The wound healing effect of herb PA on diabetic rats. Wu CW1*, Wu YY2 ,Hsiao CW2,Wu RY2 and Chen YP1.1.Southern Taiwan University of technology, 2.Development center for biotechnology. 草藥 PA 對糖尿病鼠傷口癒合的促進效果。 吳俊緯 1 ，吳玉園 2 ，蕭景文 2 ，吳瑞鈺 2 ，陳翼鵬 1 。 1. 南台科技大學 2. 財團法人生物技術開發中心

Introduction

Traditional Chinese herbal medicine (CHM) was used in wound healing treatment for thousands of years and showed excellent reaults. However, only very limited CHM are still used to treat wound in today’s morden medicine. The CHM for the wound healing has recently been paid attention as the limitation of western medicine has reached especially in patients with complications, e.g. diabetes.It has been well established that patients with diabetes suffer from wound problem greater than normal patients. This study, thus, wish to clarify two well known Chinese herbals for diabetic patients with wound problems.

S1 and PA were shown to help wound patients in the old time. Therefore, they are good candidates for our study. In addition, the combination of S1 and PA may have greater results than S1 or PA alone.

This study used animal model to examine the ability of S1 and PA. The results showed here demonstrate that the wound healing time was shorten in diabetic rats with S1 and the healing speed was faster in diabetic rars with S1 and PA.

Materials and Mathods Animal model. Wistar rats which 8~10 weeks old and weighing 250~300 g were used at the time of study. Animals received food and water ad libitum. Streptozotocin (65 mg/kg) was injected to induced diabetes. Wounds were made on the rats showing elevated blood glucose (more than 300 mg/dl). Wounding. Animals were anesthetized with Pento, the dorsal regions were shaved and the surgical area was disinfected with 70% alcohol. Three circular wounds (fullthickness) of 1-cm diameter were made in the medial plane (Fig. 1) . The distance between two pieces is longer than 1 cm.

Rats with PA of CHCl3 extract. Animals were divided into four groups. After wounded, placebos were used on rats of group1. Rats of group2 used 3% S1, animals of group3 and group4 used 3% S1+ PA and 3% S1+ 2x PA, respectively.

Rats with PA of ethanol extract and eluted. Six groups of animals were used for this test. After wounded, placebos were used on rats of group1. Rats of group2 used 1% S1, animals of group3, group4, group5 and group6 used 3% S1+ 0.25% PA (EA and EtOH eluted), 0.5% S1+ 0.25% PA (EA eluted), 1% S1+ 0.25% PA (EA eluted) and nanometer gold, respectively.

Wound contraction assessment. The wound is taken drug right after pictured and covered with bandage to protect from infection of acratch and bite. The wound is taken picture at 0, 4, 14, 18 days. We used image pro plus 4.5 to measure area. On the measuring days the percentage of wound healing was calculated.

Immunocytochemical evaluation. After sacrificed, newly epithelialized tissues was obtained from rats in each group. 4 μm-thick sections of tissues were made and placed on glass slides. After treatment with 3% hydrogen peroxide, the samples were incubated with blocking medium. The antibodies against MHCII and CD71 were used for primary antibodis. After washing in PBS, second antibodies and peroxidase incubation, DAB was used for visualization. The tissues were then counterstained with hematoxylin after immunoreaction.

Results and Discussion Rats with PA of CHCl3 extract. Several kinds of solvent were used at experiment. PA of different solvent revealed different healing effect. Although the wound of placebo showed the slowest healing effect, when combined with S1, PA extract of CHCl3 didn’t show better healing effect compared with S1 alone (Fig. 2).

Rats with PA of ethanol extract and elution. PA extract of ethanol showed better healing effect than extract CHCl3, and healing effect was much better when PA extract of ethanol was eluted with column. When combined with S1, all the PA of ethanol extract and eluted by ethanol or ethyl acetate revealed higher healing efficiency than S1 alone. Aithough S1 concentration of group3 was higher than group4, group5 and PA of group3 was eluted with two elutent (ethyl acetate and ethanol), the wound healing effect was worse than group4 and group5 (Fig.3, day 9). We found that 1% S1 combined with 0.25% PA will reveal very good effect on wound healing.

Immunocytochemical evaluation. The cells with MHCII and CD71 were as index of wound healing. Rats of non-diabete showed rare immunoreaction against anti MHCII-antibody (Fig.4) and anti CD71-antibody (Fig.7). Diabetic rats also showed less MHCII when they were wouned because bad blood circulation caused by diabetes mellitus (Fig.5). S1 and PA used on diabetic rats could change the condition and showed more MHCII (Fig.6). The cells with CD71 were sparse on diabetic rats with placebo (Fig.8) and this phenomenon could be improved when we used S1 and PA on the skin of diabetic rats. The skin of diabetic rats that used herb showed intensive CD71 (Fig.9) than other rats.

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Fig.1. The experimental model. Three circular full-thickness 1 cm diameter wounds were made.

Fig.2. the healing effect of S1 and PA of CHCl3

Fig.3. the healing effect of S1 and eluted PA of ethanol

Fig.4. immunohistochemical expression of MHC II of non-diabetic rat—showing rare MHCII cells.

Fig.5. immunohistochemical expression of MHC II of diabetic rat with placebo—showing poor reaction with MHCII antibodies.

Fig.6. immunohistochemical expression of MHC II of diabetic rat with 3% S1 and 0.25% PA—intensive MHCII cells occured.

Fig.7. immunohistochemical expression of CD71 of non-diabetic rat–-showing rare CD71 cells.

Fig.8. immunohistochemical expression of CD71 of diabetic rat with placebo–- showing poor reaction with CD71 antibodies.

Fig.9. immunohistochemical expression of CD71 of diabetic rat with 3% S1 and 0.25% PA–-intensive CD71 cells occurred.

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