aloe gel asmiha
TRANSCRIPT
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EFFECT OF ALOE GEL IN ENHANCING
DIABETIC WOUND HEALING
Djanggan Sargowo, Yudha Handaya,
Askandar Tjokroprawiro
Brawijaya Medical School
Malang1
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DM
2003 (197 millions)2010 ( 50%)
2025 (333 millions/6,3% population)
77% Developed Country
(Lauderdale 2006, Goycheva,et al 2006)
WHOIndonesia : 4th largest population with DM
2000(5,6 millions)2006(14 millions); 2025 (6,5% ofpopulation)
(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)
BACKGROUNDS
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3
Grade 0pre-ulcerative lesion, healed
ulcers, presence of bony
deformity
Grade 1superficial ulcer withoutsubcutaneous tissue
involvement
Grade 2penetration through the
subcutaneous tissue (may
expose bone, tendon, ligamentor joint capsule)
Grade 3osteitis, abscess, or
osteomyelitis
Grade 4gangrene on the forefoot
Grade 5 gangrene on the entire foot
WAGNER CLASSIFICATION
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# Micro/Macroangiopaty
diabetic foot ulcers
Wound Healing
Wound healing complexmediated by :
vascular, neuronal, cellular & immunefactors,
Wound healing on DM
grow th factor &pro-angiogenic cytokinesmatrix deposition & remodel l ing(Brownlee2005,Ralf et al 2005)
AngiopathyNeuropathy
combination(David et al 2002)
Imbalance Wound Healing Phases
4
WOUND HEALING DISORDERS
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DMHyperglycemia ROS apoptosis angiogenesis dysfunction (#tube formation)WH diabetic foot ulcers (DFU) progression amputation
(David et al2002,Hesham et al2006 ,Beem 2007, Tjokropawiro 2010)
DM VEGF Ca cytosolic activity + eNOS Homing EPCAngiogensis # WH ulkus DM(Costanza et al2002, Pradhan et al2007,Singhan et al2007)
DFU proper managementHerbal therapy enhance angiogenesis
(Larijani et al2008)
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HIPERGLIKEMIA (DM)
bAGE
>1 week HEKSOSAMINE PATWAY (H) ARPKCROSTNF
NFB
NO NFB, eNO, TGFNAD(P)H OXIDASE
ET-1, PAI-1, VEGF
TGF,MMPs
CELL PROLIFERATION
SORBITOLGSH
TNF
OH+
TNF
GFAT
P A H A
STRESS OXIDATIVEINSULIN SENSITIVITY -CELL FUNCTION
PAHAM:PKC,AGE,HEKSOSAMINE,AR,MITOCONDRIAL DYSFUNCTION
MIKROANGIOPATI & MAKROANGIOPATI
TRAUMA VASCULOPATHY NEUROPATHY
MOTORIC SENSORY AUTONOM MICROPATHY MACROPATHY
WEAKNESS
ATROPHY
DEFORMITY
PLANTAR PRESSURE
CLAVUS
PROTECTION
SENSATION
ANHIDROSIS
DRY SKIN
FISSURE
SYMPATHY PRESSURE
ATEROSCLEROSIS
ISCHEMIA
BM CAPILLARY THICKENNING
A-V SHUNT
BLOOD FLOW
EDEMA NEUROPATHY
OSTEOARTHROPATHYRESPONSE 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
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ALOE VERA
herbalhigh potency for diabetic woundhealing(Wendell, 2000).
Easy to find, antiinflammation (C-glukosylchromone), immune modulator, wound
nutrition (Wendell, 2000)
clinical Aloe gel modulate immunity,blood glucose stabilizer, pain ; arteriolardiameter (Somboonwong et al., 2000).
Aloe (gel): 98% water ;Acetylatedmannan>60% active compound 1 mg aloegel (acemanan 0,6mg) (Somboonwong et al,2000; Kusmawati,2007 )
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Acemannan-(1-4)-linked glukomanan acetylated and
manosaasetilasi residu
Polysaccharides doses 20 g/mL enhance WH, stimulatecellular proliferation,migration and cytokines production
Stimulate oxygen consumption, angiogenesis & sintesis (Lauraet al., 2002).
oral WH fibroblast proliferation and induceexpresssion ofKGF-1 and VEGF (Ramamoorthy, 1996; Hamman, 2008).
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Problem of the Study
herbal Aloe gelangiogenesisWH DM
wound healing VEGF expression
eNOS
EPC
CEC
EPC/CEC ratio
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Aim of the Study
PROVE :herbal Aloe gelangiogenesisWH DM
wound healing
VEGF expression
eNOS,
EPCCEC
EPC/CEC ratio
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ERK-1/2
ALOE GEL
VEGF
eNOS
EPC CEC
ANGIOGENESIS
DIABETIC WOUND HEALING
Cellular proliferation
CONCEPTUAL THEORY
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Aloe gel
Dose of30,60,120
(mg/day)
Diabetic wound
healing
wounddiameter
Angiogenesis
VEGF,eNOS ,EPC,CEC ,Rasio EPC/CEC
Expression and
amount
Hipotesis
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MATERIALS AND METHODS
Subject of study
Male strain wistar rats induced bySTZto makeDiabetic rats and wounded and treated by Aloe
veragel orally
Study Design
True experimental in vivo study designed by posttest only control grup design
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SCHEME OF STUDY PROTOCOL(ROCKET SYSTEM MODIFICATION, TJOKROPRAWIRO, 1978)
14
R
E
P
O
R
T
WOUND + ALOE
GEL 120 MG
WOUND + ALOE
GEL 60 MG
A
N
A
L
I
S
I
S
D
A
T
A
DAY
0 14
DM 0
DM120
DM 60
B0R
A
N
D
O
M
STRAI
N
WIS
TAR+ALOE
GEL
VEGF,eNOS,
EPC,CEC
WOUND
PRA
PENELITIAN
A : NORMAL RATSB : UNTREATED RATSC : DIABETIC RATS+TREATMENTD : DIABETIC RATS+TREATMENTE : DIABETIC RATS+TREATMENTPRE-STUDY: 0-14 days bedore treatment
STUDY : 0-14 hari after treatmentAle gel : glycoprotein and polysacchaarides aloe vera
VEGF :Vascular Endothelial Growth Factor
eNOS :endothelial Nitric Oxide Synthase
EPC :Endothelial Progenitor Cells
CEC :Circulating Endothelial Cells
VEGF : immunohistochemistry
eNOS : immunohistochemistryEPC : CD34 (Flowcitometri)
CECs : CD45(FITC)+CD146(PE)
(Flowcitometri)
wound : wound dimeter
Injection of STZ 60mg/kg (IP)
for 14 days (2 weeks)
Diabetes if blood glucose level >200mg/dl
NON
DMWOUND
WOUND + ALOE
GEL 30 MG
D14
PENELITIAN
140
280 14
N:15
n:3
n:3
MAIN PARAMETER
ketamin (10mg/kg/iv)
Wound diameter 2,5 cm depth 0,2 cm
Wash the wound with Nacl 0,9%
n:3
n:3
A0
D0
E0
VEGF,eNOS,
EPC,CEC
WOUND
HASILDATA
DATA
DATA
DATAA14
PRE- STUDY STUDY
DAY
MAKING OF DIABETIC RATS
MAKING OF WOUND
PARAMETER DATA ANALYSIS
C14
E14
SAMPLE POPULATION
p(t-1)(r-1) 15
t=treatmentr=repetition
DM 30C0
WOUNDB14 DATAn:3
Linear Regression
Anova (simple anova)
PRE- STUDY STUDY
D
A
T
A
A
N
A
LY
S
I
S
RESULT
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STUDY TIMELINE
PREMAKING OF ALOE GEL
MAKING OF DIABETIC RATS
studyStage I study
Effect of Aloe Gel wounddiameter
stage II study
(IMMUNOHISTOCHEMISTRY)
Effect of Aloe Gel Expression ofVEGF 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
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
Diabetic rats + 60 mg aloe
gel
3 0,270,02(b
Diabetic rats + 120 mg
aloe gel
3 0,410,02(b
17p = 0.000
Normal Rats DM + 120 mgDiabetic rats DM + 30 mg DM + 60 mg
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Vascu lar Endo thel ial Grow th Factor(VEGF) Expression
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
18p = 0.676
Normal Rats Diabetic Rats DM+30mg DM+60mg DM+120mg
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Endo thel ial Nitr ic Oxide Synthase(eNOS) Expression
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
19p = 0.000
Normal RatsDM + 120
mgDiabetic rats DM + 30 mg DM + 60 mg
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Amoun t of Endothelial Progeni tor Cel ls(EPCs) CD 34
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
Non DMhari ke-14(kontrol -)
DM hari ke-14(kontrol +)
DM + aloe gel 30 mghari ke-14
DM + aloe gel 60 mghari ke-14
DM + aloe gel 120 mghari ke-14
p = 0.457
n kelompok = 3
20
Normal RatsDM + 120
mgDiabetic rats DM + 30 mg DM + 60 mg
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Amount of Circulat ing Endo thel ial Cel ls(CECs) CD 45
Non DMhari ke-14(kontrol -)
DM hari ke-14(kontrol +)
DM + aloe gel 30 mghari ke-14
DM + aloe gel 60 mghari ke-14
DM + aloe gel 120 mghari ke-14
21
p = 0.000
Normal Rats DM + 120 mgDiabetic rats DM + 30 mg DM + 60 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
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PATH ANALYSIS
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DISCUSSION
Eff t d i i t ti f Al l d
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Aloe gel
Dosis
(mg/day) Diabeticwound healing
Wounddiameter
wound contraction
Effect administration ofAloe gel on wound
diameter
24
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)
Eff t d i i t ti f Al l E i f VEGF d
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Aloe gel
Dosis(mg/day) Diabeticwound healing
Wounddiameter
Angiogenesis
Effect administration ofAloe gel onExpression 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).
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VEGFeNOS
EPC mobilization
Eff t d i i t ti f Al l t f
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Aloe gel
Dosis
(mg/day)
Diabeticwound healing
Wounddiameter
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
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migration/Homing EPCCECs
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EPC/CEC RATIO AFTER ADMINISTRATIONOF ALOE GEL
aloe gel herbal pro-angiogenic keep survival
EPCs during mobilization and hom ing into woundincrease amount of EPCs to damage
endothelial cells from vascular wall (CECs).
Treatment Mean SD (p 0,05)
Normal rats 6,53,41 (cDiabetic rats 0,720,1(a
Diabetic rats + 30 mg aloe
gel
2,180,16(ab
Diabetic rats + 60 mg aloegel
2,510,84(ab
Diabetic rats + 120 mg aloe
gel
6,351,99(c
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Aloe gel
Dose
120(mg)
wound healing
wounddiameter
cm2
Angiogenesis
VEGF,eNOS,EPC,CEC
Exresion (ng/ml)
and amount
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Ratio
EPC/CEC Hipotesisstatisticallyproved
(P
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HEALTHY WOUND DIABETIC WOUNDTHE ROLES OF HSP 70 / HSP 72
on Wound Healing
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Mekanisme Penyembuhan luka Normal dibanding Diabetes (Beem et al 2007, Tjokropawiro 2011)
DM WITH 5 DYSFUNCTION : KERATINOCYTE, PLATELET,ENDOTHELIUM, MACROPHAGE, MYOFIBROBLAST
Blood Glukose
Should be