| ObjectiveTo establish the rat model of diabetic skin ulcer and observe the influences of Huiyang Shengji ointment and its two different modified formulae on wound healing and angiogenesis associated cytokines VEGF&VEGFR-2at different time points. Analyse the mechanism of Huiyang Shengji ointment on curing diabetic skin ulcer and elaborate the theory of "Huiyang and Shengji".Methods1.Fifty-six SD rats were divided randomly into normal control group(Control group),Model group,Huiyang Shengji ointment group(Whole formula group),Wenyang Yiqi group(Wenyang group) and Huoxue Shengji group(Huoxue group).The rat model of diabetic skin ulcer was established by injecting STZ(streptozocin,STZ) via abdominal cavity,hormone intervention, artificial skin defects and embedment of plastic rings.Each group was offered to the corresponding petrolatum gauze and ointment gauze.The random blood glucose of each group was measured after72hours,one week and two weeks after injecting STZ.The wound healing rates of each rat at3rd day,7th day and14th day after medication were counted. The variation of wound form in each group was observed.The wound granulation tissues were collected for HE staining,Masson staining and CD34immunohistochemistry staining at14th day after medication in order to find out the alteration of skin tissue,the distribution of collagen and the microvessel density.2.One hundred-twenty SD rats were divided randomly into normal control group(Control group),Model group,Huiyang Shengji ointment group(Whole formula group),Wenyang Yiqi group(Wenyang group) and Huoxue Shengji group(Huoxue group).The rat model of diabetic skin ulcer was established by injecting STZ(streptozocin,STZ) via abdominal cavity, hormone intervention, artificial skin defects and embedment of plastic rings.Each group was offered to the corresponding petrolatum gauze and ointment gauze.At3rd day,7th day and14th day after medication each group was chosen6rats to be put to death by drawing blood in abdominal aorta,and then the granulation tissues were preserved in liquid nitrogen.The concentration of VEGF&VEGFR-2in serum was detected by enzyme linked immunosorbent assay (ELISA). The expression of VEGF&VEGFR-2in granulation tissue were tested by the Western Blot methods.Resultsl.The random blood glucose of the diabetic groups were all above16.7mmol/l,obviously higher than the Control group(P<0.01).The mutual comparison in the diabetic groups had no satistical significance(P>0.05),indicating the diabetic groups were comparable.2.The wound healing rates of the Model group were all lower than that of the Control group at3rd day,7th day and14th day after medication(P<0.01).The wounding healing rates of the Wenyang group were higher than that of the Model group,Whole formula group and Huoxue group at3rd day after medication(P<0.05, P<0.05, P<0.05).The wound healing rates of Whole formula group were higher than that of Model group, Wenyang group and Huoxue group at7th day and14th day after medication(P<0.05, P<0.05, P<0.05).The wound healing rates of all the treated groups were higher than that of Model group at10th day after medication (P<0.05).3.The variation of wound form in each group shows that in Control group the granulation tissue was bright red with the secretion of yellow viscous pyogenic fluids and the wound was moistening, centripetally growing, healing fast, covered by dried blood and approaching to complete epithelization.Whereas in Model group the wound turned pale with little and thin secreta and the granulation tissue was white or relusterly reddish in small quantities leading to slow coalescence.To the contrary in Whole formula group the granulation tissue was red as well as grew vigorously and the wound secreted little with fast healing and nearly epirhelial. In Wenyang group the granulation tissue was reddish and covered by yellow purulent or dried blood therefore the wound healing relatively fast.Finally in Huoxue group the granulation tissue was scarlet and the wound secreted little and covered by dull-red dried blood with rather slow healing.4.The H&E staining showed in Control group the skin was structurally complte with thick collagenous fiber and the derma was infiltrated with numerous inflammatory cells without hair follicle and gland. In Model group the newborn epidermis was thin and the derma was filled with masses of inflammatory cells accompanying sporadic fibroblast, little collagenous fiber and few blood capillaries.In Whole formula group the newborn epidermis was considerably thick and the derma consisted of sporadic fibroblast and inflammatory cells, multitudinous blood capillaries and the numerous thick collagenous fiber connectting with each other to form a network.In Wenyang group there was newborn epidermis and the fibroblast and inflammatory cells were aggregated distribution in the dermis where the amount of the collagenous fiber was quite much while the shape was fairly thin.In Huoxue group the wound was covered by thick crusta formed by plasma and tissue fluild and the derma contained many inflammatory cells,plenty of fibroblast,abundant blood capillaries and little thin collagenous fiber.5.The Masson staining showed in Control group the collagenous fiber was relatively thick and interweaved a net.In Model group the collagenous fiber was sparse and arranged irregularly. In Whole formula group the collagenous fiber was distributed evenly,thick and arranged regularly.In Wenyang group the collagenous fiber was relatively thick and arranged fairly regularly.In Huoxue group the collagenous fiber was quite thin and arranged mussily.6.The comparison of microvessel density marked by CD34showed the IOD of the Model group was evidently lower than that of the Control group(P<0.01).The IOD of the Whole formula group,Wenyang group and Huoxue group was all distinctly exceeding that of the Model group(P<0.01, P<0.01, P<0.01).The IOD of the Wenyang group and Huoxue group was both inferior to that of the Whole formula group(P<0.05, P<0.05).The IOD of the Huoxue group was under that of the Wenyang group(P<0.05).7.The comparison of content of VEGF showed that at3rd,7th and14th day after medication the quantity of VEGF in blood serum in Model group was sharply lower than that in Control group(P<0.01).It was higher in all the treated groups than that in Model group(P<0.01).And it was lower in both the Wenyang group and the Huoxue group than that in the Whole formula group(P<0.01,P<0.01).At3rd and7th day after medication the quantity of VEGF in blood serum in Huoxue group was lower than that in Wenyang group(P<0.01).At14th day after medication the quantity of VEGF in blood serum in Huoxue group was exceeding that in Wenyang group(P<0.01).At3rd,7th and14th day after medication the quantity of VEGF in granulation tissue in Model group was clearly lower than that in Control group(P<0.01).It was higher in all the treated groups than that in Model group (P<0.01).At3rd day after medication the quantity of VEGF in granulation tissue in Huoxue group was lower than that in Whole formula group and Wenyang group (P<0.01, P<0.01).At7th day after medication the quantity of VEGF in granulation tissue in Wenyang group and Huoxue group was inferior to that in Whole formula group(P<0.01, P<0.05),moreover Huoxue group was lower than Wenyang group(P<0.01).At14th day after medication the quantity of VEGF in granulation tissue in Wenyang group was lower than that in Whole formula group and Huoxue group (P<0.01,P<0.01).8.The comparison of the content of VEGFR-2showed that at3rd,7th and14th day after medication the quantity of VEGFR-2in blood serum in Model group was obviously lower than that in Control group(P<0.01).It was higher in all the treated groups than that in Model group(P<0.01).At3rd day after medication the quantity of VEGFR-2in blood serum in Huoxue group was lower than that both in Whole formula group and in Wenyang group (P<0.01,P<0.01).At7th day after medication the comparion of the quantity of VEGFR-2in blood serum among Wenyang group,Huoxue group and Whole formula group had no statistical significance(P>0.05).At14th day after medication the quantity of VEGFR-2in blood serum in Wenyang group was lower than that in Whole formula group and Huoxue group(P<0.05, P<0.05).At3rd,7th and14th day after medication the quantity of VEGFR-2in granulation tissue in Model group was distinctly lower than that in Control group(P<0.01).It was higher in all the treated groups than that in Model group(P<0.01).At3rd day after medication the quantity of VEGFR-2in granulation tissue in Huoxue group was lower than that in Whole formula group and Wenyang group (P<0.01, P<0.01).At7th day after medication the comparion of the quantity of VEGFR-2in granulation tissue among Wenyang group,Huoxue group and Whole formula group had no statistical significance(P>0.05).At14th day after medication the quantity of VEGFR-2in granulation tissue in Wenyang group was lower than that in Whole formula group and Huoxue group (P<0.01, P<0.01). Conclusion1.Huiyang Shengji ointment could validly promote wound healing in rats with diabetic skin ulcer.In addition,Wenyang Yiqi disassembled prescription worked better in the early phase of ulcer healing,while Huoxue Shengji one in the mid-to-late stage.Futhermore the exclusive application of its two disassembled prescription failed to reach excellent effects.2.Huiyang Shengji ointment could facilitate the well growth of granulation tissue,enrich the blood capillaries and collagenous fiber, promote angiogenesis to accelerate the wound healing rates.3.The decline in the quantity of VEGF&VEGFR-2in blood serum and the granulation tissue may be one of the mechanisms which resulted in the diabetic skin ulcer difficult to heal. Huiyang Shengji ointment could increase the quantity of VEGF&VEGFR-2to accelerate the wound healing in diabetic skin ulcer,and even bring forward the peak of both proteins. |
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