基于Akt/mTOR信号通路探讨丹黄散在糖尿病大鼠皮肤创面愈合中的作用机制

Study on the mechanism of Danhuang powder on skin wound healing in diabetic rats based on ATM/TOR signaling pathway

目的基于Akt/mTOR信号通路探讨丹黄散对糖尿病大鼠皮肤创面愈合的影响及作用机制。方法从100只SD大鼠中随机选取75只给予腹腔注射链脲佐菌素(STZ)构建糖尿病模型,另外25只注射等体积无菌生理盐水作为对照组。造模成功3 d后,将糖尿病模型大鼠随机分为模型组、西药组、丹黄散组。各组大鼠背部两侧剪下2 cm×2 cm皮肤,伤后对照组、模型组不进行药物干预,西药物、丹黄散组分别敷用表皮生长因子和丹黄散,观察各组大鼠伤后第5天、第14天、第28天创面愈合情况。于第29天处死大鼠,分别采用HE染色、电镜、免疫荧光观察创面组织的形态、超微结构、α-平滑肌肌动蛋白(α-SMA)表达情况,采用Western blot法检测创面组织中p-Akt、p-mTOR蛋白表达情况。结果模型组伤后第5天、第14天、第28天创面愈合率均明显低于同期对照组(P均<0.05),西药组、丹黄散组创面愈合率均明显高于同期模型组(P均<0.05),西药组与丹黄散组各时间点创面愈合率差异均无统计学意义(P均>0.05)。HE及电镜观察,模型组中炎性细胞明显增多,未见毛细血管新生;西药组、丹黄散组均出现成纤维细胞致密分布,少量新生毛细血管。与对照组比较,模型组大鼠创面组织中α-SMA和p-Akt、p-mTOR蛋白表达水平均明显降低(P均<0.05);与模型组比较,西药组、丹黄散组大鼠创面组织中α-SMA和p-Akt、p-mTOR蛋白表达水平均明显升高(P均<0.05),西药组与丹黄散组各指标比较差异均无统计学意义(P均>0.05)。结论丹黄散可促进糖尿病大鼠皮肤创面愈合,作用机制可能与上调Akt/mTOR信号通路中Akt和mTOR磷酸化水平有关。

Objective It is to investigat the effect and its mechanism of Danhuang powder on skin wound healing in diabetic rats based on the Akt/mTOR signaling pathway.Methods A total of 75 SD rats were randomly selected from 100 SD rats to be intraperitoneally injected with streptozotocin(STZ)to establish the diabetes models,and another 25 rats were injected with equal volume of sterile normal saline as control group.After 3 days of successful modeling,the diabetic model rats were randomly divided into model group,western medicine group and Danhuang powder group.A piece of skin(2 cm×2 cm)on both sides of the back of the rats in each group were cut off.The control group and the model group were not treated with drug intervention after injury,while the western medicine group and Danhuang powder group were treated with epidermal growth factor(EGF)and Danhuang powder respectively by external application.The healing of skin were observed in each group on the 5 th,14 th 28 th day after injury.The rats were sacrificed on the 29 th day.The morphology,ultrastructure,and expression ofα-smooth muscle actin(α-SMA)of the wound tissue were observed by HE staining,electron microscopy,and immunofluorescence respectively.Western blot method was used to detect the expression of p-Akt and p-mTOR protein in the wound tissue.Results The wound healing rate of the model group on the 5 th,14 th,and 28 th day after injury was significantly lower than that of the control group in the same period(P<0.05).The wound healing rate of the western medicine group and the Danhuang powder group was significantly higher than that of the model group in the same period(P<0.05).There was no significant difference in wound healing rate between the western medicine group and Danhuang powder group at each time point(P>0.05).According to HE and electron microscopy,the inflammatory cells in the model group were significantly increased,and no capillary angiogenesis was found;fibroblasts were densely distributed and a small amount of new capillaries were found in the western medicine group and Danhuang powder group.Compared with the control group,the expression levels ofα-SMA,p-Akt and p-mTOR protein in the wound tissue of the model group were significantly reduced(P<0.05);compared with the model group,the expression levels ofα-SMA,p-Akt and p-mTOR protein in the wound tissue of the western medicine group and Danhuang powder group were significantly increased(P<0.05),and there was no significant difference in the indicators between the western medicine group and the Danhuang powder group(P>0.05).Conclusion Danhuang powder can promote and improve the healing of skin wounds in diabetic rats,and the mechanism may be related to the up-regulation of Akt and mTOR phosphorylation levels in the Akt/mTOR signaling pathway.