7. 정해영

Hae Young Chung, Ph. D.

Molecular Inflammation Research Center

for Aging Intervention (MRCA)

College of Pharmacy

Pusan National University

Busan, South Korea

Molecular Inflammation as the Underlying

Mechanism of the Aging Process and its

Intervention

The young The oldAging process

Modulation of NF-B by Age and CR

MAPK

(JNK, p38, Erk)

ROS

p65

B-site

p50 COX-2, iNOS, HO-1, TNFα, IL-1,6, AMs

IBa, IB

Degradation

p50 p65

IBa

P

IBa

p50 p65

NIK

IKKP

P

CR

CR

CR

CR

CR

CR

CR

Inflammatory

Gene

Transcription

Nucleus

Inflammatory process

Aging process

CR

Redox state Reactive oxygen speciesReactive nitrogen speciesCatalase, Superoxide dismutaseGSH peroxidase, GSH/GSSG

┣

┣

┣

┣

Proinflammatory

enzymes

Inducible NO SynthaseHeme oxygenase-1Cyclooxygenase-2Conversion of Xanthine Dehydrogease to Xanthine Oxidase

┣

┣

┣

┣

Proinflammatory

cytokines

IL-1IL-6TNF-a

┣

┣

┣

Adhesion

molecules

E-selectin

P-selectin

VCAM-1

ICAM-1

┣

┣

┣

┣

NF-B activation NF-B DNA binding activity

NIK/IKK activation

Phosphorylation of IBa

Degradation of IBa and IB in cytoplasm

Nuclear translocation of p65 and p50

NF-B-dependent gene expression

Active MAPKs (ERK, JNK, p38 MAPK)

┣

┣

┣

┣

┣

┣

┣

, Increased ; , decreased ; ┣, blunted

Changes of Inflammatory Parameters during Aging Process

(Microsc Res Techniq, 2002)

Parameters

Biochemical Changes Leading to Inflammatory Response

Intracellular Changes Tissue Damage Inflammatory Responses

Molecular Events

Redox

imbalance,

Oxidized

molecules

etc.

NF-B

Pro-inflammatory

Gene Activation

• Endogenous oxidative damage

• Redox imbalance

• Activation of pro-inflammatatory

reaction

• Ca2+ release

• Pro-inflammatory gene expression

• Migration of surveillance cells

• Activation of pro-inflammatory cells

• Release of pro-inflammatory

mediators

• Production of pro-oxidants

• Necrosis/Apoptosis

• Altered permeability

• Modified ion balance

• Protein leakage

• Homodynamic changes

• Edema

•COX-2 PGs + ROS

•iNOS NO ONOO-

·O2-

•Cytokines, Adhesion molecules

• M o l e c u l a r i n f l a m m a t i o n :l o w - g r a d e i n f l a m m a t i o nemphasizing the importance ofthe molecular mechanisms thatac t as precurs ive eventsleading to ful ly expressedinf lammatory phenomena.

(Microsc Res Techniq, 2002)

Ⅰ. Molecular Inflammation

Cytokines (IL-1, IL-6, TNFa)

NADPH oxidaseImmune Cell

•O2-

NF-B activation

COX-2 iNOS

•O2- NO •O2

-

Molecular Inflammatory Process

Chronic Inflammation,

Tissue Response

Aging(Chung, HY Rev Clin Gerontol, 2000)(Chung, HY Ann N Y Acad Sci, 2001)(Chung, HY Microsc Res Techniq, 2002)(Chung, HY Antioxid Redox Signal, 2006)

ONOO-

Molecular Inflammation Hypothesis of Aging

IKK MAPK

XDH

XOD

Redox Imbalance

Epithelial cell

NIK= CR

Physiological AgingPathological Aging

CR/Exercise

Redox Imbalance

Molecular

Inflammation

Obesity, Sarcopenia

Metabolic, Syndrome

Dementia, Cancer

Atherosclerosis,

Osteoporosis

Functional Decline

Life Span

Chung HY et al .: Aging Res Rev, 8,18~30 (2008)

Molecular

inflammation

VitalityCell death

20yrs 40yrs 80yrs

Aging

Aging-related diseases

Ⅱ.Vascular inflammation is fundamental to

whole body aging

O2/Nutrient

ATP

productionATP

depletion

Importance of Vascular Aging

Abnormal AgingNormal Aging

LO. LOO.L.

.O2- .OH H2O2

1O2

ONOO-

NO

Hypoxia

MolecularInflammation

Disease

ROS / RNS generators

ROS / RNS Scavengers

Urate, Vt C, E, GSH,

SOD, CAT, GSHPx

Cell Damage

Vascular alteration

Cell death

ATP depletion

Lipid Accumulation

①

②

Increased expression of AMs by aging in aorta and

serum

aorta

serum

↑: endothelial cell layer; *: smooth muscle cells

scale = 100 mm

(months)

J Gerontol 61, 232 (2004)FASEB J 18, 320 (2004)

What is responsible for up-regulation of AMs during

aging?

10mM 2mM 10mM 1mM 10mM

0

50

100

150

200

250

300

350

400

UTC TC LPC BSO t-BHP DEM AMVN AAPH HNE MDA PMA LPS IL-1

Rela

tive R

LU

(%

of

co

ntr

ol)

10mM 10mM 10mM 10mM 100ng/

mL

10ng/

mL

luciferase assay for

VCAM gene promoter

Proinflammatory

cytokines

Bioactive

lipids

Inflammatory

mediators

EndotoxinLipid

peroxides

OxidantsAdhesion Molecules

AngⅡUTC TC LPC BSO BHP DEM AMVN AAPH HNE MDA LPS 1L-1

1) Accumulation of LPC during aging

Production of LPC

0

0.5

1

1.5

2

2.5

3

3.5

6mon 12mon 18mon 24mon

Le

ve

l o

f L

PC

in

se

rum

(uM

/ u

g s

eru

m p

rote

in)

AL

CR

#

####

** **

*: P<0.05 vs. 6AL; #: P<0.05 vs. age-matched CR

Rejuv Res 12, 15 (2009)

Lysophosphatidylcholine (LPC)

↑ VCAM-1

↑ P-selectin

GPR4 activation

↑ cAMP

↑ PKA activation

↑ CREB

activation

MDL 12,330A

GPR4+

H89

↑ NF-B

↑ Oxidative stress

CAPE

SN-50

NAC

ACREB

AA861

Adenylyl cyclase

activation

Lipoxygenase

activation

Action Mechanism of LPC

FEBS J 274, 2573 (2007)

ROS can attack membrane lipids and initiate lipid peroxidation. The

lipid peroxidation productions include radical and aldehyde-derivative

such as MDA, HNE and HHE

4-hydroxynonenal (HNE)

4-hydroxyhexenal (HHE)

2) Increased HNE/HHE during aging

IKK

HNE

HHE

NIK

MAPK

(ERK, p38,)

PI3K

MEK1/2

Ras

Raf

Action Mechanism of HHE and HNE in endothelial cells

P

P

P

p50 p65

B-site

ApoptosisiNOS

COX-2

IBa

p50 p65p50 p65

IBa

ONOO-

HNE

HHE

FEBS Lett 566, 183 (2004)

TLR4

LPS

MD2

CD

14

MyD88

IRAK4

TRAF6

TAK1

P

IRAK1P

IKKa IKK

IKKP

P P

IBap50

p65

p38,JNK

p50 p65 COX-2, iNOS, TNF, IL6

Src PTKs

(LCK)

3) Involvement of Endogenous LPS in Vascular aging

Y

pIBa

Yp

p

p

Aging?

Am J Physiol Lung Cell Mol Physiol

(Okutani D, 2006)

0

0.2

0.4

0.6

0.8

1

1.2

1 2

AL

CR

#

LP

S le

vels

in

seru

m

(EU

/ml)

6 24 (mon)

**p < 0.01 vs. 6 months old

# p < 0.05 vs. same aged AL rats

**

Effects of Aging and CR on LPS levels

intestine

endotoxin

blood

bacteria

TLR4

LPSMD2

TAK1

PIKKa IKK

IKKP

P P

IBap50

p65

p50 p65 COX-2, TNF, IL6, iNOS

Possible mechanism of aging in TLR4/Lck/NF-B

pathway

p65p50

Aging

CR

Inflammation

Lck

P Y

YP

ONOO-

p Ser

CR

CR

Ser

Redox Imbalance

COX-2, iNOS,

AMs, TNFa

Vascular Alteration

CR

NF-B

NIK/IKK, MAPKs

AgingHypoxia

Cell Death Systemic Inflammation

Ⅲ.Supporting Evidences for Age-

related Inflammation

1) Microarray data in aging process

2) Systems-biological approach

Screening of age-related genes

CR transcriptome

N =586

Aging transcriptome

N = 478380 48898

N=37

N=44

Aging CR GO description p-value

lipid metabolism0.0182

Aging CRGO description p-value

phosphate transport 0.018

cell adhesion 0.003

immune response 0.004

Patterns # of genes

Aging CR 7

Aging CR 44

Aging CR 37

Aging CR 10

(SREBP, PPAR)

(NF-B)

Ⅳ.Aging Intervention strategies

1) CR

2) Exercise

3) PPAR agoinst

4) Antioxidants

Calorie Restriction

FOXO

Insulin, GF

IRS-1

PI3K

Akt

NF-κB

MAPKs

SIRT1TRX/GSH

System

NAD+/NADH

SOD/CAT

IKK

COX2/iNOS/LOX/AMs

ROS

PPARs

Chronic Inflammation

Aging

1)Molecular Mechanism of CR

Aging

ROS

MAPK/IKK

NF-κB

COX-2, iNOS

Voluntary Exercise

(Seo et al. ARS, 8, 529, 2006)

(Radak et al. Faseb J, 2004)

2) Modulation of age-related inflammation by exercise

Age-related Upregulation

XDH, Ferritin, ET-1, SOD3, RAGE,

-GT, THP, uPA, TGF-1, HSP70, NUO,

TLR 4, Calbindin, Cathepsin, CD81, uPAR

CD37, complementary factor 1, COX-2,

iNOS, Adhesion molecules, cytokines,

chemokines, etc

aging

CR-related Upregulation

FATP-1, FAT/CD36, ACS, CPT-1, ApoA1,

Lipoprotein lipase, HMG-CoA synthase,

Acyl-CoA binding protein, etc

NF-B PPARs

(Mech Ageing Dev, 2005)

3) PPAR agonist effects

Inflammatory process

Aging process

CR PPAR

agonist

Redox state Reactive oxygen speciesReactive nitrogen speciesCatalase, Superoxide dismutaseGSH peroxidase, GSH/GSSG

┣

┣

┣

┣

┣

┣

┣

┣

Proinflammatory

enzymes

Inducible NO SynthaseHeme oxygenase-1Cyclooxygenase-2Conversion of Xanthine Dehydrogease to Xanthine Oxidase

┣

┣

┣

┣

┣

┣

┣

┣

Proinflammatory

cytokines

IL-1IL-6TNF-a

┣

┣

┣

┣

┣

┣

Adhesion

molecules

E-selectin

P-selectin

VCAM-1

ICAM-1

┣

┣

┣

┣

┣

┣

┣

┣

NF-B activation NF-B DNA binding activity

NIK/IKK activation

Phosphorylation of IBa

Degradation of IBa and IB in cytoplasm

Nuclear translocation of p65 and p50

NF-B-dependent gene expression

Active MAPKs (ERK, JNK, p38 MAPK)

┣

┣

┣

┣

┣

┣

┣

┣

┣

┣

┣

┣

┣

┣

, Increased ; , decreased ; ┣, blunted

Changes of Inflammatory Parameters during Aging Process

(Antioxid Redox Signal, 2006)

CBP

p50 p65

p50 p65

pi

IκB

activ

atio

n

IκBpi

Aging/ROS

Nucleus

baicalein

PPAR

Possible mechanism of baicalein

iNOS, COX-2, VCAM-1 RXR

SRC-1

Glucose,

lipid metabolism

PPAR

BI/BE

Based on Molecular Inflammation Hypothesis of

Aging:

To screen active herbs and the active compounds from Nelumbo

nucifera, orange, and Goji berries against ROS and ONOO-

To confirm ROS/RNS scavenging effects of active

compounds, kaempferol, hesperetin, baicalein, and betaine

To elucidate action mechanism of the active compounds focusing

on NF-B activation and proinflammatory gene expressions in

aged rat

4) Antioxidants

MKK 3/6

p38

NIK

IKKIB

NF-B

NF-B

IBP

PP

NF-B

Nucleus

COX-

2, iNOS, VCAM-

1, ICAM-1

Homocysteine

Met

Betaine

DMG THF

5-Me-THF

CYSTEINE

GSH (Thiols: SH)

MEK 1/2

ERK JNK

Vascular

Aging

MSBHMT

Possible mechanism of Betaine

Systemic approaches to find drug target molecules and

their modulators for anti-aging

:Integrating protein-protein interaction network

and docking simulation

박대의 (박사 3년)

Ⅴ. New drug for aging intervention

in the future

The scheme of systemic approaches

OMICs data

Genome

NGS

Transcriptome

Microarray

Proteome

2D/Mass

Protein-protein interaction network

Degree HUBs , Centrality HUBs

Protein Structure

Modeling

Protein-Ligand

Docking

Experimental evaluation (Wet Lab)

Testing value as drug (Wet Lab)

• Protein-protein interaction network

• Analysis of biological network

• Protein structure modeling

• Docking simulation

NameAutodock(Kcal/Mol)

708 -8.88

778 -8.23

602 -8.06

rosiglitazone -8.03

866 -7.76

900 -7.75

915 -7.66

667 -7.57

455 -7.54

Structural modeling and docking simulation for PPAR

rosiglitazone

Candidates

Docking Energy

PPAR-gamma

Pharmacophore

Conclusion

Large-scale microarray data

Protein-protein Interaction analysis

Target Validation

Differentially Expressed Genes

-15

-10

-5

0

5

10

-6 -4 -2 0 2 4 6

Ob

se

rve

d S

co

re

Expected Score

Significant: Median False

Tail strength se (%): 26.9

Docking Simulation

Biological activity

Optimization of leader compds

Drug candidate

DRUG THERAPY

(Wald and Law, 2003)• Everyone aged 55+ years take a Polypill

(to reduce cardiovascular disease)

- a statin to lower blood cholesterol

- 3 antihypertensive drugs

- aspirin to reduce platelet aggregation

- folic acid to reduce serum homocysteine

• Life extension of about 10 years

Conclusion

1. Molecular inflammation during aging

: Balance between NF-B and PPARs

2. Modulation of age-related inflammation by CR and exercise

3. Flavonoid, kaempferol and baicalein

: through modulation of PPAR, SIRT1, and redox

4. Sulfurhydryl inducer, betaine

: Modulation of redox status (-SH/-S-S)

5. Genomic, proteomic and systems-biological approaches

: Powerful tools for integration and overview of scientific

knowledge and development of new drugs