EFFECT OF ALOE GEL IN ENHANCING DIABETIC WOUND HEALING

Djanggan Sargowo, Yudha Handaya, Askandar Tjokroprawiro Brawijaya Medical School Malang
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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)

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
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# 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
(Brownlee2005,Ralf et al 2005)

Angiopathy Neuropathy combination

(David et al 2002)

Imbalance Wound Healing Phases

WOUND HEALING DISORDERS

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)

HIPERGLIKEMIA (DM) HYPERGLYCEMIA

SCHIFF BASE <1 day AMADORI 1-7 days

ROS TNF NFB NO

PKC

bAGE >1 week

HEKSOSAMINE PATWAY (H) GFAT

AR
SORBITOL GSH TNF

A
OH+ TNF

NFB, eNO, TGF NAD(P)H OXIDASE ET-1, PAI-1, VEGF

TGF,MMPs CELL PROLIFERATION

INSULIN SENSITIVITY

OXIDATIVE STRESS STRESS OXIDATIVE

-CELL FUNCTION

PAHAM:PKC,AGE,HEKSOSAMINE,AR,MITOCONDRIAL DYSFUNCTION MIKROANGIOPATI & MAKROANGIOPATI MACRO-MICROANGIOPATHY

NEUROPATHY MOTORIC SENSORY AUTONOM TRAUMA MICROPATHY
BM CAPILLARY THICKENNING A-V SHUNT BLOOD FLOW EDEMA NEUROPATHY

VASCULOPATHY MACROPATHY
ATEROSCLEROSIS ISCHEMIA

WEAKNESS ATROPHY DEFORMITY PLANTAR PRESSURE CLAVUS

PROTECTION SENSATION

ANHIDROSIS DRY SKIN FISSURE SYMPATHY PRESSURE

OSTEOARTHROPATHY

RESPONSE TO INFECTION

NUTRITION CAPILLARY BLOOD FLOW

#INFLAMMATION NEUTROPHIL MONOCYTE

ENDOTHELIAL DYSFUNCTION # ANGIOGENESIS KERATINOCYTES PROLIFERATION #PROLIFERATIOM 2-20 DAYS

FIBROBLAST GF CITOKINES
#REMODELLING 6WEEK-3 MONTHS

HEMOSTASIS 1 HOUR AMPUTASI AMPUTATION

#INFLAMMATION 2-7 DAYS

PENYEMBUHAN ULKUS KAKI DM #GAMGGUAN WOUND HEALING

AMPUTASI AMPUTATION

Gambaran Kegagalan Patofisiologi Ulkus diabetes dan Patologi Penyembuhan ulkus diabetes (Robert et al 2006, Gugliucci 2007, Pradhan et al 2007, Tjokroprawiro 2010).

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 )

Acemannan
-(1-4)-linked glukomanan acetylated and manosaasetilasi residu

Polysaccharides doses 20 g/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).

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

EPC ANGIOGENESIS

CEC

ERK-1/2

Cellular proliferation

DIABETIC WOUND HEALING

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Hipotesis
Dose of 30,60,120 (mg/day)

wound diameter

Aloe gel

Angiogenesis

Diabetic wound healing

VEGF, eNOS ,EPC ,CEC , Rasio EPC/CEC

Expression and amount

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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

S T R A I N
W I S T A R + A L O E G E L
MAIN PARAMETER
VEGF eNOS EPC CEC
A

(ROCKET SYSTEM MODIFICATION, TJOKROPRAWIRO, 1978)

0
PRA PENELITIAN PRE- STUDY

DAY 14 0
PENELITIAN STUDY

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VEGF,eNOS, EPC,CEC WOUND

A14 DATA

A0

NON DM DM 0

WOUND

n:3

N:15

R A N D O M

B0

WOUND
WOUND + ALOE GEL 30 MG WOUND + ALOE GEL 60 MG WOUND + ALOE GEL 120 MG
PRE- STUDY STUDY

n:3

B14

DATA

C0 DM

30

n:3

C14

DATA

D0

DM 60

n:3

D14

DATA

E0

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

R E P O R T

DM120

n:3

E14

DATA

14 DAY
MAKING OF DIABETIC RATS

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VEGF,eNOS, EPC,CEC WOUND

PARAMETER VEGF eNOS EPC CECs : immunohistochemistry : immunohistochemistry : CD34 (Flowcitometri) : CD45(FITC)+CD146(PE) (Flowcitometri) wound : wound dimeter SAMPLE POPULATION p (t-1)(r-1) 15 t=treatment r=repetition

DATA ANALYSIS
Anova (simple anova) Linear Regression

:Vascular Endothelial Growth Factor :endothelial Nitric Oxide Synthase :Endothelial Progenitor Cells :Circulating Endothelial Cells

Injection of STZ 60mg/kg (IP) for 14 days (2 weeks) Diabetes if blood glucose level >200mg/dl MAKING OF WOUND ketamin (10mg/kg/iv) Wound diameter 2,5 cm depth 0,2 cm Wash the wound with Nacl 0,9%

: 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

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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

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RESULTS
14th Day, measurement of :
Wound diameter Immunohistochemistry of dermal tissue Blood for Flowcitometry

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Measurement of Wound Diameter

Normal Rats Diabetic rats DM + 30 mg DM + 60 mg DM + 120 mg

Treatment
Normal Rats Diabetic Rats Diabetic rats + 30 mg aloe gel Diabetic rats + 60 mg aloe gel Diabetic rats + 120 mg aloe gel

n
3 3 3 3 3

Mean SD (p 0,05)
1,100,14 (a 1,190,12(a 0,320,01(b 0,270,02(b 0,410,02(b

p = 0.000

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Vascular Endothelial Growth Factor (VEGF) Expression

Normal Rats Diabetic Rats DM+30mg DM+60mg DM+120mg

treatment

Mean SD (p 0,05) 24,672,00 (a 25,334,51(a 28,335,86(a 34,675,03(a 25,674,73(a

Normal Rats Diabetic Rats

3 3

Diabetic rats + 30 mg aloe gel Diabetic rats + 30 mg aloe gel Diabetic rats + 30 mg aloe gel

p = 0.676

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Endothelial Nitric Oxide Synthase (eNOS) Expression Normal Rats Diabetic rats DM + 30 mg DM + 60 mg

DM + 120 mg

Treatment

Mean SD (p 0,05) 24,671,73 (b 30,672,08(a 30,001,00(a 34,332,08(a 42,001,00(b

Normal Rats

Diabetic Rats

Diabetic rats + 30 mg aloe gel Diabetic rats + 60 mg aloe gel Diabetic rats + 120 mg aloe gel

p = 0.000

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Amount of Endothelial Progenitor Cells (EPCs) CD 34

Non DM hari ke-14 (kontrol -)

Normal Rats

DM hari ke-14 (kontrol +)

Diabetic rats DM + 30 mg

DM + aloe gel 30 mg hari ke-14

DM + aloe gel 60 mg hari ke-14

DM + 60 mg

DM + aloe gel 120 mg hari ke-14

DM + 120 mg

n kelompok = 3

Treatment

Mean SD (p 0,05)

Normal Rats
Diabetic Rats Diabetic rats + 30 mg aloe gel Diabetic rats + 60 mg aloe gel

3
3 3 3

16,880,59 (a
10,471,14(a 10,649,34(a 12,111,16(a

Diabetic rats + 120 mg aloe gel

13,751,63(a

p = 0.457

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Amount of Circulating Endothelial Cells (CECs) CD 45

Non DM hari ke-14 (kontrol -)

Normal Rats

DM hari ke-14 (kontrol +)

Diabetic rats DM + 30 mg

DM + aloe gel 30 mg hari ke-14

DM + aloe gel 60 mg hari ke-14

DM + 60 mg

DM + aloe gel 120 mg hari ke-14

DM + 120 mg

Treatment
Normal Rats Diabetic Rats Diabetic rats + 30 mg aloe gel

n
3 3 3

Mean SD (p 0,05)
3,161,68(bc 14,762,28(a 7,361,22(b

Diabetic rats + 60 mg aloe gel

Diabetic rats + 120 mg aloe gel

3
3

4,890,83(bc
2,200,74(c

p = 0.000

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PATH ANALYSIS

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DISCUSSION
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Effect administration of Aloe gel on wound diameter

Dosis (mg/day)

Wound diameter

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)

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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).

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Effect administration of Aloe gel on amount of EPCs and CECs circulation

Dosis (mg/day)

Wound diameter

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

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EPC/CEC RATIO AFTER ADMINISTRATION OF ALOE GEL

Mean SD (p 0,05)

Treatment Normal rats

6,53,41 (c
0,720,1(a
2,180,16(ab 2,510,84(ab

Diabetic rats
Diabetic rats + 30 mg aloe gel Diabetic rats + 60 mg aloe gel Diabetic rats + 120 mg aloe gel

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).

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Dose 120(mg)

Aloe gel

wound diameter cm2

Angiogenesis

wound healing

Hipotesis statistically proved (P<0,05)

Ratio EPC/CEC VEGF, eNOS ,EPC ,CEC

Exresion (ng/ml) and amount

EPC/CEC ratio
Novel (new) Base of therapy Prognostic factor

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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- DAG PKC Raf-1 MEK
ERK1/2

IP3 C a 2+
CaM

cSrc
FAK

eNOS p38MAPK
Calpain

PI3K
PIP3

HSP27

NO
HIF-1, AP-1, NFB, Ets-1
Paxilli n

Akt/PKB

Migration

MMPs, uPA, PAI-1

prolifera tion

EPC Homing, CEC

vascular permeability

Angiogenesis

Diabetic wound healing

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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 Pure Mannan ,

mannanasetilasi, glucomannan

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 Alkaline phosphatase, amylase, carboxypeptidase, catalase,cyclooxidase, cyclooxygenase, lipase, oxidase, phosphoenolpyruvate carboxylase, superoxide dismutase Calcium, chlorine, chromium, copper, iron, magnesium, manganese, potassium, phosphorous, sodium, zinc Asam Arachidonic,asam linolenic, steroids (campestrol,cholesterol, sitosterol), triglicerides, triterpenoid, gibberillin, lignins,Potassium sorbate, salicylic acid, uric acid Alanine, arginine, asam aspartic,asam glutamic, glycine,histidine,hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tyrosine, valine Lectins, lectin-like substance

Enzymes Inorganic substances Other substances (Organic and fat)

Asam Amino Non-essential and essential Protein Saccharides Vitamin

Mannose, glucose, L-rhamnose, aldopentose

B1, B2, B6, C, -carotene, choline, folic acid-tocopherol

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