1. Molecular Inflammation as the Underlying
Mechanism of the Aging Process and its
Intervention
Hae Young Chung, Ph. D.
Molecular Inflammation Research Center
for Aging Intervention (MRCA)
College of Pharmacy
Pusan National University
Busan, South Korea

2. The young Aging process The old

3. Modulation of NF-B by Age and CR
CR
ROS
CR
P
NIK
CR CR
P
IKK CR MAPK
IBa
p50 p50
(JNK, p38, Erk)
p65 p65
P
IBa
IBa, IB
Inflammatory
Degradation CR Gene
CR Transcription
p50 p65 COX-2, iNOS, HO-1, TNFα, IL-1,6, AMs
Nucleus B-site

4. Changes of Inflammatory Parameters during Aging Process
Inflammatory Aging
Parameters CR
process process
Redox state Reactive oxygen species   ┣
Reactive nitrogen species   ┣
Catalase, Superoxide dismutase   ┣
GSH peroxidase, GSH/GSSG   ┣
Proinflammatory Inducible NO Synthase   ┣
Heme oxygenase-1   ┣
enzymes Cyclooxygenase-2
  ┣
Conversion of Xanthine Dehydrogease to
Xanthine Oxidase   ┣
Proinflammatory IL-1   ┣
IL-6   ┣
cytokines TNF-a ┣
 
Adhesion E-selectin   ┣
P-selectin   ┣
molecules ┣
VCAM-1  
ICAM-1   ┣
NF-B activation NF-B DNA binding activity   ┣
NIK/IKK activation   ┣
Phosphorylation of IBa   ┣
Degradation of IBa and IB in cytoplasm   ┣
Nuclear translocation of p65 and p50   ┣
NF-B-dependent gene expression   ┣
Active MAPKs (ERK, JNK, p38 MAPK)   ┣
, Increased ; , decreased ; ┣, blunted
(Microsc Res Techniq, 2002)

5. Ⅰ. Molecular Inflammation
Biochemical Changes Leading to Inflammatory Response
Intracellular Changes Tissue Damage Inflammatory Responses
• Endogenous oxidative damage • Migration of surveillance cells • Altered permeability
• Redox imbalance • Activation of pro-inflammatory cells • Modified ion balance
• Activation of pro-inflammatatory • Release of pro-inflammatory • Protein leakage
reaction mediators • Homodynamic changes
• Ca2+ release • Production of pro-oxidants • Edema
• Pro-inflammatory gene expression • Necrosis/Apoptosis
•Molecular inflammation:
low-grade inflammation
emphasizing the importance of
the molecular mechanisms that
Molecular Events act as precursive events
leading to fully expressed
inflammatory phenomena.
Redox
imbalance,
NF-B
•COX-2 PGs + ROS
Oxidized
·O2-
molecules •iNOS NO ONOO-
Pro-inflammatory
etc.
Gene Activation
•Cytokines, Adhesion molecules
(Microsc Res Techniq, 2002)

6. Molecular Inflammation Hypothesis of Aging
Molecular Inflammatory Process
Cytokines (IL-1, IL-6, TNFa)
= CR
NIK NADPH oxidase
Immune Cell
IKK MAPK
•O2-
NF-B activation
XDH Epithelial cell
COX-2 iNOS XOD
•O2- NO •O2-
ONOO-
Redox Imbalance
Chronic Inflammation,
Tissue Response
(Chung, HY Rev Clin Gerontol, 2000)
Aging (Chung, HY Ann N Y Acad Sci, 2001)
(Chung, HY Microsc Res Techniq, 2002)
(Chung, HY Antioxid Redox Signal, 2006)

7. Redox Imbalance
CR/Exercise Molecular
Inflammation
Pathological Aging Physiological Aging
Obesity, Sarcopenia Functional Decline
Metabolic, Syndrome
Dementia, Cancer
Atherosclerosis,
Osteoporosis
Life Span
Chung HY et al .: Aging Res Rev, 8,18~30 (2008)

8. Ⅱ.Vascular inflammation is fundamental to
whole body aging
20yrs 40yrs 80yrs
O2/Nutrient
ATP
ATP production
depletion
Cell death Vitality
Molecular Aging
inflammation Aging-related diseases

9. Normal Aging ①
Abnormal Aging
Vascular alteration
Disease
Urate, Vt C, E, GSH,
Hypoxia
SOD, CAT, GSHPx
ATP depletion
Cell death ②
ROS / RNS Scavengers Lipid Accumulation
Molecular
Inflammation
Cell Damage
ONOO- NO
LO. LOO. L.
.O - .OH H 2 O2 1O
2 2
ROS / RNS generators
Importance of Vascular Aging

10. Increased expression of AMs by aging in aorta and
serum
aorta
serum
(months)
↑: endothelial cell layer; *: smooth muscle cells
scale = 100 mm
FASEB J 18, 320 (2004) J Gerontol 61, 232 (2004)

11. What is responsible for up-regulation of AMs during
aging?
Proinflammatory Bioactive
cytokines lipids
Lipid Endotoxin
peroxides
Adhesion Inflammatory
400 Oxidants Molecules mediators
Relative RLU (% of control)
350
luciferase assay for
300 VCAM gene promoter
250
200
150
100
50
0
UTC
UTC TC
TC LPC
LPC BSO
BSO BHP
t-BHP DEM
DEM AMVN
AMVN AAPH
AAPH HNE
HNE MDA
MDA AngⅡ
PMA LPS
LPS 1L-1
IL-1
10mM 2mM 10mM 1mM 10mM 10mM 10mM 10mM 10mM 100ng/ 10ng/
mL mL

12. 1) Accumulation of LPC during aging
3.5 Production of LPC
AL
3 CR
**
(uM/ ug serum protein)
**
Level of LPC in serum
2.5
##
2 ##
1.5
#
1
0.5
0
6mon 12mon 18mon 24mon
*: P<0.05 vs. 6AL; #: P<0.05 vs. age-matched CR
Rejuv Res 12, 15 (2009)

13. Action Mechanism of LPC
Lysophosphatidylcholine (LPC)
GPR4 activation + GPR4
AA861 Lipoxygenase
activation
Adenylyl cyclase
activation MDL 12,330A
NAC
↑ Oxidative stress ↑ cAMP
CAPE ↑ PKA activation H89
SN-50
↑ CREB ACREB
↑ NF-B activation
↑ VCAM-1
↑ P-selectin FEBS J 274, 2573 (2007)

14. 2) Increased HNE/HHE during aging
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)

15. Action Mechanism of HHE and HNE in endothelial cells
HNE HNE Ras
PI3K HHE HHE Raf
P
NIK MEK1/2
P MAPK
IKK (ERK, p38,)
IBa
p50 p65
p50 p65 P
IBa
iNOS p50 p65
Apoptosis ONOO-
COX-2
B-site
FEBS Lett 566, 183 (2004)

16. 3) Involvement of Endogenous LPS in Vascular aging
LPS
MD2 ? Aging
CD14
TLR4
Y p MyD88
p Src PTKs IRAK4
(LCK)
P
IRAK1
TRAF6
P
P IKK P
P
IKKa IKK TAK1
p
Y
IBa Am J Physiol Lung Cell Mol Physiol
p38,JNK
(Okutani D, 2006)
p50
p65
p p50 p65
IBa COX-2, iNOS, TNF, IL6

17. Effects of Aging and CR on LPS levels
LPS levels in serum
1.2 **
1
(EU/ml)
0.8 #
AL
0.6
CR
0.4
0.2
0
61 2
24 (mon)
intestine blood
endotoxin bacteria **p < 0.01 vs. 6 months old
# p < 0.05 vs. same aged AL rats

18. Possible mechanism of aging in TLR4/Lck/NF-B
pathway
Aging
LPS CR
MD2
TLR4
CR
P Y
Lck ONOO- CR
P
P IKK
p Ser P P Ser
P Y
IKKa IKK
TAK1
IBa
p50
p65
p65
p50
p50 p65 COX-2, TNF, IL6, iNOS Inflammation

19. Redox Imbalance CR
NIK/IKK, MAPKs
NF-B
COX-2, iNOS,
AMs, TNFa
Vascular Alteration
Hypoxia Aging
Cell Death Systemic Inflammation

20. Ⅲ.Supporting Evidences for Age-
related Inflammation
1) Microarray data in aging process
2) Systems-biological approach

21. Screening of age-related genes
Aging CR GO description p-value
(SREBP, PPAR)
lipid metabolism 0.0182
N=37
Patterns # of genes
Aging CR 7
Aging transcriptome CR transcriptome
380 98 488
N = 478 N =586 Aging CR 44
Aging CR 37
Aging CR 10
GO description p-value
Aging CR phosphate transport 0.018
N=44 cell adhesion 0.003
immune response 0.004 (NF-B)

22. Ⅳ.Aging Intervention strategies
1) CR
2) Exercise
3) PPAR agoinst
4) Antioxidants

23. 1)Molecular Mechanism of CR
Calorie Restriction
Insulin, GF
ROS NAD+/NADH IRS-1
PI3K
TRX/GSH SIRT1 Akt
System
MAPKs
IKK FOXO
SOD/CAT
PPARs NF-κB
COX2/iNOS/LOX/AMs
Chronic Inflammation
Aging

24. 2) Modulation of age-related inflammation by exercise
Aging
ROS Voluntary Exercise
MAPK/IKK
NF-κB
COX-2, iNOS (Radak et al. Faseb J, 2004)
(Seo et al. ARS, 8, 529, 2006)

25. 3) PPAR agonist effects
Age-related Upregulation
CR-related Upregulation
XDH, Ferritin, ET-1, SOD3, RAGE,
-GT, THP, uPA, TGF-1, HSP70, NUO,
TLR 4, Calbindin, Cathepsin, CD81, uPAR FATP-1, FAT/CD36, ACS, CPT-1, ApoA1,
CD37, complementary factor 1, COX-2, Lipoprotein lipase, HMG-CoA synthase,
iNOS, Adhesion molecules, cytokines,
Acyl-CoA binding protein, etc
chemokines, etc
NF-B PPARs
aging
(Mech Ageing Dev, 2005)

26. Changes of Inflammatory Parameters during Aging
Process
Inflammatory Aging PPAR
CR
process process agonist
Redox state Reactive oxygen species   ┣ ┣
Reactive nitrogen species   ┣ ┣
Catalase, Superoxide dismutase   ┣ ┣
GSH peroxidase, GSH/GSSG   ┣ ┣
Proinflammatory Inducible NO Synthase   ┣ ┣
Heme oxygenase-1   ┣ ┣
enzymes Cyclooxygenase-2
  ┣ ┣
Conversion of Xanthine Dehydrogease to
Xanthine Oxidase   ┣ ┣
Proinflammatory IL-1   ┣ ┣
IL-6   ┣ ┣
cytokines TNF-a ┣ ┣
 
Adhesion E-selectin   ┣ ┣
P-selectin   ┣ ┣
molecules ┣ ┣
VCAM-1  
ICAM-1   ┣ ┣
NF-B activation NF-B DNA binding activity   ┣ ┣
NIK/IKK activation   ┣ ┣
Phosphorylation of IBa   ┣ ┣
Degradation of IBa and IB in cytoplasm   ┣ ┣
Nuclear translocation of p65 and p50   ┣ ┣
NF-B-dependent gene expression   ┣ ┣
Active MAPKs (ERK, JNK, p38 MAPK)   ┣ ┣
, Increased ; , decreased ; ┣, blunted
(Antioxid Redox Signal, 2006)

27. Possible mechanism of baicalein
Aging/ROS
baicalein
activation
pi PPAR
IκB IκB
p50 p65
Nucleus
CBP
SRC-1
BI/BE
pi
p50 p65 iNOS, COX-2, VCAM-1 PPAR RXR Glucose,
lipid metabolism

28. 4) Antioxidants
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

29. Possible mechanism of Betaine Met
CYSTEINE DMG THF
BHMT MS
GSH Betaine 5-Me-THF
(Thiols: SH)
Homocysteine
NIK
MKK 3/6 IKK
MEK 1/2 IB
NF-B
P
P P
p38
ERK JNK IB
NF-B
Nucleus
Vascular
NF-B COX-
2, iNOS, VCAM- Aging
1, ICAM-1

30. Ⅴ. New drug for aging intervention
in the future
Systemic approaches to find drug target molecules and
their modulators for anti-aging
:Integrating protein-protein interaction network
and docking simulation
박대의 (박사 3년)

31. The scheme of systemic approaches
Genome Transcriptome Proteome
NGS Microarray 2D/Mass
OMICs data
Protein-protein interaction network • Protein-protein interaction network
Degree HUBs , Centrality HUBs • Analysis of biological network
• Protein structure modeling
• Docking simulation
Experimental evaluation (Wet Lab)
Protein Structure Protein-Ligand
Modeling Docking
Testing value as drug (Wet Lab)

32. Structural modeling and docking simulation for PPAR
Docking Energy
Autodock
Name
(Kcal/Mol)
708 -8.88
778 -8.23
rosiglitazone 602 -8.06
rosiglitazone -8.03
866 -7.76
900 -7.75
915 -7.66
667 -7.57
455 -7.54
Pharmacophore
PPAR-gamma
Candidates

33. Conclusion
Large-scale microarray data Target Validation
Significant:
Median
10 Tail strength
se (%): 26.9
False
5
Observed Score
0
-6 -4 -2 0 2 4 6
-5
-10
-15
Docking Simulation
Expected Score
Differentially Expressed Genes Protein-protein Interaction analysis Biological activity
Optimization of leader compds
Drug candidate

34. 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

35. 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