Formononetin ameliorates DSS induced ulcerative colitis in ...

Formononetin ameliorates DSS-induced ulcerative colitis in mice through induction of Nrf2 in colons

Qian Yang1,2#, Gang Chen2#, Yang Yang2, Xueting Cai2, Zhonghua Pang2, Chunping Hu2, Shuangquan Zhang1*, Peng Cao2*

1. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University,

Nanjing 210046, China

2. Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, Nanjing 210028,

Jiangsu, China

Abstract: Isoflavone formononetin (FN) is a main active component of red clover (Trifolium pratense L.), a medicinal plant

possessing antitumorigenic and antioxidant properties. In the present study, we aimed to examine the effect of FN on dextran

sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. The results showed that FN (25, 50 mg/kg) markedly attenuated

the loss of body weight, the disease activity index (DAI), shortening of colon length and tissue injury induced by DSS treatment.

In addition, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) were also significantly

reduced in FN treatment group compared with the DSS group. Moreover, several representative oxidative stress parameters

in colorectum, including superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), myeloperoxidase (MPO) and

8-oxoguanine, were markedly ameliorated. In this study, we also found that the expression of Nrf2 was increased by FN treatment.

However, symptoms of UC were not ameliorated in Nrf2 knockout mice. Taken together, FN could prevent the development

of UC through activating of Nrf2 axis, and the protective effect was Nrf2 dependent. Our results demonstrated that FN

might be a potential therapeutic agent in the treatment of UC.

Keywords: Nrf2, Ulcerative colitis, Isoflavone formononetin, Flammatory cytokines, Oxidative stress

CLC number: R965 Document code: A Article ID: 1003–1057(2016)3–178–11

Received: 2015-10-23, Revised: 2015-12-04, Accepted: 2016-01-11.

Foundation items: National Natural Science Foundation of China

(Grant No. 81274150, 81573680 and 81470179). #These authors contributed equally to this work. *Corresponding author. Tel./Fax: +86-25-85891053,

Tel./Fax: +86-25-85608666,

E-mail: [email protected], [email protected]

http://dx.doi.org/10.5246/jcps.2016.03.021

1. Introduction

As an aidiopathic, chronic inflammatory illness,

inflammatory bowel disease (IBD) includes two main

clinicopathological subtypes, principally ulcerative

colitis (UC) and the chronic relapsing inflammatory

disorder Crohn’s disease (CD)[1]. UC begins in the

rectum, proximally spreads in a continuous fashion and

frequently involves the periappendiceal region, and it

is characterized by acute pain, vomiting, weight loss,

diffuse mucosal inflammation, diarrhea and bloody

stool symptoms[2–4]. In contrast, CD is related to any

part of the gastrointestinal tract–most commonly the

terminal ileum or the perianal region[3]. The population

of UC patients has increased in Asia as well as in

Western countries, and this is associated with an

increased risk for colorectal cancer. Currently, a lot of

experiments have been conducted to study UC, however

the common molecular mechanisms provoking UC have

not been entirely explained. Accumulated studies have

suggested that the major UC-triggering factors are

immunization, apoptosis, heredity, physical environment

and infection. Among them, intestinal microbiota is

considered to be a significant factor in their etiology[5].

Clinical studies have shown the relationship between

quiescent IBD and peripheral polyneuropathy and

indicated the existence of axonal and demyelinating

polyneuropathy in the IBD patients[6]. Currently, some

specific drugs, including mesalamine agents, steroids,

thiopurines, and antibodies against tumor necrosis factor

alpha (TNF-α), are the mainstay of therapeutics for UC.

In the last several years, agents like budesonide-Multi-

Matrix System (MMX), adalimumab, golimumab, and

178 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn

Copyright © 2016 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University http://www.jcps.ac.cn

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179 Yang, Q. et al. / J. Chin. Pharm. Sci. 2016, 25 (3), 178–188

vedolizumab have also been used as a novel therapy,

provided new treatment options for UC patients [2].

Recent investigations have presented that the available

treatment therapies for UC include nutritional therapy,

leukocyte extract technique, hyperbaric oxygen therapy

and Chinese herb therapy, especially traditional Chinese

medicine with precise effect, lower recurrent rate, and

lower rate of adverse reactions[7].

In general, host defense against microbial antigen is

driven by immune system, eventually which is followed

by immune imbalance. Histologically, UC displays the

superficial inflammatory changes limited to mucosa

and submucosa with cryptitis and crypt abscesses. At

present, the mechanism against UC is bound up with

anti-inflammation, antibiosis and antioxidant. Oxidative

stress has been related to some clinical features in

IBD, such as tissue injury and fibrosis, and also to the

UC-associated colorectal cancer[8]. Similarly, a significant

increase in inflammation and DNA damage are also

associated with UC induced by dextran sulfate sodium

(DSS)[9]. Nuclear factor-erythroid 2-related factor-2 (Nrf2)

is a key transcription factor playing a central role in

cellular defense against oxidative and electrophilic

insults by induction of antioxidative and phase-2

detoxifying enzymes as well as related stress-response

proteins. Recent studies have demonstrated that Nrf2 is

also involved in attenuation of inflammation-associated

pathogenesis, as well as suppression of pro-inflammatory

signaling pathways[10,11]. Many studies have shown that

the expression of Nrf2 is decreased in DSS-induced

chronic colitis model[12,13]. Recently, the chemopreventive

effect of dietary digitoflavone on colitis-associated colon

tumorigenesis has been determined, which is correlated

with a induction of Nrf2 signaling pathway[14]. Therefore,

Nrf2 would be a key target for treatment of UC.

Isoflavone formononetin (FN) is a major isoflavonoid

constituent extracted from the inflorescence and branches

of red clover (Trifolium pratense L.). Red clover has

a long history of medicinal use in traditional Chinese

medicine, and it possesses diverse pharmacological

benefits such as anti-inflammation, anticancer, and

anti-oxidation activities[15]. Studies have proved that

FN significantly suppresses arachidonic acid release in

HT-29 human colon cancer cells[16]. In addition, FN

plays an important role in lung cancer suppression,

breast cancer prevention, prostate cancer prevention and

osteoperosis reduction[17,18]. However, the data of the

effect of FN on colitis remain insufficient. In this study,

we investigated the effect of FN on UC induced by DSS.

Furthermore, we assessed the relationship between FN

and Nrf2 on curative effect of DSS-induced acute and

chronic UC model. Our results provide evidence

for the broader use of FN as a chemotherapeutic agent

against UC.

2. Materials and methods

2.1. Chemicals and reagents

FN was purchased from Zelang Medical Technology

(Nanjing, China). DSS with a molecular mass between

36–50 kDa was obtained from Sigma-Aldrich chemical

Co. (St. Louis, MO, USA). Primary antibodies (COX-2,

8-oxoguanine, Nrf2, and γ-GCSc) were provided by

Santa Cruz Biotechnology (CA, USA). Immunohisto-

chemistry kits, such as SOD, MDA, and MPO (m)

ELISA Kit were obtained from Boster, China. ELISA

kits (TNF-α, and IL-10) were supplied from AMEKO,

China.

2.2. Animal model of colitis and treatment

Pathogen-free 8-week-old C57BL/6 male mice (Nrf2+/+)

and Nrf2 knockout (Nrf2–/–) mice were allowed to adapt

to the laboratory for 1 week prior to the experiment.

All animal experiments were performed in accordance

with the NIH Guidelines for the Care and Use of

Laboratory Animals. Acute colitis was induced by oral

administration of 3% DSS in autoclaved drinking water

for 7 d. In two FN groups, the mice were received the

dosage of 25 mg/kg and 50 mg/kg three days before

and during DSS treatment via oral gavage per day, a

total of 10 d. Three days later, the mice in acute colitis

experiment groups were sacrificed. In contrast, chronic

colitis in Nrf2+/+ and Nrf2–/– mice were induced by


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three cycles of 2% DSS in drinking water for 5 d,

followed by normal water for 5 d[12]. In FN groups, the

mice were given 25 mg/kg and 50 mg/kg for 5 d before

and during DSS treatment via oral gavage per day. Mice

in vehicle group and DSS group were given the same

volume of water.

2.3. Colitis assessment

During the period of all experiments, daily routine

clinical evaluations, including body weight, stool characters

and hematochezia, were monitored every day. The evalua-

tion of disease activity index (DAI) was performed using

Murano’s methods as previously described, which was

calculated by combining scores of weight loss, stool

consistency and rectal bleeding divided by 3 [12].

Animals were sacrificed by cervical dislocation under

anesthesia. The colorectum was removed and the length

was determined. The distal colorectum was fixed in

4% paraformaldehyde overnight, embedded in paraffin

and stained with Hematoxylin and Eeosin (H&E)

and Periodic acid Schiff (PAS) according to standard

protocols.

2.4. Enzyme-linked immunosorbent assay

The concentration of inflammatory cytokines in

colorectal tissue, such as TNF-α and IL-10, was

quantified using enzyme-linked immunosorbent assay

(ELISA) kits. The levels of superoxide dismutase (SOD)

and MDA were used as indicators of oxidation resistance

and oxidative stress, respectively[12,19]. Myeloperoxidase

(MPO), which is identified as a specific marker for

leukocyte infiltration that can produce HOCl to resist

bacteria by catalyzing the conversion of H2O2 and

Cl–. In this study, colorectum from each group was

homogenized with tissue lysis buffer to extract total

protein at 4 °C. The homogenate was centrifuged at

12 000 g at 4 °C for 15 min. Protein sample concentration

was determined by BCA Protein Assay Kit. Ultimately,

the concentration of TNF-α, IL-10, SOD and MPO

in colorectal tissue was quantified according to the

manufacturer’s instructions.

2.5. Immunohistochemical analyses

Paraffin-embedded sections (4–6 µm) of colorectum

were prepared to assess the expression levels of COX-2,

8-oxoguanine, γ-GCSc and Nrf2. After dewaxing

hydration and retrieving of antigens, the expression

was respectively detected using 100 µL primary

antibodies at 4 °C overnight. Each section was washed

with phosphate buffer saline (PBS) three times and

then rinsed with PBS containing 0.05% Triton X-100

for 3 min. The section was thereafter incubated with

50 µL biotin-labeled secondary antibodies at room

temperature for 40 min, co-cultured with SABC at 37 °C

for 20 min, and washed with PBS five times for 20 min.

Then, redyed with immunoreactions were detected using

3,3-diaminobenzidine (DAB) for 5 min followed by

counterstaining with haematoxylin.

2.6. Statistical analysis

Statistical analysis was performed using SPSS soft-

ware version 15.0. Data were presented as mean±SD.

Unpaired Student t-tests were used to compare the means

of two groups. A level of P<0.05 was considered as

statistically significant.

3. Results

3.1. FN ameliorates DSS-induced acute and chronic

UC in Nrf2+/+ mice

Acute UC was induced by administration of 3%

DSS in the drinking water for 7 d. During modeling,

no difference in body weight change was detected

between the FN groups and the vehicle group in the first

4 days. Then a significant body weight decrease in body

weight was observed in DSS-treated mice. The mice in

FN groups exhibited a less weight loss compared with

the DSS group mice at 11–13 d (Fig. 1A). In contrast to

acute colitis, 2% DSS in drinking water induced chronic

colitis with marked symptoms as human UC[12]. In the

DSS-treated mice, the body weight was significantly

decreased in the third cycle (Fig. 1B). Generally, DAI


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exhibited features of loose feces, hematochezia and

body weight reduction, and it was used to evaluate

inflammation severity in colitis mice. The score of DAI

was significantly increased after DSS intake, whereas

it was markedly attenuated in FN-treated group. Colon

shortening is another index to reflect the disease severity

of colorectal inflammation. Figures 1A and B show that

a significant shortening of colorectum was observed in

DSS group compared with vehicle group and FN groups.

H&E-stained colorectum sections showed that the DSS

group exhibited distortion of crypts, loss of goblet cells,

severe epithelial injury and inflammatory cell infiltration in

mucosa and submucosa. However, FN groups exhibited

obvious protection of the colon crypt structures and

less severe histologic inflammation. The result of PAS

showed that goblet cells were significantly reduced in

DSS-treated group, and they were obviously increased

in FN groups (Fig. 2). These results suggested that

FN was capable of preventing DSS-induced colitis

in a dose-dependent manner.

Figure 1. Improvement role of FN against DSS-induced acute and chronic colitis in Nrf2+/+ mice. (A) Body weight changes after 3% DSS induction of colitis, DAI, intestines photograph and statistics of colorectum length of each group. (B) Body weight changes, DAI, intestine photograph and statistics of colorectum length in each group of 2% DSS induced chronic coliti s.

130

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t (d)

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DSS + FN-2

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Colo

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(m

m) ***

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0 DSS – + + + FN (mg/kg) 0 0 25 50

Colo

rect

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length

(m

m) ***

*

***

**

*

(B)

***

*

*


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3.2. FN regulates cytokine expression levels in

colorectum of Nrf2+/+ mice with DSS-induced acute

and chronic colitis

TNF-α, IL-10, and COX-2 are known to play a pivotal

role in the inflammation of colitis. Compared with the

vehicle group, the levels of TNF-α and COX-2 were

significantly increased in the colorectum of DSS group.

However, the administration of FN-2 (50 mg/kg/day)

significantly suppressed the accumulation of TNF-α and

COX-2 in the colonic tissues of mice with DSS-induced

acute and chronic colitis, but there was no significant

difference between FN-1 (25 mg/kg/day) and DSS

group in chronic colitis model. Additionally, the

anti-inflammatory cytokine IL-10 was decreased to

some extent after DSS induction, but IL-10 level was

significantly improved in the FN-2-treated mice. While

the level of IL-10 showed no significant difference

between FN-1 and DSS group in chronic colitis model

(Fig. 3). Taken together, our data suggested that FN

exerted an anti-inflammatory action in DSS-induced

acute and chronic colitis.

3.3. Effects of FN on oxidative stress in colorectum of

Nrf2+/+ mice with DSS-induced acute and chronic colitis

Oxidative stress parameters, such as SOD, MDA,

MPO and 8-oxoguanine, were determined in the present

work. The results showed that SOD activity was decreased

in DSS group, but it was dramatically increased in FN-2

group. The concentration of MDA, MPO and 8-oxoguanine

was significantly increased in DSS group, and FN-2 could

significantly inhibit the increase of these oxidative

stress parameters. However, no difference between

FN-1 and DSS group was found in the level of SOD

and MDA (Fig. 4). These results indicated that FN

possessed anti-oxidant properties and could prevent

DNA damage to some extent.

Figure 2. Protection of FN against intestinal lesion during acute and chronic colitis induced by DSS in Nrf2+/+ mice. (A) Representative H&E-stained and PAS colorectum sections (magnification ×400) in acute colitis mice. (B) H&E-stained and PAS colorectum sections in chron-ic colitis mice.

Vehicle DSS DSS + FN-1 DSS + FN-2 (A)

PA

S

H

&E

Acute

co

liti

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Vehicle DSS DSS + FN-1 DSS + FN-2 (B)

PA

S

H

&E

Chro

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liti

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3.4. FN can improve Nrf2 expression and protect

DSS-induced colitis in Nrf2+/+ mice

Nrf2 and γ-GCSc play a key role in antioxidant

mechanism. Based on expression level of the inflamma-

tory cytokines and antioxidants, Nrf2 and γ-GCSc

were also measured[19]. The protein levels of Nrf2

and γ-GCSc were increased in DSS-treated group

and could be reversed by the administration of FN,

suggesting that FN could increase Nrf2 expression and

reduce the oxidative stress (Fig. 5).

3.5. Effect of FN on DSS-induced chronic colitis in

Nrf2–/– mice

Nrf2–/– mice were used to verify the modulation of Nrf2 in

DSS-induced chronic colitis. The expression level of Nrf2 in

Nrf2+/+ and Nrf2–/– mice was determined and presented in

Figure 6. Figure 7 shows that no significant change in

weight, DAI, the length of colorectum and histopathology

was observed in Nrf2–/– mice treated by 2% DSS and FN.

These data suggested that FN had no protective effect

against DSS induced chronic colitis in Nrf2–/– mice.

Figure 3. Effects of FN on the production of inflammatory cytokines in colorectums of Nrf2+/+ mice with DSS-induced acute and chronic ulcerative colitis. (A) TNF-α, IL-10 in colorectums of acute ulcerative colitis model were determined by ELISA, and levels of COX-2 were examined by immunohistochemistry from colon sections (magnification ×100). (B) The concentration of TNF-α, IL-10 and the expression level of COX-2 in chronic ulcertive colitis model. *P<0.05; **P<0.01; ***P<0.001, versus DSS-treated group. Data are presented as mean SD of 7 mice in each group.

25

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nic

TN

F-α

(p

g/m

g p

rote

in)

***

* (A)

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L-1

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pg/m

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in)

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0 (

pg/m

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in)

***

**

(B)

Vehicle DSS

CO

X-2

DSS + FN-1 DSS + FN-2

Vehicle DSS

CO

X-2

DSS + FN-1 DSS + FN-2


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Figure 5. The effects of FN on Nrf2 and γ-GCSc expression in colorectums of Nrf2+/+ mice with DSS-induced acute and chronic ulcerative colitis. (A) Expression of Nrf2 and γ-GCSc in colorectums of acute ulcerative colitis. (B) Levels of Nrf2 and γ-GCSc in colorectums of chronic ulcerative colitis.

40

30

20

10

0

Figure 4. Protection of FN against oxidative stress in colorectums during acute and chronic ulcerative colitis induced by DSS in Nrf2+/+ mice. (A) SOD activity, MDA MPO content and 8-oxoguanine levels (magnification ×100) in colorectal tissue of acute ulcerative colitis. (B) SOD activity, MDA and MPO content, and 8-oxoguanine levels in colorectal tissue of chronic ulcerative colitis. *P<0.05; **P<0.01; ***P<0.001, versus DSS-treated group. Data are presented as mean SD of 7 mice in each group.

DSS – + + + FN (mg/kg) 0 0 25 50

SO

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U/m

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CS

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Vehicle DSS DSS + FN-1 DSS + FN-2 (B)

γ-G

CS

c

Nrf

2


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Figure 6. The expression levels of Nrf2 in Nrf2+/+ and Nrf2–/– mice. (A) The levels of Nrf2 exermined by Immunohistochemistry (magnification ×100). (B) The Nrf2 mRNA expression levels were determined by real-time PCR.

Figure 7. No improvement of FN against DSS-induced chronic colitis in Nrf2–/– mice. (A) Body weight changes after DSS induction of colitis. (B) DAI. (C) Statistics of colorectum length. (D) Representative H&E-stained and PAS-stained colorectum sections (magnification ×400).

4. Discussion

UC is associated with genetic and environment leading

to impairment of the intestinal mucosal barrier. Previous

studies have reported that IL-10–/–, TRAF6IEC-KO and

TMF–/– mice are susceptible to colitis[21,22]. Moreover,

attenuation of the Bin1 (Bridging integrator 1) gene

can limit UC pathogenicity in the mouse by supporting

mucosal barrier function and protecting integrity of the

lymphoid follicle, offering a novel strategy to treat UC

and possibly limiting risks of colorectal cancer[23]. At

present, an increasing number of studies are focusing on

the relationship between UC and gut microorganism[22,24].

The balanceable state would be destroyed as these

invasive antigens are introduced into the organism,

then the immune cells are activated and cytokines are

produced. When the anti-inflammation mechanisms are

not able to resolve the acute mocosal inflammation, pro-

inflammation responds to organism, showing up-regulation

of pro-inflammation cytokines, and inflammatory cellular

infiltration. More seriously, chronic inflammation may

cause tissue destruction such as fibrosis, abscess, fistula

and even cancer, which are driven by mucosal cytokine

infiltration[25]. UC is generally treated with anti-inflammatory

Nrf2+/+ Nrf2–/– (A)

Nrf+/+ Nrf–/–

(B) 5

4

3

2

1

0

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2 m

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A e

xpre

ssio

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in c

olo

rect

um

Vehicle DSS DSS + FN-1 DSS + FN-2 (D)

PA

S

H

&E

Nrf

2–/–

DSS – + + + FN (mg/kg) 0 0 25 50

Co

lore

ctal

len

gth

(m

m)

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80

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Nrf2–/–

NS NS

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(A) Nrf2–/–

NS NS

6

4

2

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DA

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1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 t (d)


Nrf2–/–

NS NS

(B)


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or immunosuppressive drugs. In recent years, more and

more studies have been conducted to develop effective

treatment for colitis. A new study has found that glucagon-

like peptide-2 (GLP-2) microspheres, a proper candidate

for the treatment of UC, is resistant to degradation and

can decrease the severity of DSS-induced UC[26].

Enoxaparin, a widely used antithrombotic agent, has been

reported to possess anti-inflammatory properties, and

it can significantly reduce the inflammatory pathology

associated with DSS-induced colitis in mice therefore

representing a novel therapeutic option for the manage-

ment of UC[27]. However, current therapies can only

reduce the inflammatory process of IBD, and improved

treatment modalities for the complex IBD are still

evolving. Therefore, haematopoietic stem cells (HSC)

and mesenchymal stromal cells (MSC) therapies are

both being investigated for the IBD treatment [28].

Although clinical drugs for the treatment of colitis have

been reported, most of these treatments often have

various side effects. Consequently, many patients turn

to alternative therapies, such as traditional plant-based

remedies. Geraniol has been proved as a potential

therapeutic agent for IBD by inhibiting pro-inflammatory

cytokines and NF-κB signaling[29].

In this study, we investigated the anti-inflammation

mechanism of FN in the vivo model of acute and

chronic colitis induced by DSS in C57BL/6 mice. After

treated with DSS, the mice exhibited clinical symptoms

and pathalogic change corresponding to those of human

UC, including loss of body weight, diarrhoea, bloody

faeces, mucosal ulceration and shortening of colon.

Two different concentrations of FN along with DSS

were used to evaluate its therapeutic effect on UC. The

results demonstrated that FN significantly suppressed

DSS-induced colitis in a dose-dependent manner by

decreasing DAI scores, weight loss and colonic

shortening. H&E and PAS findings were consistent with

DAI data. As shown in Figure 2, the colons of mice

treated with DSS presented more severe inflammatory

cell infiltration, mucosal erosion, and both distortion

and loss of crypts. In contrast, administration of FN

reduced all these above mentioned inflammatory changes,

suggesting that FN could ameliorate the inflammatory

condition induced by DSS. In order to further explore

the therapeutic mechanism of FN, the production of

pro-inflammatory cytokines (TNF-α, COX-2) and

anti-inflammatory (IL-10) were measured in colonic

tissue[30]. ELISA and immunohistochemical results

confirmed the up-regulation of pro-inflammatory

cytokines TNF-α and COX-2, and down-regulation of

IL-10 in DSS induced group. In contrast, a distinct

decrease in concentrations of TNF-α and COX-2 and

an increase in IL-10 concentration were observed

when FN was administered along with DSS.

UC is associated with oxidative stress. SOD, MDA

and MPO are important markers in oxidative stress.

Previous reports have proved a significant drop in SOD

activity and an obvious increase in the concentrations

of MDA and MPO in DSS-induced chronic colitis,

which were consistent with our experimental results.

However, increased MDA level and MPO activity

as well as reduced SOD activity in colonic tissue of

DSS-colitis group were significantly improved after oral

FN administration. Besides, the level of 8-oxoguanine,

a DNA damage marker, was significantly increased in

colorectum of DSS model, and it was set to normal in

DSS along with FN. Therefore, our results obviously

showed that FN could exert an anti-oxidative effect by

improvement of SOD activity and reducing peroxidation

of polyunsaturated fatty acids and DNA damage.

Nrf2 is a key transcription factor that protects cells

against oxidative stress by producing phase-2 detoxifying

enzymes and anti-oxidative enzymes[8,31,32]. Previous

studies have shown that Nrf2–/– mice are sensitive to

DSS-induced colitis, displaying significant increase in

incidence, colonic crypt loss, inflammatory cell infiltration

and so on[33]. Above all, inflammatory cytokines, such as

IL-1β, IL-6, TNF-α, iNOS and COX-2, are increased

in Nrf2–/– mice. Nrf2 signaling is also involved in

inflammation associated pathogenesis, such as gastritis,

colitis, rheumatoid arthritis and atherosclerosis. In our

study, DSS-induced chronic colitis in Nrf2–/– showed

no significant distinction compared with those of FN

treated group. However, the expression Nrf2 in mice


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without genetic defect was significantly improved with

FN treatment. Above results suggested that the doses

of 50 mg/kg FN was superior to 25 mg/kg, and the two

doses could ameliorate DSS-induced acute and chronic

ulcerative colitis in mice.

Based on the findings of this study, we confirmed

that FN exhibited anti-inflammatory and anti-oxidant in

vivo by improving Nrf2 expression, activating anti-oxiditive

and anti-inflammatory gene expression. In the process

of colitis treatment, FN might be an effective candidate

for the treatment of UC.

Acknowledgements

This work was supported by the National Natural

Science Foundation of China (Grant No. 81274150,

81573680 and 81470179) and Jiangsu Province’s

Outstanding Leader Program of Traditional Chinese

Medicine.

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刺芒柄花素通过诱导Nrf2表达缓解DSS诱导的小鼠溃疡性结肠炎

杨倩1,2#, 陈刚2#, 杨洋2, 蔡雪婷2, 庞中化2, 胡春萍2, 张双全1*, 曹鹏2*

1. 南京师范大学生命科学学院分子生物技术与医学实验室, 江苏南京 210046

2. 江苏省中医药研究院细胞与分子生物学实验室, 江苏南京 210028

摘要: 异黄酮刺芒柄花素是从红车轴草中提取的一种主要的活性成分。红车轴草是一种具有抗炎和抗癌功能的药用

植物。在本研究中, 我们旨在研究刺芒柄花素对DSS诱导的急性和慢性小鼠溃疡性结肠炎的作用。研究结果发现刺芒柄

花素 (25, 50 mg/kg) 可以明显地减弱DSS引起的体重下降, 疾病活动指数评分, 肠度缩短和组织损伤。进一步研究发现, 在

给予刺芒柄花素的保护组中, 肿瘤坏死因子(TNF-α), 白介素-6(IL-6) 和环氧合酶(COX-2)的含量都会明显降低; 结肠组织

中具有代表性的氧化压力参数, 包括超氧化物歧化酶和髓过氧化物酶的活性, 以及丙二醛和8-羟基鸟嘌呤的含量明显得到

改善;同时, 研究中还发现刺芒柄花素保护组中Nrf2表达量明显升高, 但是Nrf2基因敲除小鼠慢性溃疡性结肠炎症状没有

得到缓解。综上所述, 我们推断刺芒柄花素可以通过激活Nrf2的表达保护溃疡性结肠炎的发生。结果证明刺芒柄花素

可能对治疗溃疡性结肠炎具有潜在的作用价值。

关键词: Nrf2; 溃疡性结肠炎; 异黄酮刺芒柄花素; 炎症因子; 氧化应激


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