THE EFFECT OF ALOE GEL IN ENHANCING ANGIOGENESIS IN DIABETIC WOUND HEALING

Djanggan Sargowo*, Adeodatus Yuda Handaya, Askandar Tjokroprawiro

Postgraduate Program, Brawijaya Medical School

Malang-Indonesia

Correspondence email : djanggan@yahoo.com

ABSTRACT

Background: Diabetic micro and macroangiophaty will lead to the diabetic foot ulcers (DFU) incident that characterized by an increasing the number of circulating endothelial cells (CECs) and decreasing the number and function of endothelial progenitor cells (EPCs). This fact is correlates with ischemia and diabetic wound healing failure. Aloe vera gel is known able to stimulate VEGF expression and activity through enhancing of the NO production as result of NOS enzyme activity. It is to be a potential target to enhancing angiogenesis in wound healing.

Objective: The goal of this study is exploring the major role of vera gel on wound healing diabetic ulcers through increasing level of EPCs, VEGF, and eNOS, also reducing level of CECs that involved in angiogenesis process of diabetic ulcers healing.

Materials and Methods: The experimental groups is divided into five groups consist of non diabetic wistar rats, diabetic rats without peroral therapy aloe gel, and treatment groups (diabetic rats) with aloe gel doses 30, 60, and 120 mg/day during 14 day. All of this groups are wounded and also daily observations of wounds area was conducted. The measurement of EPC (CD 34) and CEC (CD45 and CD146) number by flowcytometry and followed with the measuring of VEGF and eNOS expression on dermal tissue by Imunohistochemistry method was observed in 0 day and 14 day after treatment. The quantitative data were analyzed by computerized software by One-way ANOVA and Linear Regression with a confidence interval 5% significance level (p <0.05) using Statistical Software Products and Solutions Servive 16 PS (SPSS 16 PS) to compare the difference and correlation between wound diameter, the number of EPC and CEC as well as the levels of VEGF and eNOS.

Results: The results research showed that aloe gel oral treatment in diabetic wistar rats able to accelerate the wound healing process. It is marked by reduction of wound diameter (0.27 ± 0.02), the increasing of EPCs number (13.75 ± 1.63), elevated levels of VEGF (34,67 ± 5,03) and eNOS (42,00 ± 1,00) and also the decreasing of CECs number (0.42 ± 0.57) significantly (p <0.05). In other hand, the wound diameter and eNOS indicators showed a significantly different at doses 60 mg while the number of EPC, CEC and VEGF levels showed significantly different results in a dose 120 mg. Aloe gel peroral therapy was shows a positive trend in wound healing acceleration on the optimum dose range 60-120 mg per day.

Conclusion: Aloe gel is potential to be candidates for diabetic wound healing herbal therapies through enhancing EPC homing, decreasing the CECs number, and stimulate the increasing of VEGF and eNOS level that already proved are involved as dominant factor in the angiogenesis process.

Key Word: aloe gel, diabetes, wound healing, angiogenesis

*) Prof. Dr. Djanggan Sargowo, MD, FIHA, FACC, FESC, FCAPC, FASCC

Introduction

Diabetes is a metabolic disease that occurs almost in various countries around the world. The incidence of diabetes continues to experience significant increases. In 2003, diabetes affects 197 million people worldwide, and is expected to rise more than 50% in 2010. This figure is predicted to be 333 million by 2025 or 6.3% of the total world population. Some 77% predicted to occur in developing countries (Goycheva et al 2006). According to WHO, Indonesia ranks fourth largest in the number of diabetics in the world, from the 5.6 million diabetics in 2000 to 14 million people in 2006. In 2025 Indonesia prevalence of diabetes is predicted to be 6.5%. International Diabetes Federation (IDF) said that the prevalence of diabetes aged 20-79 years in Indonesia as many as 7 million cases and ranked 9th in 2010 from 10 countries in the world with the highest rate of diabetes cases, and is predicted to rise to the rank-6 in 2030 with 12 million cases (Tjokropawiro 2010).

Complications of diabetes led to the emergence and makroangiopati microangiopathic disorders and neuropathy, which will cause interference with the process of angiogenesis, epitelialisasi, deposition of collagen and some other essential processes in wound healing stages. Failure of healing of diabetic ulcers occur due to disruption of a number of processes in the stages of wound healing. The process of inflammation, cell proliferation, cell migration, vascular permeability and angiogenesis due to deficiency of growth factors and cytokines proangiogenik, matrix deposition and tissue remodeling (Ralf et al 2005, Pradhan et al 2007).

Endothelial progenitor cells (EPCs) play an important role in the re-endotelialisasi of blood vessel damage caused by ischemia. EPCs potentially contribute to endothelial repair by homing into the damaged endothelial and able to maintain endothelial integrity. EPCs dysfunction is closely linked with the incidence of vascular ischemia (Cindy et al 2004).

Decrease in the number and function of EPCs associated with the pathogenesis of early vascular complications of diabetes both type 1 and 2. Increased oxidative stress (increased ROS) due to chronic hyperglycemia via activation of four major pathways of diabetic complications will also affect the expression and phosphorylation of endothelial nitric oxide synthase (eNOS). Enos known to be important for the survival, migration, and angiogenesis of EPCs or endothelial cells. Nitric oxide (NO) produced by eNOS activity started mobilizing EPCs from the bone marrow through nitrosilasi and increased expression of vascular endothelial growth factor (VEGF) (Chen et al 2007).

In normal individuals the number of circulating endothelial cells (CECS) is low. The amount will be increased in case of endothelial cell injury, as in the case of microbial infection triggers diabetes. Although there is no real proof that the CEC with endothelial damage associated with elevated levels of CEC in blood flow, the results of the study found that in patients with type 1 diabetes compared with normal individuals is known to occur is a significant difference to the amount of circulating CEC in the circulation of these patients (McClung et al 2005).

Management of diabetic ulcers need to be done in a comprehensive manner through efforts to cope with comorbid disease, reducing the load pressure (offloading), in order to always keep the wound moist (moist), treatment of infection, debridement, revascularization and elective surgery, prophylactic, curative or emergency. One of the other herbal ingredients that have great potential to cure cases of diabetic wounds is Aloe vera.

Aloe vera is one of the plants are easy to find and contain the active ingredient. Aloe vera a lot of therapy known to be involved in various diseases, especially in wound healing. Aloe vera gel in the capacity as an immune modulator and a source of nutrients that will likely help increase the rate of wound healing. Aloe gel is able to control the inflammatory phase as well as provide supply of nutrients to the wound so it can immediately complete the process of wound healing.

Recent research and clinical use of mentioning that Aloe gel has an effect pemacuan immunity, balance blood glucose and minimizes pain. Only in the group with aloe gel treatment showed an increase in arteriolar diameter toward normal conditions. Wound area with aloe gel treatment showed better result than without Aloe gel (Somboonwong et al 2000). Penetration into the gel network will reduce inflammation and increase blood supply by inhibiting the synthesis of a potent vasoconstrictor thromboxane A2 (Gallagher 2003). Besides aloe gel contains active ingredients of potential Acemannan (Ramamoorthy 1996, Hamman 2008).

Acemannan as one of the aloe polysaccharide gel at a dose of 20 mg / ml can stimulate wound healing speed, capable of stimulating cellular proliferation, migration and cytokine production. Acemannan the aloe gel is capable of stimulating oxygen consumption, increased angiogenesis and increased synthesis of collagen in the wound area (Laura et al 2002). Giving oral Acemannan accelerates wound healing by inducing fibroblast proliferation and stimulate expression kgf-1 and VEGF. Acemannan have the ability to stimulate hematopoiesis and antioxidant effects. Acemannan also able to reduce inflammation through the synthesis of prostaglandins and increased infiltration of leukocytes (Ramamoorthy 1996, Hamman 2008).

Based on some of the above facts, it still needed further research on the potential of the active ingredients in Aloe vera as a candidate therapeutic herbal healing diabetic wounds. This study was conducted to prove that the administration of Aloe gel herbal therapy can accelerate wound healing in animal models of diabetes by increasing the number of EPC and CEC lowers the amount of circulation in the wound healing diabetic wistar rat dermal tissue.

Materials and Methods

Sampling

The rats used in this experiment are male wistar strain with the age more or less 2 months with body weight 150-200g. For preparing normal rats (negative control), normal rats are wounded in dermal area then are injected with pH 7,4 PBS. Next, for normal rats group with treatment is orally aloe gel therapy given rats, with the dose 30, 60, and 120 mg per day. For the manufacture of diabetic mice (positive control), rats injected with STZ dose of 60-70 mg / dl has been considered intraperitoneal and diabetes if fasting blood glucose levels 200-300 mg / dl 3 days after injection of Streptozotocin (Gokhale et al 1998, Kaneez et al 2003, Wei et al 2003, Haddad et al 2005). On day 14 after diabetes, rats were then made cuts on the dermal area, divided into five groups: positive control, negative control, and the group treated with therapeutic doses of peroral aloe gel 30, 60 and 120 mg / day for 14 days.

Animal Preparation

Before rats are treated with condition control during preservation in experiment cage. For wound making, rats are then anesthetized before wounded. The process is conditioned surgery under sterile conditions using ketamine anesthesia (10 mg / kg body weight). Rats are anesthetized intraperitoneally with 1 ml of ketamine hydrochloride (10 mg / kg body weight) and the fur was shaved on both sides of the shavers. For the wound area was made by making the outer lines on the back of rats with methylene blue using a marker / ring-shaped pattern. Wound diameter (Ø) is made with a size of 2.5 cm and a depth of 0.2 cm from the dermal surface using tweezers, scalpel number 15, and scissors with pointed tips. Wound diameter and sterility is maintained by sewing on the tool and compressed with a gauze cover and 0.9% (Nayak et al 2006).

Extraction and Purification of Aloe Vera

Gel (Aloe vera Linn.)

Collection of Aloe vera leaves is performed to the healthy leaves which are mature enough from Aloe vera farming in Malang, East Java. These leaves are then cleaned, given the nontoxic disinfectant (calcium hypochlorate 98%) and brushed, then are rinsed with aquadest and dried on their surface. the cut Aloe vera leaves then are put in a suitable container perpendicularly to the bottom of the container, and let the exudate of the leaves flow out for about 30-60 minutes. The base of the leaves are then cut for more or less 1 cm, peeled thoroughly breaking the outside part of parenchyme cell. The leaf pulps (gel) then are rinsed with flowing water several times, peeled and chopped for further processing with juicer. The washing step is done to rinse out dirts and bacteria on lidah buaya leaves surface. The obtained Aloe vera juice then added with ethanol 96% with 1:4 ratio, in this case 50cc aloe vera juice added with 200 cc ethanol 96%. Aloe vera and ethanor mixture juice after that stirred for 10 minutes in maximal temperature 30oC. Then left for 10 hour to let the precipitation process begin. The obtained precipitation result, separated with the whole solution using inhalator filter, then the sediment dried with vacuum dryer under themperature 50oC (Padmadisastra et al 2003).

Wound Healing Mesurement

The measurement area of wound healing is done by drawing on transparent plastic and then described to the graph paper with carbon paper and counted after 14 days post treatment.

Flowcytometry Measurement of CEC and EPC

Peripheral blood sample obtained using syringe. Blood is incubated with anti-CD146-FITC. Blood then flown and isolated with flowcytometry. Cells gathered and spinned with the speed 2500 rpm within 5 minutes under 4oC temperature. Cell sedimentation is washed with phosphate buffer saline (PBS) then shaken slowly. Wash the cells twice. The last sedimentation added with 100 µl PBS and anti-CD146-FITC and anti CD45-PE (1:100). Cells are then incubated with antibody for 30 minutes under roo temperature. Then diluted until 1mL and read with flowcytometry (FACS Callibur, BD) (Blann et al, 2005).

Immunohistochemistry Measurement of VEGF and eNOS

After staining with immunohistochemistry techniques, preparations for each treatment and each repetition was photographed with Olympus Brand fotomikroskop with 400x magnification. Results from the photo shoot and then calculated the percentage of cells that express VEGF and eNOS protein in rat dermal tissue slice preparations from all treatment groups.

Results

The negative control group of non-diabetic wistar wistar and positive control of diabetes (glucose 200-300mg/dl) showed that aloe gel significantly affects the healing of diabetic wounds (p> 0.05) on the diameter of the wound, the number of EPC and CEC as well as levels of VEGF and eNOS. After peroral therapy for 14 days of any change of significant value (positive trend) in all indicators.

Figure 1. Results of the observation area of Injury in Normal and DM rats treated with the Aloe gel

Description: The surface wounds on day-0 is created with a diameter of 2.5 cm. Measurement of wound area day-14 in the administration of 60 mg dose group (0.27 ± 0.02) showed a narrowing of the wound area significantly different to the negative control (1.10 ± 0.14) and positive (1.19 ± 0.08 ).

Based on Figure 1. above, after extensive wound measurement using millimeter paper shows that there is a significant difference in the average value of the total area of the wound area in each treatment group. In the DM group rats with the treatment dose of Aloe gel 30 and 120 mg showed significantly different results to the control group on the average area of the same injury. While the DM group treated Aloe gel 60 mg dose showed the most significant result of the control group. Based on statistic examination with one way Anova, it was shown that wound area width performed a differently significant result (p = 0,000) according to measuring result with graphic paper and computer analyzed. The 60 mg/kg BW treatment group (0,27+0,02) performs a significantly different result to the negative control (0,92+0,12) with confidence level (p<0,05). Average value diversity as shown in table1:

Table 1. The mean value of Wound Size Measurements Based on the results

Treatment Group

n

Mean ± SD

Negative control

3

1.10 ± 0.14a)

Positive control

3

1.19 ± 0.08a)

DM dose of 30

3

0.32 ± 0.01b)

DM dose of 60

3

0.27 ± 0.02b)

DM dose of 120

3

0.41 ± 0.02b)

Description: The same notation indicates not significantly different while different notations indicate significantly different.

Endothelial Progenitor Cells (EPCs) Amount Measuring Result with Flowcytometry Method

According to Endothelial Progenitor Cells (EPCs) amount measuring result with flowcytometry method, it obtained the average value comparison description of EPCs amount in all treatment group, as performed in figure 2 :

Figure 2. calculated number of endothelial progenitor cells (EPCs) marker CD 34 using flowcytometri method

Based on statistic evaluation with one way Anova, the amount of EPC performed different result but is not statistically significant. The 120 mg/kg BW treatment group (13,75+1,63) shows an obviously different result to the negative control (16,88+0,59) and the positive one (10,47+1,14). The average result diversity is shown in table 2 :

Table 2. The mean number of EPC based measurement

Treatment Group

n

Mean ± SD

Negative control

3

16.88 ± 0.59a)

Positive control

3

10.47 ± 1.14a)

DM dose of 30

3

10.64 ± 9.34a)

DM dose of 60

3

12.11 ± 1.16a)

DM dose of 120

3

13.75 ± 1.63a)

Description: The same notation indicates not significantly different while different notations indicate significantly different.

Calculated Expression and Levels of Vascular Endothelial Growth Factor (VEGF) by Immunohistochemistry Methods

Based on the calculation results obtained by the method of comparative picture Immunohistochemistry average value of VEGF expression between the treatment groups as in Figure 3 below:

Figure 3. Result of expression of VEGF using immunohistochemistry method on rats

Description : calculated expression of VEGF by immunohistochemistry method showed the group a target dose of 60 mg of epithelial cells in stratum basale cells that expressed more than other dose groups (1=negative control; 2=positive control;3=dose of 30 mg DM; 4=60 mg; 5=120 mg).

One-way ANOVA test showed that expression of VEGF showed that no significant statistical different (p = 0.676) based on the measurement results with immunohistochemical methods. From figure 3. above, the results showed differences in the amount of staining imunositokimia percent of cells that expressed VEGF in the control group had cells that expressed the amount of percent higher than the treatment group. At doses of 60 mg treatment group were significantly different but significantly different to the control group. Based on figure 3. above it appears that the expression of VEGF increased in the three treatment groups dose 30mg, 60mg and 120mg compared to positive controls that look of total percent of his cell. Comparison of VEGF expression based on the average number of percent of cells that expressed VEGF between groups as shown in table 3.

Table 3. The mean expression of VEGF using Immunohistochemistry method

Treatment Group

n

Mean ± SD

Negative control

3

24.67 ± 2.00a)

Positive control

3

25.33 ± 4.51a)

DM dose of 30

3

28.33 ± 5.86a)

DM dose of 60

3

34.67 ± 5.03a)

DM dose of 120

3

25.67 ± 4.73a)

Description: The same notation indicates not significantly different while different notations indicate significantly different.

Calculated Expression of Endothelial Nitric Oxide Synthase (eNOS) by Immunohistochemistry Methods

Based on the calculation results obtained by immunohistochemical methods picture of comparison the average value of the expression of eNOS between the treatment groups such as the chart of Figure 4 below :

Figure 4. Result of expression of eNOS using immunohistochemistry methods on rats wound

Description : calculated expression of eNOS by immunohistochemistry method showed the group a target dose of 60 mg of epithelial cells in stratum basale cells that expressed more than other dose groups (1=negative control; 2=positive control;3=dose of 30 mg DM; 4=60 mg; 5=120 mg).

One-way ANOVA showed that expression of eNOS showed different results are statistical significant (p = 0.000) based on the measurement results with immunohistochemical methods. From the figure 4. above, the results showed differences in the amount of staining imunositokimia percent of cells that expressed eNOS. At doses of 120 mg treatment group were significantly different weight but differ significantly against the control group. Based on Figure 9. above it appears that the expression of eNOS increased in the three treatment groups dose 30mg, 60mg, 120mg is evident from the number percent increase compared to control cells. Comparison of eNOS expression based on the average color intensity of eNOS expression between groups as in table 4. follows:

Table 4. The mean expression of eNOS using Immunohistochemistry method

Treatment Group

n

Mean ± SD

Negative control

3

24.67 ± 1.73b)

Positive control

3

30.67 ± 1.08a)

DM dose of 30

3

30.00 ± 1.00a)

DM dose of 60

3

34.33 ± 2.08a)

DM dose of 120

3

42.00 ± 1.00b)

Description: The same notation indicates not significantly different while different notations indicate significantly different.

To find out more picture of the relationship of several variables previous studies it conducted the test with linear regression and obtained the results of path analysis as the scheme below:

Figure 5. Path analysis

Linear Regression Based on the test results to illustrate the path analysis (path analysis) that there is a strong mutual relationship in the pathway wound healing mechanisms of diabetes associated with the role of the EPC, CEC, VEGF and eNOS that impact on the diameter of the wound as an indicator morphology diabetes wound healing process. From the regression test results it appears that there is a strong relationship between VEGF on EPC number (R 2 = 0.119) levels of eNOS on wound diameter (R2 = 0.208), eNOS to total CEC (R 2 = 0.357), VEGF to the wound diameter (R2 = 0.038 ) and the EPC to the wound diameter (R2 = 0.079).

Discussion

The Corellation of Aloe Gel Administration into Cutaneous Wound Diameter

The main invented result in the experiment of diabetic wistar rats wound which are administered with aloe gel orally shows that wound diameter improvement takes place, compared to control which is accompanied by the presence of VEGF, eNOS increased level, increased EPC emount and reduced CEC level which shows the importance of the role of herbal aloe gel into the angiogenesis in diabetic wound healing. From the previous experiment, it is mentioned that anti inflammation effect and wound healing stimulator from aloe gel is capable of providing significant effect allowable daily intake (ADI) and Upper Intake Level (UIL) in the range of 60-120 mg/kg body weight with assumption that the administration is done within 1 month (Kwack et al 2009 ; Jia et al 2008).

Aloe gel administration in wounded diabetic wistar mice shows wound improvement with the shortening of the wound diameter in aloe gel administration with dose range 60-120 mg/kg body weight. This acceleration in wound diameter change is probably as the result of aloe gel with polysaccharide substance accelerates fibroblast proliferation and hyaluronic acid production and hydroxypylene significantly compared to the group without aloe gel, which indicates that aloe gel polysaccharide owns central role in remodeling extracellular matrix during wound healing period (Liu et al , 2010). Micronutrient therapy and Aloe vera topically can enhance tissue regeneration process. The mannose-6-phosphate content in Aloe vera is considered will bind to fibroblast receptor and antivate this cell’s proliferation (Fernanda et al., 2009).

The effect of aloe gel administration is also enhance wound diameter reducing acceleration through enhancing wound healing capability compared to the group without aloe gel followed by immune cells increasing such as neutrophils and macrophage and fibroblast cell quantity in the wound cleft. This is parallel to the previous experiment result where 30 g dose of aloe gel administered twice a day is able to enhance wound healing progression in normal mice (Nasrin et al., 2009). Wound diameter reducing is also resulted by aloe gel stimulation to the mice dermal wound through increasing collagen and glycosaminoglycan synthesis (Jettanacheawchankit et al 2009)

Aloe vera is effective in accelerating wound contraction either in oral administration or topical application. Wound contraction is a crucial factor in healing process resulted from wound healing shift to the middle. Other experiment reports that orally administered Aloe vera accelerates wound healing in acute radiation exposed rats model.

The amount of EPCs and CECs undergo such a significant change in aloe gel administration group in the dose of 120 mg/kg body weight per day. This shows oral therapy of aloe gel is highly potent to accelerate wound healing through promoting EPCs mobilization, its lifetime in peripheral circulation in diabetic condition, and also neovascularization trough stabilizing the role of EPC in cutaneous wound vascular area of the diabetic rats. In the other hand, it enhances EPCs amount accompanied by cellular marker of another vascular dysfunction that is CECs. In this experiment, in the dose range 60-120 mg/kb body weight, aloe gel is capable of significantly reducing CECs amount in diabetic rats. Wound healing acceleration in this experiment is also supported by the significantly increased EPCs amount in 120 mg/kg BW dose and obviously reduced CEC amount in circulation in 60 mg/kb BW. Neovascularization failure in diabetic wound makes it possible for the increasing of eNOS phosphorylation in bone marrow after therapy with aloe gel.

Generally, in the case of diabetic healing failure caused by granule tissue reducing and apoptosis increasing with the increased apoptosis due to circulated EPC level. In diabetic rats, circulated EPC doesn’t change until 4 weeks after diabetic modified or skin wound made. After 4 weeks, it is monitored that there has been a significant reduction in the EPCs amount in diabetic rats which is shown by the increase of apoptosis and the proliferation reduction on the wound area in the diabetic mice. It is concluded to be closely related to the damage in recruiting, lifetime, and BM derived progenitor cells. Therapy administration is expected to be capable of restoring EPC homing and these progenitor cells lifetime for increasing wound healing in diabetic tissue. In the other hands, it is mentioned that progenitor cells dysfunction in diabetic individual will influence vasculogenesis process (adhesion, proliferation, migration, and tube formation failure take place) (Albiereo et al., 2011)

The newest experiment result explains a consideration that in diabetic animal and human, wound healing failure is extremely related to the failure of migration in several vasculogenetic progenitor in circulation (Oren et al., 2010). However, in this experiment Aloe gel administration in treatment applied diabetic rats in this experiment is capable of keeping EPCs capability to migrate and accelerating wound healing followed by wound diameter narrowing acceleration. It is true that until these time, whether the decrease of these vasculogenic progenitor cells which also decrease in type 1 diabetic rats model is involved in the pathogenesis of wound healing failure, remains poorly understood. However, this experiment result proves that it is shown that in wounded type 1 diabetic rats model, significantly increased EPCs amount has occurred, compared to the group given oral therapy with aloe gel in range of dose 60-120 mg/kg BW.

In the other hand, it is known that type II diabetes animal model has significantly decreased progenitor cells during homeostasis process after cutaneous ischemia takes place or wound formed (Oren et al., 2010). Generally, from this experiment, there’s an obtained description that the correlation in increased level/amount stem cell (vasculogenic progenitor) is significant towards the improvement of wound reepithelialization in diabetic animal model. EPCs amount and function change occurs in Diabetes Mellitus and wound healing (Sibal et al., 2009).

Spinetti et al. (2008) states that oxidative stress resulted from high glucose exposure in circulation will trigger EPC dysfunction. That experiment result assumes that DM induces EPC destruction during its route when migrates from bone marrow. It also comes up a speculation that EPC amount increasing released from bone marrow to circulation occurs to the diabetic individual is caused by high amount of dead EPC along its vascular route. Diabetic EPC also shows integrative ability failure in neovascularization in ischemic organ and undergoes reendothelialization function decline after vascular damage occurs. Oral therapy administration of aloe gel is able to prove that it can increase circulated EPC amount through keeping these survival progenitor to the vascular in wound area to begin neovascularization. Aloe gel therapy also stimulates repair and re-endothelialization in wound area.

In diabetic wistar rat wound, EPC level showsn its raising in 14th day after aloe gel administration compared to the control group through expressing CD34 antigen marker in flowcytometry test. Clinically, EPC is capable of improving disease conditions begun by endothelial cells damage. , either anatomically/structurally or functionally, through neovascularization mechanism. The result of previous experiments are convinced that inside bone marrow and peripheral blood flow, there are cells which are able to multiply and differentiate to be endothelial cells and improve ischemic tissue caused by damaged blood vessel wall.

Aloe gel therapy is predicted to be able to accelerate Endothelial cells line sticks to basal membrane. The lost in blood flow cells are very low with consequence to be destructed by reticuloendothelial system. Aloe gel can suppress pathophysiologic process in diabetic mice which causes endothelial cells release to basal membrane, and will results in increased CECs amount in blood flow.

In diabetic wistar rats, the level of CEC seen declining in 14th day after aloe gel administration compared to the EPC control through expressing CD 45 and CD 146 antigen marker. On the other hand, in normal individuals, the amount of circulating endothelial cells (CECs) is low. The amount will increase if there’s an endothelial cell injury. From the previous experiment result, it is found that in diabetes mellitus patient, compared to the normal ones, there’s such a significant different in the amount of circulating CEC in that patient circulation. (McClung et al., 2006)

Based on experiment result data, it is understood that there’s a VEGF increased level in the group with 120 kg/mg BW dose, followed by eNOS level increase within 60-120 mg/kg BW dose range. In diabetic rats wound, VEGF level shows an increasing in 14th day after aloe gel administration compared to the control, tested with ELISA examination.

This indicates that oral therapy administration of aloe gel in the wound healing model in diabetic animal model can enhance the wound healing through increasing level of EPC and VEGF and activation of NO synthase enzyme which becomes downstream part of angiogenesis molecular pathway under VEGF.

Aloe vera treatment shows positive effect in wound healing after 7th-14th day from the beginning of the wound (Heggers, 1993). Aloe gel is suspected to stimulate phagocytosis activity formation and induce NO production activity, enhances synthesis, cross linking, and collagen maturation, stimulates cell proliferation, stimulates fibroblast function and proliferation, inhibits arachidonic acid oxidation, possess anti-inflammatory effect, reduces TNF-α level and antioxidant activity (Innan et al., 2007). Besides, oral aloe gel therapy which contains beta-Sitosterol also enhances angiogenesis related protein expression, that is vascular endothelial growth factor (VEGF (Choi et al., 2002)

Aloe gel in Aloe vera in this experiment is capable of accelerating NO production, so it will accelerate eNOS enzyme in endothelial as well, and VEGF activity in enhancing diabetic wound healing angiogenesis. Aloe gel stimulates endothelial nitric oxide synthase (eNOS) dominant role and NO production in endothelial, upon 2 process, which are angiogenesis (new blood vessel development from the already existing blood vessel) and vasculogenesis (de novo blood vessel formation from progenitor cells) (Duda et al., 2004)

Besides, this experiment result shows positive effect where oral aloe gel therapy administration can increase eNOS level. eNOS is the central factor for EPC mobilization either in human or diabetic animal model. Aloe gel is predicted to be able to suppress some molecules involved in eNOS activity inhibition. It is according to previous experiment where dimethyl-arginine increasing as NOS endogen inhibitor in DM case, becomes supporting factor in EPC function failure in diabetic individual. Protein glycooxidation is suspected to decline EPC capability. Diabetic EPC also release some unidentified factor which accelerate vascular endothelial cell ageing (Spinetti et al., 2008). That’s why this aloe gel therapy is expected to be able to be an effort for damaged vascular improvement by mobilizing EPC for re-stabilizing the damaged vascular function caused by DM complication.

In fact, aloe gel can increase VEGF level in wound area. It will give good effect in diabetic wound healing acceleration, where VEGF production reduction and angiogenesis declining process contribute to the tissue improvement in diabetic patients. VEGF expression is strongly induced by hypoxia condition through transcription factor hypoxia inducible factor-1. In diabetic mice, VEGF topical application performs acceleration with skin wound improvement as well as mobilization and recruiting vascular progenitor (Pradhan et al., 2007). With stimulation effect into VEGF activity, so aloe gel is predicted to be able to accelerate wound healing by increasing growth factor local regulation., it is important for tissue repair and will systematically mobilize precursor cell from bone marrow, especially progenitor cell contributes in blood vessel formation that will accelerate with wound repair (Stuart et al, 2006).

Other factor is, aloe gel can probably regulate proteolysis effect which is reported to begin VEGF activity reduction in diabetic wound (Saaristo et al,2006; Costanza et al., 2002). Aloe gel performs stimulation effect into wound healing involved cells, such as keratinocyte, and macrophage, plays an important role in this growth factor during normal wound healing (Domenica et al., 2001) through accelerate VEGF as growth factor with proangiogenic effect by increasing cellular proliferation (Singhan et al., 2007; Young et al., 2004)

Other thing to be considered, it comes up a suspicion that in this experiment, aloe gel is able to promote angiogenesis by increasing the pathways of PI3K/Akt, MAPK, ERK, and aNOS/NO signal route. PI3K/Akt pathway activation stimulates eNOS activation through phosphorylation in serin 1177, which will begin NO production increasing. Aloe gel may also be able to decline PI3K/Akt pathway inhibition or eNOS activity will inhibit growth factor activity and angiogenesis mediating cytokine. Aloe gel can acceleratewound healing process with probability of promoting PI3K/Akt/eNOS signaling pathway activation, takes the crucial role for angiogenesis. And the pathway of ERK1/2 holds vital role in growth mechanism, differentiation, cell survival and death during vascular protection process, as well as angiogenesis in wound healing. It is parallel to the result of the previous experiment where Korean red ginseng extract exposure under HUVECs culture can activate Akt, ARK and eNOS pathways as well as increasing NO production related in angiogenesis process (Kim et al., 2007). Other comes up probability is aloe gel possess a potential to regulate cytosolic calcium level. Even though eNOS activity can be accelerated by VEGF that will accelerate Akt and PKC pathway activation, the main stimulus for NO production will be enhanced if it is induced by increasd cytosolic calcium level (Dawson et al 2006, Aramoto et al 2004). Therefore, there’s a strong prediction that aloe gel has angiogenic effect mediated by NO as the resut of eNOS induction in vascular cell (Tanimoto et al 2002).

If aloe gel can stabilize VEGF activity, thus in the other part, expression and phosphorylation of endothelial nitric oxide synthase (eNOS) whis is mentioned is important for survival, migration and angiogenesis of EPCs or endothelial cell will be stable. The Nitric Oxide (NO) resulted from eNOS activity begins EPCs mobilization from bone marrow trough nitrosylation and vascular endothelial growth factor (VEGF) expression increase (Chen et al 2007)

Conclusion

The conclusion of this experiment is Aloe gel oral therapy (optimal dose range 60-120 mg/kg BW per day) correlates with to the wound healing in diabetic wistar mice, that is the presence of wound diameter improvement in positive control and treatment compared to the negative control. Besides, aloe gel has potential capability to be diabetic wound healing herbal therapy candidate through acceleration of EPC homing, reducing CEC amount and accelerating VEGF and eNOS pathway increasing in neovascularization process within diabetic dermal area wound healing.

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