EFFECT OF ALOE GEL IN ENHANCING DIABETIC WOUND HEALING Djanggan Sargowo, Yudha Handaya, Askandar Tjokroprawiro Brawijaya Medical School Malang 1

EFFECT OF ALOE GEL IN ENHANCING DIABETIC WOUND HEALING

Djanggan Sargowo, Yudha Handaya,

Askandar Tjokroprawiro

Brawijaya Medical School

Malang11

BACKGROUNDS

22

33

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 forefootGrade 5 gangrene on the entire foot

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 forefootGrade 5 gangrene on the entire foot

WAGNER CLASSIFICATION

44 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)

55

HIPERGLIKEMIA (DM)

b↑AGE>1 week ↑HEKSOSAMINE PATWAY (H) ↑AR ↑PKC↑ROS

↑TNF↑NFκB↓NO ↑NFκB, ↓eNO, ↑TGFβ

↑NAD(P)H OXIDASE↑ET-1, ↑PAI-1, ↑VEGF

↑TGFβ,↑MMPs↓CELL PROLIFERATION

↑SORBITOL↓GSH↑TNFα

↑OH+↑TNFα

GFATP A H A

STRESS OXIDATIVE STRESS OXIDATIVE INSULIN SENSITIVITY -CELL FUNCTIONΒ

“PAHAM”:PKC,AGE,HEKSOSAMINE,AR,MITOCONDRIAL DYSFUNCTION

MIKROANGIOPATI & MAKROANGIOPATI

TRAUMA VASCULOPATHYNEUROPATHY

MOTORIC SENSORY AUTONOM MICROPATHY MACROPATHY

WEAKNESSATROPHYDEFORMITY↑PLANTAR PRESSURE CLAVUS

↓PROTECTION↓SENSATION

ANHIDROSISDRY SKINFISSURE↑SYMPATHY PRESSURE

ATEROSCLEROSISISCHEMIA

BM CAPILLARY THICKENNINGA-V SHUNT↑BLOOD FLOWEDEMA NEUROPATHY

OSTEOARTHROPATHY ↓RESPONSE TO INFECTION↓NUTRITION ↓CAPILLARY BLOOD FLOW

HEMOSTASIS 1 HOUR #INFLAMMATION 2-7 DAYS #PROLIFERATIOM 2-20 DAYS #REMODELLING 6WEEK-3 MONTHS

GAMGGUAN PENYEMBUHAN ULKUS KAKI DMAMPUTASI AMPUTASI

#INFLAMMATION↓NEUTROPHIL↓MONOCYTE

ENDOTHELIAL DYSFUNCTION# ANGIOGENESIS↓KERATINOCYTES↓PROLIFERATION

↓FIBROBLAST↓GF↓CITOKINES

SCHIFF BASE <1 day

AMADORI 1-7 days

Gambaran Kegagalan Patofisiologi Ulkus diabetes dan Patologi Penyembuhan ulkus diabetes (Robert et al 2006, Gugliucci 2007, Pradhan et al 2007, Tjokroprawiro 2010). 66

HYPERGLYCEMIA

MACRO-MICROANGIOPATHY

# WOUND HEALING

OXIDATIVE STRESS OXIDATIVE STRESS

AMPUTATIONAMPUTATION

77

88

99

1010

1111

ERK-1/2ERK-1/2

ALOE GEL

VEGF

eNOS

EPC CEC

ANGIOGENESIS

DIABETIC WOUND HEALING

Cellular proliferation

CONCEPTUAL THEORY

Aloe gelAloe gel

Dose of Dose of 30,60,12030,60,120(mg/day)(mg/day)

Diabetic wound Diabetic wound healinghealing

wound wound diameterdiameter

Angiogenesis

VEGF, VEGF, eNOS eNOS ,,EPCEPC,,CEC , CEC ,

Rasio EPC/CECRasio EPC/CEC

Expression and Expression and amountamount

Hipotesis

1212

MATERIALS AND METHODSMATERIALS AND METHODS

1313

SCHEME OF STUDY PROTOCOL (ROCKET SYSTEM MODIFICATION, TJOKROPRAWIRO,

1978)

1414

REPORT

WOUND + ALOE GEL 120 MG


ANALISIS

DATA

DAY0 1

4

DM 0

DM120

DM 60

B0RANDOM

STRAIN

WISTAR+ALOE

GEL

VEGF,eNOS, EPC,CEC Ø WOUND

PRA PENELITIAN

A : NORMAL RATSB : UNTREATED RATSC : DIABETIC RATS+TREATMENTD : DIABETIC RATS+TREATMENTE : DIABETIC RATS+TREATMENTPRE-STUDY: 0-14 days bedore treatmentSTUDY : 0-14 hari after treatmentAle gel : glycoprotein and polysacchaarides aloe vera

VEGF :Vascular Endothelial Growth Factor

eNOS :endothelial Nitric Oxide Synthase

EPC :Endothelial Progenitor CellsCEC :Circulating Endothelial Cells

VEGF : immunohistochemistryeNOS : immunohistochemistryEPC : CD34 (Flowcitometri)CECs : CD45(FITC)+CD146(PE)

(Flowcitometri)Ø wound : wound dimeter

Injection of STZ 60mg/kg (IP)for 14 days (2 weeks)Diabetes if blood glucose level >200mg/dl

NON DM

WOUND


D14

PENELITIAN

14

0

28

0 14

N:15N:15

n:3n:3

n:3n:3

MAIN PARAMETERMAIN PARAMETER

ketamin (10mg/kg/iv)Wound diameter 2,5 cm depth 0,2 cmWash the wound with Nacl 0,9%

n:3n:3

n:3n:3

A0

D0

E0

VEGF,eNOS,EPC,CEC Ø WOUND

HASILHASILDATADATA

DATADATA

DATADATA

DATADATAA14

PRE- STUDY STUDY

DAY

MAKING OF DIABETIC RATS

MAKING OF WOUND

PARAMETER DATA ANALYSIS

C14

E14

SAMPLE POPULATIONp(t-1)(r-1)≥ 15

t=±treatmentr=±repetition

DM 30C0

WOUND B14 DATADATAn:3n:3

Linear Regression

Anova (simple anova)

PRE- STUDY STUDY

DATA

ANALYSIS

RESULTRESULT

STUDY TIMELINE

1515

RESULTS

14th Day, measurement of :•Wound diameter

•Immunohistochemistry of dermal tissue•Blood for Flowcitometry

1616

Measurement of Wound Diameter

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

3 0,320,01(b


3 0,270,02(b


3 0,410,02(b

1717p = 0.000

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

Vascular Endothelial Growth Factor (VEGF) Expression

treatment n Mean SD (p ≤ 0,05)




3 28,335,86(a


3 34,675,03(a


3 25,674,73(a

1188

p = 0.676

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

Endothelial Nitric Oxide Synthase (eNOS) Expression


Normal Rats 3 24,671,73 (b



3 30,001,00(a


3 34,332,08(a


3 42,001,00(b

1919p = 0.000

Normal RatsDM + 120

mgDiabetic rats DM + 30 mg DM + 60 mg

Amount of Endothelial Progenitor Cells (EPCs) CD 34





3 10,649,34(a


3 12,111,16(a


3 13,751,63(a

p = 0.457

n kelompok = 3

2200

Normal RatsDM + 120

mgDiabetic rats DM + 30 mg DM + 60 mg

Amount of Circulating Endothelial Cells (CECs) CD 45

2121p = 0.000

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


Normal Rats 3 3,161,68(bc



3 7,361,22(b


3 4,890,83(bc


3 2,200,74(c

PATH ANALYSIS

2222

2323

DISCUSSION

Aloe gelAloe gel

DosisDosis(mg/day)(mg/day)

Diabetic Diabetic wound healingwound healing

Wound Wound diameterdiameter

↑ wound contraction

Effect administration of Aloe gel on wound diameter

Effect administration of Aloe gel on wound diameter

2424

• 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)

Aloe gelAloe gelDosisDosis

(mg/day)(mg/day) Diabetic Diabetic

wound healingwound healing


↑ Angiogenesis

Effect administration of Aloe gel on Expression of VEGF and eNOS Dermal Tissue

Effect administration of Aloe gel on Expression of VEGF and eNOS Dermal Tissue

(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).

2525

VEGF eNOS

EPC mobilization

Aloe gelAloe gel

DosisDosis(mg/day)(mg/day)

Diabetic Diabetic wound healingwound healing


↑ Angiogenesis

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

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

2266

migration/Homing EPC

CECs

2727

EPC/CEC RATIO AFTER ADMINISTRATION OF ALOE GELEPC/CEC RATIO AFTER ADMINISTRATION OF ALOE GEL

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).

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).

Aloe gelAloe gelDose Dose

120(mg)120(mg)

wound healingwound healingwound healingwound healing

wound wound diameter diameter

cmcm22

Angiogenesis

VEGF, VEGF, eNOS eNOS ,,EPCEPC

,,CEC CEC

Exresion (ng/ml) Exresion (ng/ml) and amountand amount

2288

Ratio Ratio EPC/CECEPC/CEC

Ratio Ratio EPC/CECEPC/CEC

• Hipotesis statistically proved (P<0,05)

• Hipotesis statistically proved (P<0,05)

EPC/CEC ratio Novel (new)Base of therapyPrognostic factor

EPC/CEC ratio Novel (new)Base of therapyPrognostic factor

2929

3030

3131

Mekanisme Penyembuhan luka Normal dibanding Diabetes (Beem et al 2007, Tjokropawiro 2011)•

DM WITH 5 DYSFUNCTION : KERATINOCYTE, PLATELET, ENDOTHELIUM, MACROPHAGE, MYOFIBROBLAST

DM WITH 5 DYSFUNCTION : KERATINOCYTE, PLATELET, ENDOTHELIUM, MACROPHAGE, MYOFIBROBLAST

3232

Blood Glukose Should be <

200

Blood Glukose Should be <

200

HEALTHY WOUND DIABETIC WOUNDTHE ROLES OF HSP 70 / HSP 72

on Wound HealingTHE ROLES OF HSP 70 / HSP 72

on Wound Healing

-catenin-catenin

c-mycc-myc

LIMITEDEPC HOMING

IMPROVED BY LOCAL SDF-1

INJECTION

IMPROVED BY LOCAL SDF-1

INJECTION

LIMITED NEOVASCULOGENESIS AND WOUND HEALING

IMPROVED BY HBO THERAPY

IMPROVED BY HBO THERAPY

DecreasedeNOS Activation

VEGF

Conceptual TheoryConceptual Theory

33

Active Compound of Aloe Gel (Hamman, 2008)

34

Documents

EFFECT OF ALOE GEL IN ENHANCING DIABETIC WOUND HEALING Djanggan Sargowo, Yudha Handaya, Askandar Tjokroprawiro Brawijaya Medical School Malang 1