Transcript Aloe gel
EFFECT OF ALOE GEL IN ENHANCING DIABETIC WOUND HEALING Djanggan Sargowo, Yudha Handaya, Askandar Tjokroprawiro Brawijaya Medical School Malang 1 BACKGROUNDS DM 2003 (197 millions)2010 ( 50%) 2025 (333 millions/6,3% population) 77% Developed Country (Lauderdale 2006, Goycheva,et al 2006) WHO Indonesia : 4th largest population with DM 2000(5,6 millions)2006(14 millions); 2025 (6,5% of population) (sidartawan 2007) 2010 ( 7 jt) no 92030 (12jt)No 6 (IDF) (Tjokropawiro 2010) DM (15% DFU) amputation ( 84% DFU) Surabaya3,8% Fountain IVDFU (Beem et al 2007,Tjokropawiro 2011) 2 WAGNER CLASSIFICATION Grade 0 pre-ulcerative lesion, healed ulcers, presence of bony deformity Grade 1 superficial ulcer without subcutaneous tissue involvement Grade 2 penetration through the subcutaneous tissue (may expose bone, tendon, ligament or joint capsule) Grade 3 osteitis, abscess, or osteomyelitis Grade 4 gangrene on the forefoot Grade 5 gangrene on the entire foot 3 # Micro/Macroangiopaty diabetic foot ulcers Wound Healing Wound healing complex mediated by : vascular, neuronal, cellular & immune factors, Wound healing on DM ↓growth factor & pro-angiogenic cytokines matrix deposition & remodelling Angiopathy Neuropathy combination (David et al 2002) (Brownlee2005,Ralf et al 2005) Imbalance Wound Healing Phases WOUND HEALING DISORDERS 4 DMHyperglycemia↑ ROS ↑ apoptosis angiogenesis dysfunction (#tube formation) ↓ WH ↑ diabetic foot ulcers (DFU) progression amputation (David et al 2002, Hesham et al 2006 ,Beem 2007, Tjokropawiro 2010) DM↓ VEGF ↓ Ca cytosolic activity + ↓eNOS ↓Homing EPC ↓ Angiogensis # WH ulkus DM (Costanza et al 2002, Pradhan et al 2007,Singhan et al 2007) DFU proper management Herbal therapy enhance angiogenesis (Larijani et al 2008) 5 HIPERGLIKEMIA (DM) HYPERGLYCEMIA SCHIFF BASE <1 day AMADORI 1-7 days P ↑PKC ↑ROS ↑TNF ↑NFκB ↓NO b↑AGE >1 week A H ↑HEKSOSAMINE PATWAY (H) A ↑AR GFAT ↑NFκB, ↓eNO, ↑TGFβ ↑NAD(P)H OXIDASE ↑ET-1, ↑PAI-1, ↑VEGF ↑TGFβ,↑MMPs ↓CELL PROLIFERATION OXIDATIVE STRESS STRESS OXIDATIVE INSULIN SENSITIVITY ↑OH+ ↑TNFα ↑SORBITOL ↓GSH ↑TNFα Β-CELL FUNCTION “PAHAM”:PKC,AGE,HEKSOSAMINE,AR,MITOCONDRIAL DYSFUNCTION MIKROANGIOPATI & MAKROANGIOPATI MACRO-MICROANGIOPATHY NEUROPATHY MOTORIC WEAKNESS ATROPHY DEFORMITY ↑PLANTAR PRESSURE CLAVUS SENSORY ↓PROTECTION ↓SENSATION TRAUMA AUTONOM #INFLAMMATION 2-7 DAYS MACROPATHY BM CAPILLARY THICKENNING A-V SHUNT ↑BLOOD FLOW EDEMA NEUROPATHY ↓RESPONSE TO INFECTION #INFLAMMATION ↓NEUTROPHIL ↓MONOCYTE AMPUTASI AMPUTATION MICROPATHY ANHIDROSIS DRY SKIN FISSURE ↑SYMPATHY PRESSURE OSTEOARTHROPATHY HEMOSTASIS 1 HOUR VASCULOPATHY ENDOTHELIAL DYSFUNCTION # ANGIOGENESIS ↓KERATINOCYTES ↓PROLIFERATION #PROLIFERATIOM 2-20 DAYS PENYEMBUHAN ULKUS KAKI DM #GAMGGUAN WOUND HEALING ATEROSCLEROSIS ISCHEMIA ↓NUTRITION ↓CAPILLARY BLOOD FLOW ↓FIBROBLAST ↓GF ↓CITOKINES #REMODELLING 6WEEK-3 MONTHS AMPUTASI AMPUTATION Gambaran Kegagalan Patofisiologi Ulkus diabetes dan Patologi Penyembuhan ulkus diabetes (Robert et al 2006, Gugliucci 2007, Pradhan et al 2007, Tjokroprawiro 2010). 6 ALOE VERA herbal high potency for diabetic wound healing (Wendell, 2000). Easy to find, antiinflammation (C-glukosyl chromone), immune modulator, wound nutrition (Wendell, 2000) clinical Aloe gel modulate immunity, blood glucose stabilizer,↓ pain ; ↑ arteriolar diameter (Somboonwong et al., 2000). Aloe (gel): 98% water ;Acetylated mannan>60% active compound 1 mg aloe gel (acemanan 0,6mg) (Somboonwong et al, 2000; Kusmawati,2007 ) 7 Acemannan Β-(1-4)-linked glukomanan acetylated and manosaasetilasi residu Polysaccharides doses 20 µg/mL ↑ enhance WH, stimulate cellular proliferation,↑ migration and cytokines production Stimulate oxygen consumption, ↑ angiogenesis & ↑ sintesis (Laura et al., 2002). oral ↑WH fibroblast proliferation and induce expresssion of KGF-1 and VEGF (Ramamoorthy, 1996; Hamman, 2008). 8 Problem of the Study herbal Aloe gel angiogenesis WH DM • • • • • • wound healing VEGF expression eNOS EPC CEC EPC/CEC ratio 9 Aim of the Study PROVE : herbal Aloe gelangiogenesisWH DM wound healing VEGF expression eNOS, EPC CEC EPC/CEC ratio 10 CONCEPTUAL THEORY ALOE GEL VEGF eNOS CEC EPC ANGIOGENESIS ERK-1/2 Cellular proliferation DIABETIC WOUND HEALING 11 Hipotesis wound diameter Dose of 30,60,120 (mg/day) Aloe gel Angiogenesis Diabetic wound healing VEGF, eNOS ,EPC ,CEC , Rasio EPC/CEC Expression and amount 12 MATERIALS AND METHODS Study Design True experimental in vivo study designed by post test only control grup design Subject of study Male strain wistar rats induced by STZ to make Diabetic rats and wounded and treated by Aloe vera gel orally 13 SCHEME OF STUDY PROTOCOL (ROCKET SYSTEM MODIFICATION, TJOKROPRAWIRO, 1978) S T R A I N W I S T A R + A L O E DAY 14 0 0 PRA PENELITIAN PRE- STUDY A0 N:15 R A N D O M B0 E0 WOUND n:3 DM 0 WOUND n:3 B14 DATA 30 WOUND + ALOE GEL 30 MG n:3 C14 DATA DM 60 WOUND + ALOE GEL 60 MG n:3 D14 WOUND + ALOE GEL 120 MG n:3 E14 DM120 G E L PRE- STUDY 0 A14 STUDY 14 DAY 28 A :Vascular Endothelial Growth Factor :endothelial Nitric Oxide Synthase :Endothelial Progenitor Cells :Circulating Endothelial Cells : NORMAL RATS B : UNTREATED RATS C : DIABETIC RATS+TREATMENT D : DIABETIC RATS+TREATMENT E : DIABETIC RATS+TREATMENT PRE-STUDY: 0-14 days bedore treatment STUDY : 0-14 hari after treatment Ale gel : glycoprotein and polysacchaarides aloe vera DATA DATA MAKING OF DIABETIC RATS Injection of STZ 60mg/kg (IP) for 14 days (2 weeks) Diabetes if blood glucose level >200mg/dl D A N A T A L A I A S N I A S L D Y A S T I A S RESULT HASIL DATA VEGF,eNOS, EPC,CEC Ø WOUND PARAMETER MAIN PARAMETER VEGF eNOS EPC CEC VEGF,eNOS, EPC,CEC Ø WOUND PENELITIAN STUDY NON DM C0 DM D0 14 VEGF eNOS EPC CECs : immunohistochemistry : immunohistochemistry : CD34 (Flowcitometri) : CD45(FITC)+CD146(PE) (Flowcitometri) Ø wound : wound dimeter DATA ANALYSIS Anova (simple anova) Linear Regression MAKING OF WOUND ketamin (10mg/kg/iv) Wound diameter 2,5 cm depth 0,2 cm Wash the wound with Nacl 0,9% SAMPLE POPULATION p (t-1)(r-1)≥ 15 t=±treatment r=±repetition 14 R E P O R T STUDY TIMELINE PRE MAKING OF ALOE GEL MAKING OF DIABETIC RATS study Stage I study Effect of Aloe Gel wound diameter stage II study (IMMUNOHISTOCHEMISTRY) Effect of Aloe Gel Expression of VEGF and eNOS stage III study (FLOWCYTOMETRI) Effect of Aloe Gel amount of EPC, CEC and EPC/CEC ratio 15 RESULTS 14th Day, measurement of : • • Wound diameter Immunohistochemistry of dermal tissue • Blood for Flowcitometry 16 Measurement of Wound Diameter Normal Rats p = 0.000 Diabetic rats DM + 30 mg DM + 60 mg DM + 120 mg Treatment n Mean SD (p ≤ 0,05) Normal Rats 3 1,100,14 (a Diabetic Rats 3 1,190,12(a Diabetic rats + 30 mg aloe gel Diabetic rats + 60 mg aloe gel Diabetic rats + 120 mg aloe gel 3 0,320,01(b 3 0,270,02(b 3 0,410,02(b 17 Vascular Endothelial Growth Factor (VEGF) Expression Normal Rats Diabetic Rats DM+30mg DM+60mg DM+120mg treatment n Mean SD (p ≤ 0,05) Normal Rats 3 24,672,00 (a Diabetic Rats 3 25,334,51(a Diabetic rats + 30 mg aloe gel 3 28,335,86(a Diabetic rats + 30 mg aloe gel 3 34,675,03(a Diabetic rats + 30 mg aloe gel 3 25,674,73(a p = 0.676 18 Endothelial Nitric Oxide Synthase (eNOS) Expression Normal Rats p = 0.000 Diabetic rats DM + 30 mg DM + 60 mg DM + 120 mg Treatment n Mean SD (p ≤ 0,05) Normal Rats 3 24,671,73 (b Diabetic Rats 3 30,672,08(a Diabetic rats + 30 mg aloe gel 3 30,001,00(a Diabetic rats + 60 mg aloe gel 3 34,332,08(a Diabetic rats + 120 mg aloe gel 3 42,001,00(b 19 Amount of Endothelial Progenitor Cells (EPCs) CD 34 Non DM hari ke-14 (kontrol -) Normal Rats DM hari ke-14 (kontrol +) DM + aloe gel 30 mg hari ke-14 Diabetic rats DM + 30 mg n kelompok = 3 DM + aloe gel 60 mg hari ke-14 DM + 60 mg DM + aloe gel 120 mg hari ke-14 DM + 120 mg Treatment n Mean SD (p ≤ 0,05) Normal Rats 3 16,880,59 (a Diabetic Rats 3 10,471,14(a Diabetic rats + 30 mg aloe gel 3 10,649,34(a Diabetic rats + 60 mg aloe gel 3 12,111,16(a Diabetic rats + 120 mg aloe gel 3 13,751,63(a p = 0.457 20 Amount of Circulating Endothelial Cells (CECs) CD 45 Non DM hari ke-14 (kontrol -) Normal Rats DM hari ke-14 (kontrol +) DM + aloe gel 30 mg hari ke-14 Diabetic rats DM + 30 mg DM + aloe gel 60 mg hari ke-14 DM + 60 mg DM + aloe gel 120 mg hari ke-14 DM + 120 mg Treatment n Mean SD (p ≤ 0,05) Normal Rats 3 3,161,68(bc Diabetic Rats 3 14,762,28(a Diabetic rats + 30 mg aloe gel 3 7,361,22(b Diabetic rats + 60 mg aloe gel 3 4,890,83(bc Diabetic rats + 120 mg aloe gel 3 2,200,74(c p = 0.000 21 PATH ANALYSIS 22 DISCUSSION 23 Effect administration of Aloe gel on wound diameter Wound diameter Dosis (mg/day) Aloe gel • • • ↑ wound contraction Diabetic wound healing Anti inflammatory fibroblast proliferation collagen dan glycosaminoglikan synthesis (Heggers 1993; Jia et al 2008;Kwack et al 2009 ,;Liu et al., 2010; Fernanda et al., 2009;Jettanacheawchankit et al 2009) 24 Effect administration of Aloe gel on Expression of VEGF and eNOS Dermal Tissue Dosis (mg/day) Aloe gel ↑ Angiogenesis Diabetic wound healing Wound diameter VEGF eNOS EPC mobilization (Heggers 1993; Choi et al 2002; Tanimoto et al 2002;Duda et al 2004; Young et al 2004;Kim et al 2007;Pradhan et al. 2007; Beem et al. 2007; Singhan et al 2007; Chen et al 2007). 25 Effect administration of Aloe gel on amount of EPCs and CECs circulation Wound diameter Dosis (mg/day) Aloe gel ↑ Angiogenesis Diabetic wound healing migration /Homing EPC CECs Spinetti et al. 2008;Albiero et al., 2011;Oren et al., 2010;McClung et al 2005;Fiorina et al. 2010;Sibal et al 2009;Spinetti et al 2008;Nababan et al., 2007;Boos et al 2006 26 EPC/CEC RATIO AFTER ADMINISTRATION OF ALOE GEL Treatment Mean SD (p ≤ 0,05) Normal rats 6,53,41 (c Diabetic rats 0,720,1(a Diabetic rats + 30 mg aloe gel Diabetic rats + 60 mg aloe gel Diabetic rats + 120 mg aloe gel 2,180,16(ab 2,510,84(ab 6,351,99(c aloe gel herbal pro-angiogenic keep survival EPCs during mobilization and homing into wound increase amount of EPCs to damage endothelial cells from vascular wall (CECs). 27 wound diameter cm2 Dose 120(mg) Aloe gel • Hipotesis statistically proved (P<0,05) Angiogenesis wound healing Ratio EPC/CEC VEGF, eNOS ,EPC ,CEC Exresion (ng/ml) and amount EPC/CEC ratio Novel (new) Base of therapy Prognostic factor 28 29 30 31 THE ROLES OF HSP 70 / HSP 72 on Wound Healing HEALTHY WOUND DIABETIC WOUND DM WITH 5 DYSFUNCTION : KERATINOCYTE, PLATELET, ENDOTHELIUM, MACROPHAGE, MYOFIBROBLAST c-myc -catenin VEGF LIMITED NEOVASCULOGENESIS AND WOUND HEALING LIMITED EPC HOMING IMPROVED BY LOCAL SDF-1 INJECTION IMPROVED BY HBO THERAPY Blood Glukose Should be < 200 Decreased eNOS Activation Mekanisme Penyembuhan luka Normal dibanding Diabetes (Beem et al 2007, Tjokropawiro 2011) Conceptual Theory Vascular Complication of Diabetes Ischemia/hypoxia VEGF VEGFR-2 ROS PLC-γ DAG IP3 PKC C a 2+ cSrc FAK CaM Raf-1 MEK eNOS PI3K p38MAPK Calpain ERK1/2 PIP3 HSP27 Akt/PKB NO HIF-1α, AP-1, NFκB, Ets-1 MMPs, uPA, PAI-1 Paxilli n prolifera tion EPC Homing, CEC ↓ Migration vascular permeability Angiogenesis Diabetic wound healing 33 Active Compound of Aloe Gel (Hamman, 2008) CLASS Anthraquinones/anthrones ACTIVE COMPOUND Aloe-emodin, aloetic acid, anthranol, aloin A and B (barbaloin), isobarbaloin,emodin, cinnamic acid ester mannanasetilasi, glucomannan Pure Mannan , asetilasi,glucogalactomannan,galactan, Chromones galactogalacturan,arabinogalactan, galactoglucoarabinomannan, pectic,xylan, cellulose 8-C-glucosyl-(2’-O-cinnamoyl)-7-O-methylaloediol A,8-Cglucosyl-(S)aloesol, 8-C-glucosyl-7-O-methyl-(S)-aloesol, 8-C glucosyl-7-O-methylaloediol, 8-C-glucosyl-noreugenin, isoaloeresin D, isorabaichromone, neoaloesin A Enzymes Alkaline phosphatase, amylase, carboxypeptidase, catalase,cyclooxidase, cyclooxygenase, lipase, oxidase, phosphoenolpyruvate carboxylase, superoxide dismutase Inorganic substances Calcium, chlorine, chromium, copper, iron, magnesium, manganese, potassium, phosphorous, sodium, zinc Other substances (Organic and fat) Asam Arachidonic,asam linolenic, steroids (campestrol,cholesterol, âsitosterol), triglicerides, triterpenoid, gibberillin, lignins,Potassium sorbate, salicylic acid, uric acid Asam Amino Non-essential and essential Alanine, arginine, asam aspartic,asam glutamic, glycine,histidine,hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tyrosine, valine Protein Saccharides Vitamin Lectins, lectin-like substance Mannose, glucose, L-rhamnose, aldopentose B1, B2, B6, C, â-carotene, choline, folicá acid-tocopherol 34