldquoThe global burden of asthmaexecutive summary of the GINA Dissemination Committeereportrdquo Allergy vol 59 no 5 pp 469ndash478 2004
[3] M Kudo Y Ishigatsubo and I Aoki ldquoPathology of asthmardquoFrontiers in Microbiology vol 4 article 263 2013
[4] Y Zhao J Yang Y-D Gao and W Guo ldquoTh17 immunity inpatients with allergic asthmardquo International Archives of Allergyand Immunology vol 151 no 4 pp 297ndash307 2010
[5] L Cosmi F Liotta E Maggi S Romagnani and F AnnunziatoldquoTh17 cells new players in asthma pathogenesisrdquo Allergy vol66 no 8 pp 989ndash998 2011
[6] A Ray A Khare N Krishnamoorthy Z Qi and P RayldquoRegulatory T cells in many flavors control asthmardquo MucosalImmunology vol 3 no 3 pp 216ndash229 2010
[7] N Ohkura Y Kitagawa and S Sakaguchi ldquoDevelopment andmaintenance of regulatory T cellsrdquo Immunity vol 38 no 3 pp414ndash423 2013
[8] S J McMillan and C M Lloyd ldquoProlonged allergen challengein mice leads to persistent airway remodellingrdquo Clinical andExperimental Allergy vol 34 no 3 pp 497ndash507 2004
[9] H-Z Wang M Hong L-L Gui Y-Q Hua and H-Q XuldquoEffect of Yupingfeng San against OVA-induced allergic asthmainmicerdquo hongguo Zhong Yao ZaZhi vol 38 no 7 pp 1052ndash10552013
[10] H-Y Shi Y Zhuang and X-Y Wang ldquoEffect of yupingfengdroppill in treatment of allergic rhinitisrdquo Zhongguo Zhong YaoZa Zhi vol 39 no 12 pp 2364ndash2366 2014
[11] Y Chen ldquoEfficacy of sodium cromoglicate eye drops combinedwith yupingfeng granules in the treatment of allergic conjunc-tivitisrdquo Eye science vol 28 no 4 pp 201ndash203 2013
[12] S Langier K Sade and S Kivity ldquoRegulatory T cells in allergicasthmardquo Israel Medical Association Journal vol 14 no 3 pp180ndash183 2012
[13] H Y Kim R H Dekruyff and D T Umetsu ldquoThe many pathsto asthma phenotype shaped by innate and adaptive immunityrdquoNature Immunology vol 11 no 7 pp 577ndash584 2010
[14] D Amsen C Helbig and R A Backer ldquoNotch in T celldifferentiation all things consideredrdquo Trends in Immunologyvol 36 no 12 pp 802ndash814 2015
[15] Y Zhou S Liao Z Zhang B Wang and L Wan ldquoAstragalusinjection attenuates bleomycin-induced pulmonary fibrosis viadown-regulating Jagged1Notch1 in lungsrdquo Journal of Pharmacyand Pharmacology 2016
[16] LMa RMao K Shen et al ldquoAtractylenolide I-mediatedNotchpathway inhibition attenuates gastric cancer stem cell traitsrdquoBiochemical andBiophysical ResearchCommunications vol 450no 1 pp 353ndash359 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(a)
8 9
350
300
250
200
150
100
50
0
(mAU
)
10 20 30 40 50 60
(min)
(min)
(mAU
)
50
40
30
20
10
0
30 35 40 45 50 55
(b)
1 23 4 5
6 7
8
9
(mAU
)
10 20 30 40 50 60
(min)
1200
1000
800
600
400
200
0
(c)12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
200250300350400 100
200
300
400
(d)
Figure 1 HPLC fingerprint of HFBP extracts and reference compounds (a) HFB extract (batch I) (b) HFB extract (batch II) (c) referencecompounds (d) 3D-HPLC fingerprint of HFBP extract (batch I) 1 prim-O-glucosylcimifugin 2 calycosin-7-O-120573-D-glucoside 3 macrotin4 5-O-methylvisammioside 5 ononin 6 calycosin 7 sec-O-glucosylhamaudol 8 formononetin 9 atractylenolide I
000
10000
20000
30000
40000
50000
IL-4
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowast
(a)
000
20000
40000
60000
80000
100000
120000
TGF-120573
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
lowastlowastlowast
lowastlowast
(b)
000
20000
40000
60000
80000
TNF-120572
(ng
mL)
Control Model Prednisone(55mgkg)
HFBP(22 gkg)
HFBP(44gkg)
(c)
Figure 2 Detection of levels of IL-4 (a) TGF-120573 (b) and TNF-120572 (c) in serumofmice by ELISA (Meanplusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt 001versus model group
6 Evidence-Based Complementary and Alternative Medicine
104
104
103
103
102
102
101
101100
100
IL-17
-PE
(YEL
-HLo
g)IL
-17
-PE
CD4-FITC (GRN-HLog)CD4-FITC
Plot P03 gated on P01R1
097
Control104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
513
Model104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
353
Prednisone(55mgkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
113
HFBP(22 gkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
127
HFBP(44gkg)
(a)
104
103
102
101
100
104103102101100
213Plot P03 gated on P01R1R2
Foxp
3-P
E (Y
EL-H
Log)
Foxp
3-P
E
CD25-APC (RED2-HLog)CD25-APC CD25-APC CD25-APC CD25-APC CD25-APC
Control
104103102101100
052Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Model
104103102101100
071Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Prednisone(55mgkg)
104103102101100
077Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(22 gkg)
104103102101100
102Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(44gkg)
(b)
Figure 3 Detection for proportion of Th17 cells (a) and Treg cells (b) in mice by flow cytometry (Mean plusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt001 versus model group
Control Model Prednisone (55mgkg) HFBP (22 gkg) HFBP (44gkg)
Figure 4 HampE staining of lung of asthma mice of each group (optical microscopy times200)
asthma and has rich pharmaceutical with low cost andsmall side effects Traditional Chinese medicine blocked achain reaction of inflammation in particular to improve themicroenvironment to eliminate airway chronic inflammationin the airway prevent the occurrence of airway remodelingand regulate the immune system by changing the immunegene expression reduce airway hyperresponsiveness stabi-lize environment within the body and enhance the adaptiveadjustment ability TCM gives full play to the advantages ofthe overall treatment
The ingredients of HFBP are fewer but better only threecomponents radix astragali radix saposhnikoviae and rhi-zoma atractylodis macrocephalae Radix astragali is especiallysuitable for the treatment of deficiency and night sweatpeople which is the main drug of the formulas Rhizomaatractylodis macrocephalae is the adjuvant drug of the for-mulas Radix saposhnikoviae called ldquoPinfengrdquo can releaseexterior and dispel wind In recent years more and more
pharmacological effects of HFBP have been revealed for itsldquoreplenishing qi and consolidating exteriorrdquo pharmacody-namics basis Previous studies have shown that HFBP hasgood immunity effect on allergic diseases such as asthmaallergic rhinitis and allergic conjunctivitis [9ndash11] Its phar-macological mechanismmay be through promotingTh1 cellsand the expression of IFN-120574 inhibition of Th2 cells and thesecretion of cytokines thus improving the Th1Th2 ratio andthe state of inflammation of the respiratory tract [9] Th17cells and Treg cells have obvious change in bronchial asthmapatients Higher levels of Th17 cells raise the inflammation oflung tissue lower level of Treg cells reduces the inhibitioneffect of inflammatory cells and inflammatory factor whichcan promote the occurrence of asthma disease [12 13] HFBPhas a good immunity effect on allergic diseases yet there is noevidence about its relationship with the balance of Th17 cellsand Treg cells
Evidence-Based Complementary and Alternative Medicine 7
Therefore we designed the experiment to investigate therelationship between Th17 cells differentiation mechanismand the onset of asthma and at the same time observeTh17 related cytokines expression with HFBP treatment inacute asthma mice The results showed that HFBP couldreduce asthma mice lung tissue bronchioles and perivascularinflammatory cell infiltration improve the state of airwayinflammation and reduce mucus secretion ELISA assay wasused to detect IL-4 TGF-120573 and TNF-120572 level in the blood ofmice According to the results IL-4 and TNF-120572 of asthmamice were increased compared with the blank control group(119875 lt 005) suggesting that IL-4 and TNF-120572 participate in theonset of asthma After treatingwith prednisone orHFBP IL-4and TNF-120572 level significantly reduced (119875 lt 005) suggestingthat HFBP could inhibit IL-4 and TNF-120572 expression in theblood of asthma mice TGF-120573 as a suppression of inflamma-tion factor reduced in the model group after treatment withprednisone or HFBP TGF-120573 level increased obviously Flowcytometry analysis showed that HFBP could decrease theproportion of Th17 cells and increase the proportion of Tregcells in bronchoalveolar lavage fluid which indicated thatHFBP could treat the asthma through impacting the balanceof Th17 cells and Treg cells as well as the levels of relatedinflammatory cytokines in mice
Differentiation of naıve T cells into effector cells isrequired for optimal protection against different classes ofmicrobial pathogen and for the development of immunememory Recent findings have revealed important roles forthe Notch signaling pathway in T cell differentiation intoall known effector subsets including Th1 Th2 and Tregs[14] Inhibiting Notch signaling has been shown to blockTh2 polarization by preventing Notch mediated upregulationof GATA-3 Zhou found that Astragalus injection exertedprotective effects on bleomycin-induced pulmonary fibrosisvia downregulating Jagged1Notch1 in lung [15] Atractyleno-lide I one of the main naturally occurring compounds ofAtractylodes macrocephala Koidz had the effect of reductionof expressions of Notch1 Jagged1 and downstream proteinHes1 Hey1 of Notch pathway [16] Above all we doubtedthat HFBP may impact the balance of Th17 cells and Tregcells through Notch signaling pathway Of course furtherexperiments needed to be done
Abbreviations
HFBP Yupingfeng PulvisTCM Traditional Chinese medicineTregs Regulatory T cellsOVA OvalbuminBALF Bronchoalveolar lavage fluid
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 81503573 81202967 and 81470179)
and Jiangsu Provincersquos Outstanding Leader Program of Tra-ditional Chinese Medicine
References
[1] J Bousquet T J H Clark S Hurd et al ldquoGINA guidelines onasthma and beyondrdquo Allergy vol 62 no 2 pp 102ndash112 2007
[2] M Masoli D Fabian S Holt R Beasley and Global Initiativefor Asthma (GINA) Program ldquoThe global burden of asthmaexecutive summary of the GINA Dissemination Committeereportrdquo Allergy vol 59 no 5 pp 469ndash478 2004
[3] M Kudo Y Ishigatsubo and I Aoki ldquoPathology of asthmardquoFrontiers in Microbiology vol 4 article 263 2013
[4] Y Zhao J Yang Y-D Gao and W Guo ldquoTh17 immunity inpatients with allergic asthmardquo International Archives of Allergyand Immunology vol 151 no 4 pp 297ndash307 2010
[5] L Cosmi F Liotta E Maggi S Romagnani and F AnnunziatoldquoTh17 cells new players in asthma pathogenesisrdquo Allergy vol66 no 8 pp 989ndash998 2011
[6] A Ray A Khare N Krishnamoorthy Z Qi and P RayldquoRegulatory T cells in many flavors control asthmardquo MucosalImmunology vol 3 no 3 pp 216ndash229 2010
[7] N Ohkura Y Kitagawa and S Sakaguchi ldquoDevelopment andmaintenance of regulatory T cellsrdquo Immunity vol 38 no 3 pp414ndash423 2013
[8] S J McMillan and C M Lloyd ldquoProlonged allergen challengein mice leads to persistent airway remodellingrdquo Clinical andExperimental Allergy vol 34 no 3 pp 497ndash507 2004
[9] H-Z Wang M Hong L-L Gui Y-Q Hua and H-Q XuldquoEffect of Yupingfeng San against OVA-induced allergic asthmainmicerdquo hongguo Zhong Yao ZaZhi vol 38 no 7 pp 1052ndash10552013
[10] H-Y Shi Y Zhuang and X-Y Wang ldquoEffect of yupingfengdroppill in treatment of allergic rhinitisrdquo Zhongguo Zhong YaoZa Zhi vol 39 no 12 pp 2364ndash2366 2014
[11] Y Chen ldquoEfficacy of sodium cromoglicate eye drops combinedwith yupingfeng granules in the treatment of allergic conjunc-tivitisrdquo Eye science vol 28 no 4 pp 201ndash203 2013
[12] S Langier K Sade and S Kivity ldquoRegulatory T cells in allergicasthmardquo Israel Medical Association Journal vol 14 no 3 pp180ndash183 2012
[13] H Y Kim R H Dekruyff and D T Umetsu ldquoThe many pathsto asthma phenotype shaped by innate and adaptive immunityrdquoNature Immunology vol 11 no 7 pp 577ndash584 2010
[14] D Amsen C Helbig and R A Backer ldquoNotch in T celldifferentiation all things consideredrdquo Trends in Immunologyvol 36 no 12 pp 802ndash814 2015
[15] Y Zhou S Liao Z Zhang B Wang and L Wan ldquoAstragalusinjection attenuates bleomycin-induced pulmonary fibrosis viadown-regulating Jagged1Notch1 in lungsrdquo Journal of Pharmacyand Pharmacology 2016
[16] LMa RMao K Shen et al ldquoAtractylenolide I-mediatedNotchpathway inhibition attenuates gastric cancer stem cell traitsrdquoBiochemical andBiophysical ResearchCommunications vol 450no 1 pp 353ndash359 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
104
104
103
103
102
102
101
101100
100
IL-17
-PE
(YEL
-HLo
g)IL
-17
-PE
CD4-FITC (GRN-HLog)CD4-FITC
Plot P03 gated on P01R1
097
Control104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
513
Model104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
353
Prednisone(55mgkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
113
HFBP(22 gkg)
104
103
102
101
100
CD4-FITC
104103102101100
CD4-FITC (GRN-HLog)
Plot P03 gated on P01R1
127
HFBP(44gkg)
(a)
104
103
102
101
100
104103102101100
213Plot P03 gated on P01R1R2
Foxp
3-P
E (Y
EL-H
Log)
Foxp
3-P
E
CD25-APC (RED2-HLog)CD25-APC CD25-APC CD25-APC CD25-APC CD25-APC
Control
104103102101100
052Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Model
104103102101100
071Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
Prednisone(55mgkg)
104103102101100
077Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(22 gkg)
104103102101100
102Plot P03 gated on P01R1R2
104
103
102
101
100
CD25-APC (RED2-HLog)
HFBP(44gkg)
(b)
Figure 3 Detection for proportion of Th17 cells (a) and Treg cells (b) in mice by flow cytometry (Mean plusmn SD 119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt001 versus model group
Control Model Prednisone (55mgkg) HFBP (22 gkg) HFBP (44gkg)
Figure 4 HampE staining of lung of asthma mice of each group (optical microscopy times200)
asthma and has rich pharmaceutical with low cost andsmall side effects Traditional Chinese medicine blocked achain reaction of inflammation in particular to improve themicroenvironment to eliminate airway chronic inflammationin the airway prevent the occurrence of airway remodelingand regulate the immune system by changing the immunegene expression reduce airway hyperresponsiveness stabi-lize environment within the body and enhance the adaptiveadjustment ability TCM gives full play to the advantages ofthe overall treatment
The ingredients of HFBP are fewer but better only threecomponents radix astragali radix saposhnikoviae and rhi-zoma atractylodis macrocephalae Radix astragali is especiallysuitable for the treatment of deficiency and night sweatpeople which is the main drug of the formulas Rhizomaatractylodis macrocephalae is the adjuvant drug of the for-mulas Radix saposhnikoviae called ldquoPinfengrdquo can releaseexterior and dispel wind In recent years more and more
pharmacological effects of HFBP have been revealed for itsldquoreplenishing qi and consolidating exteriorrdquo pharmacody-namics basis Previous studies have shown that HFBP hasgood immunity effect on allergic diseases such as asthmaallergic rhinitis and allergic conjunctivitis [9ndash11] Its phar-macological mechanismmay be through promotingTh1 cellsand the expression of IFN-120574 inhibition of Th2 cells and thesecretion of cytokines thus improving the Th1Th2 ratio andthe state of inflammation of the respiratory tract [9] Th17cells and Treg cells have obvious change in bronchial asthmapatients Higher levels of Th17 cells raise the inflammation oflung tissue lower level of Treg cells reduces the inhibitioneffect of inflammatory cells and inflammatory factor whichcan promote the occurrence of asthma disease [12 13] HFBPhas a good immunity effect on allergic diseases yet there is noevidence about its relationship with the balance of Th17 cellsand Treg cells
Evidence-Based Complementary and Alternative Medicine 7
Therefore we designed the experiment to investigate therelationship between Th17 cells differentiation mechanismand the onset of asthma and at the same time observeTh17 related cytokines expression with HFBP treatment inacute asthma mice The results showed that HFBP couldreduce asthma mice lung tissue bronchioles and perivascularinflammatory cell infiltration improve the state of airwayinflammation and reduce mucus secretion ELISA assay wasused to detect IL-4 TGF-120573 and TNF-120572 level in the blood ofmice According to the results IL-4 and TNF-120572 of asthmamice were increased compared with the blank control group(119875 lt 005) suggesting that IL-4 and TNF-120572 participate in theonset of asthma After treatingwith prednisone orHFBP IL-4and TNF-120572 level significantly reduced (119875 lt 005) suggestingthat HFBP could inhibit IL-4 and TNF-120572 expression in theblood of asthma mice TGF-120573 as a suppression of inflamma-tion factor reduced in the model group after treatment withprednisone or HFBP TGF-120573 level increased obviously Flowcytometry analysis showed that HFBP could decrease theproportion of Th17 cells and increase the proportion of Tregcells in bronchoalveolar lavage fluid which indicated thatHFBP could treat the asthma through impacting the balanceof Th17 cells and Treg cells as well as the levels of relatedinflammatory cytokines in mice
Differentiation of naıve T cells into effector cells isrequired for optimal protection against different classes ofmicrobial pathogen and for the development of immunememory Recent findings have revealed important roles forthe Notch signaling pathway in T cell differentiation intoall known effector subsets including Th1 Th2 and Tregs[14] Inhibiting Notch signaling has been shown to blockTh2 polarization by preventing Notch mediated upregulationof GATA-3 Zhou found that Astragalus injection exertedprotective effects on bleomycin-induced pulmonary fibrosisvia downregulating Jagged1Notch1 in lung [15] Atractyleno-lide I one of the main naturally occurring compounds ofAtractylodes macrocephala Koidz had the effect of reductionof expressions of Notch1 Jagged1 and downstream proteinHes1 Hey1 of Notch pathway [16] Above all we doubtedthat HFBP may impact the balance of Th17 cells and Tregcells through Notch signaling pathway Of course furtherexperiments needed to be done
Abbreviations
HFBP Yupingfeng PulvisTCM Traditional Chinese medicineTregs Regulatory T cellsOVA OvalbuminBALF Bronchoalveolar lavage fluid
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 81503573 81202967 and 81470179)
and Jiangsu Provincersquos Outstanding Leader Program of Tra-ditional Chinese Medicine
References
[1] J Bousquet T J H Clark S Hurd et al ldquoGINA guidelines onasthma and beyondrdquo Allergy vol 62 no 2 pp 102ndash112 2007
[2] M Masoli D Fabian S Holt R Beasley and Global Initiativefor Asthma (GINA) Program ldquoThe global burden of asthmaexecutive summary of the GINA Dissemination Committeereportrdquo Allergy vol 59 no 5 pp 469ndash478 2004
[3] M Kudo Y Ishigatsubo and I Aoki ldquoPathology of asthmardquoFrontiers in Microbiology vol 4 article 263 2013
[4] Y Zhao J Yang Y-D Gao and W Guo ldquoTh17 immunity inpatients with allergic asthmardquo International Archives of Allergyand Immunology vol 151 no 4 pp 297ndash307 2010
[5] L Cosmi F Liotta E Maggi S Romagnani and F AnnunziatoldquoTh17 cells new players in asthma pathogenesisrdquo Allergy vol66 no 8 pp 989ndash998 2011
[6] A Ray A Khare N Krishnamoorthy Z Qi and P RayldquoRegulatory T cells in many flavors control asthmardquo MucosalImmunology vol 3 no 3 pp 216ndash229 2010
[7] N Ohkura Y Kitagawa and S Sakaguchi ldquoDevelopment andmaintenance of regulatory T cellsrdquo Immunity vol 38 no 3 pp414ndash423 2013
[8] S J McMillan and C M Lloyd ldquoProlonged allergen challengein mice leads to persistent airway remodellingrdquo Clinical andExperimental Allergy vol 34 no 3 pp 497ndash507 2004
[9] H-Z Wang M Hong L-L Gui Y-Q Hua and H-Q XuldquoEffect of Yupingfeng San against OVA-induced allergic asthmainmicerdquo hongguo Zhong Yao ZaZhi vol 38 no 7 pp 1052ndash10552013
[10] H-Y Shi Y Zhuang and X-Y Wang ldquoEffect of yupingfengdroppill in treatment of allergic rhinitisrdquo Zhongguo Zhong YaoZa Zhi vol 39 no 12 pp 2364ndash2366 2014
[11] Y Chen ldquoEfficacy of sodium cromoglicate eye drops combinedwith yupingfeng granules in the treatment of allergic conjunc-tivitisrdquo Eye science vol 28 no 4 pp 201ndash203 2013
[12] S Langier K Sade and S Kivity ldquoRegulatory T cells in allergicasthmardquo Israel Medical Association Journal vol 14 no 3 pp180ndash183 2012
[13] H Y Kim R H Dekruyff and D T Umetsu ldquoThe many pathsto asthma phenotype shaped by innate and adaptive immunityrdquoNature Immunology vol 11 no 7 pp 577ndash584 2010
[14] D Amsen C Helbig and R A Backer ldquoNotch in T celldifferentiation all things consideredrdquo Trends in Immunologyvol 36 no 12 pp 802ndash814 2015
[15] Y Zhou S Liao Z Zhang B Wang and L Wan ldquoAstragalusinjection attenuates bleomycin-induced pulmonary fibrosis viadown-regulating Jagged1Notch1 in lungsrdquo Journal of Pharmacyand Pharmacology 2016
[16] LMa RMao K Shen et al ldquoAtractylenolide I-mediatedNotchpathway inhibition attenuates gastric cancer stem cell traitsrdquoBiochemical andBiophysical ResearchCommunications vol 450no 1 pp 353ndash359 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
Therefore we designed the experiment to investigate therelationship between Th17 cells differentiation mechanismand the onset of asthma and at the same time observeTh17 related cytokines expression with HFBP treatment inacute asthma mice The results showed that HFBP couldreduce asthma mice lung tissue bronchioles and perivascularinflammatory cell infiltration improve the state of airwayinflammation and reduce mucus secretion ELISA assay wasused to detect IL-4 TGF-120573 and TNF-120572 level in the blood ofmice According to the results IL-4 and TNF-120572 of asthmamice were increased compared with the blank control group(119875 lt 005) suggesting that IL-4 and TNF-120572 participate in theonset of asthma After treatingwith prednisone orHFBP IL-4and TNF-120572 level significantly reduced (119875 lt 005) suggestingthat HFBP could inhibit IL-4 and TNF-120572 expression in theblood of asthma mice TGF-120573 as a suppression of inflamma-tion factor reduced in the model group after treatment withprednisone or HFBP TGF-120573 level increased obviously Flowcytometry analysis showed that HFBP could decrease theproportion of Th17 cells and increase the proportion of Tregcells in bronchoalveolar lavage fluid which indicated thatHFBP could treat the asthma through impacting the balanceof Th17 cells and Treg cells as well as the levels of relatedinflammatory cytokines in mice
Differentiation of naıve T cells into effector cells isrequired for optimal protection against different classes ofmicrobial pathogen and for the development of immunememory Recent findings have revealed important roles forthe Notch signaling pathway in T cell differentiation intoall known effector subsets including Th1 Th2 and Tregs[14] Inhibiting Notch signaling has been shown to blockTh2 polarization by preventing Notch mediated upregulationof GATA-3 Zhou found that Astragalus injection exertedprotective effects on bleomycin-induced pulmonary fibrosisvia downregulating Jagged1Notch1 in lung [15] Atractyleno-lide I one of the main naturally occurring compounds ofAtractylodes macrocephala Koidz had the effect of reductionof expressions of Notch1 Jagged1 and downstream proteinHes1 Hey1 of Notch pathway [16] Above all we doubtedthat HFBP may impact the balance of Th17 cells and Tregcells through Notch signaling pathway Of course furtherexperiments needed to be done
Abbreviations
HFBP Yupingfeng PulvisTCM Traditional Chinese medicineTregs Regulatory T cellsOVA OvalbuminBALF Bronchoalveolar lavage fluid
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (nos 81503573 81202967 and 81470179)
and Jiangsu Provincersquos Outstanding Leader Program of Tra-ditional Chinese Medicine
References
[1] J Bousquet T J H Clark S Hurd et al ldquoGINA guidelines onasthma and beyondrdquo Allergy vol 62 no 2 pp 102ndash112 2007
[2] M Masoli D Fabian S Holt R Beasley and Global Initiativefor Asthma (GINA) Program ldquoThe global burden of asthmaexecutive summary of the GINA Dissemination Committeereportrdquo Allergy vol 59 no 5 pp 469ndash478 2004
[3] M Kudo Y Ishigatsubo and I Aoki ldquoPathology of asthmardquoFrontiers in Microbiology vol 4 article 263 2013
[4] Y Zhao J Yang Y-D Gao and W Guo ldquoTh17 immunity inpatients with allergic asthmardquo International Archives of Allergyand Immunology vol 151 no 4 pp 297ndash307 2010
[5] L Cosmi F Liotta E Maggi S Romagnani and F AnnunziatoldquoTh17 cells new players in asthma pathogenesisrdquo Allergy vol66 no 8 pp 989ndash998 2011
[6] A Ray A Khare N Krishnamoorthy Z Qi and P RayldquoRegulatory T cells in many flavors control asthmardquo MucosalImmunology vol 3 no 3 pp 216ndash229 2010
[7] N Ohkura Y Kitagawa and S Sakaguchi ldquoDevelopment andmaintenance of regulatory T cellsrdquo Immunity vol 38 no 3 pp414ndash423 2013
[8] S J McMillan and C M Lloyd ldquoProlonged allergen challengein mice leads to persistent airway remodellingrdquo Clinical andExperimental Allergy vol 34 no 3 pp 497ndash507 2004
[9] H-Z Wang M Hong L-L Gui Y-Q Hua and H-Q XuldquoEffect of Yupingfeng San against OVA-induced allergic asthmainmicerdquo hongguo Zhong Yao ZaZhi vol 38 no 7 pp 1052ndash10552013
[10] H-Y Shi Y Zhuang and X-Y Wang ldquoEffect of yupingfengdroppill in treatment of allergic rhinitisrdquo Zhongguo Zhong YaoZa Zhi vol 39 no 12 pp 2364ndash2366 2014
[11] Y Chen ldquoEfficacy of sodium cromoglicate eye drops combinedwith yupingfeng granules in the treatment of allergic conjunc-tivitisrdquo Eye science vol 28 no 4 pp 201ndash203 2013
[12] S Langier K Sade and S Kivity ldquoRegulatory T cells in allergicasthmardquo Israel Medical Association Journal vol 14 no 3 pp180ndash183 2012
[13] H Y Kim R H Dekruyff and D T Umetsu ldquoThe many pathsto asthma phenotype shaped by innate and adaptive immunityrdquoNature Immunology vol 11 no 7 pp 577ndash584 2010
[14] D Amsen C Helbig and R A Backer ldquoNotch in T celldifferentiation all things consideredrdquo Trends in Immunologyvol 36 no 12 pp 802ndash814 2015
[15] Y Zhou S Liao Z Zhang B Wang and L Wan ldquoAstragalusinjection attenuates bleomycin-induced pulmonary fibrosis viadown-regulating Jagged1Notch1 in lungsrdquo Journal of Pharmacyand Pharmacology 2016
[16] LMa RMao K Shen et al ldquoAtractylenolide I-mediatedNotchpathway inhibition attenuates gastric cancer stem cell traitsrdquoBiochemical andBiophysical ResearchCommunications vol 450no 1 pp 353ndash359 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
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