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7. 정해영 7. 정해영 Presentation Transcript

  • Molecular Inflammation as the UnderlyingMechanism 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
  • The young Aging process The old
  • 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, AMsNucleus B-site View slide
  • 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) View slide
  • Ⅰ. Molecular Inflammation Biochemical Changes Leading to Inflammatory ResponseIntracellular 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)
  • 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)
  • Redox ImbalanceCR/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)
  • 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
  • Normal Aging ① Abnormal Aging Vascular alteration Disease Urate, Vt C, E, GSH, Hypoxia SOD, CAT, GSHPx ATP depletion Cell death ②ROS / 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
  • 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)
  • What is responsible for up-regulation of AMs during aging? Proinflammatory Bioactive cytokines lipids Lipid Endotoxin peroxides Adhesion Inflammatory 400 Oxidants Molecules mediatorsRelative 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
  • 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)
  • Action Mechanism of LPC Lysophosphatidylcholine (LPC) GPR4 activation + GPR4AA861 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)
  • 2) Increased HNE/HHE during aging ROS can attack membrane lipids and initiate lipid peroxidation. Thelipid peroxidation productions include radical and aldehyde-derivativesuch as MDA, HNE and HHE 4-hydroxynonenal (HNE) 4-hydroxyhexenal (HHE)
  • 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)
  • 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
  • Effects of Aging and CR on LPS levelsLPS 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
  • 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 SerP Y IKKa IKK TAK1 IBap50 p65 p65 p50 p50 p65 COX-2, TNF, IL6, iNOS Inflammation
  • Redox Imbalance CRNIK/IKK, MAPKs NF-B COX-2, iNOS, AMs, TNFaVascular Alteration Hypoxia Aging 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 Aging CR GO description p-value (SREBP, PPAR) lipid metabolism 0.0182 N=37 Patterns # of genes Aging CR 7Aging 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)
  • Ⅳ.Aging Intervention strategies 1) CR 2) Exercise 3) PPAR agoinst 4) Antioxidants
  • 1)Molecular Mechanism of CR Calorie Restriction Insulin, GF ROS NAD+/NADH IRS-1 PI3KTRX/GSH SIRT1 Akt System MAPKs IKK FOXO SOD/CAT PPARs NF-κB COX2/iNOS/LOX/AMs Chronic Inflammation Aging
  • 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)
  • 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)
  • 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)
  • 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
  • 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
  • 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
  • Ⅴ. New drug for aging intervention in the futureSystemic approaches to find drug target molecules and their modulators for anti-aging :Integrating protein-protein interaction network and docking simulation 박대의 (박사 3년)
  • 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-LigandModeling Docking Testing value as drug (Wet Lab)
  • 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
  • Conclusion Large-scale microarray data Target ValidationSignificant:Median 10 Tail strength se (%): 26.9False 5Observed 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
  • 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
  • Conclusion1. Molecular inflammation during aging : Balance between NF-B and PPARs2. Modulation of age-related inflammation by CR and exercise3. Flavonoid, kaempferol and baicalein : through modulation of PPAR, SIRT1, and redox4. 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