【摘要】 目的：观察消肿止痛合剂（XZM）对大鼠糖尿病溃疡创面、磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B(PKB/Akt)/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路及内皮细胞自噬的影响。方法：将SD大鼠随机分为空白组、模型组、XZM组、XZM+PI3K抑制剂（GDC-0941）组（XZM+PI3Ki组）及XZM+mTOR抑制剂（mTOR inhibitor-1）组（XZM+mTORi组）共5组，每组10只。观察糖尿病足溃疡创面愈合情况，采用HE染色观察大鼠肉芽组织病理改变，免疫组化法检测组织中p-PI3K、p-Akt和p-mTOR水平，透射电镜观察血管内皮细胞改变，荧光TUNEL对肉芽组织凋亡细胞进行分析。结果：与正常组相比，模型组肉芽组织局部区域表皮层坏死，表皮结构缺失，表皮层不同程度增厚，真皮层内胶原纤维坏死，胶原纤维结构消失，伴大量炎症细胞浸润，真皮层内纤维组织增生，血管充血扩张，肉芽组织内有出血，内皮细胞结构紊乱，线粒体有严重肿胀（嵴断裂溶解），胞质内可见较多自噬。与模型组相比，XZM组溃疡创面面积明显缩小，肉芽组织表面大量痂皮形成，表皮层不同程度增厚，真皮层内胶原纤维交错排列，无炎症细胞浸润，无真皮层血管充血扩张、肉芽组织出血等病理现象，细胞内有极少量自噬，p-PI3K、p-Akt和pmTOR蛋白水平升高（P<0.05）,XZM组、XZM+PI3Ki组和XZM+mTORi组大鼠肉芽组织凋亡细胞率均显著下降（P<0.01）。与XZM组相比，XZM+PI3Ki组和XZM+mTORi组内皮细胞线粒体肿胀明显，细胞内有较多自噬体，内皮细胞凋亡率升高，真皮层内纤维组织均有着不同程度增生，复层扁平上皮层较薄或缺失，XZM+mTORi组真皮层内仍有大量炎症细胞浸润。此外，与模型组相比，XZM+PI3Ki组与XZM+mTORi组组织中p-Akt和p-mTOR蛋白水平显著升高（P<0.05）；与XZM组相比，XZM+PI3Ki组和XZM+mTORi组组织中p-PI3K、p-Akt和p-mTOR蛋白水平显著降低（P<0.05）。结论：XZM能够修复糖尿病溃疡大鼠模型受损创面，减轻炎症反应，促进糖尿病溃疡创面愈合，这可能与调控PI3K/Akt/mTOR信号通路、血管内皮细胞自噬和凋亡有关。更多还原

【Abstract】 AIM: To investigate the effects of Xiaozhong-Zhitong mixture(XZM) on the ulcer wounds in diabetic rats, as well as on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/Akt)/mammalian target of rapamycin(mTOR) signaling pathway and endothelial cells in rats. METHODS: Sprague-Dawley(SD) rats were randomly divided into the following 5 groups with 10 rats per group: blank group, model group, XZM group, XZM+PI3K inhibitor(PI3Ki) group and XZM+mTOR inhibitor(mTORi) group. The healing of foot ulcers in diabetic rats was evaluated. The pathological changes in the granulation tissues of rats were studied using HE staining. The levels of p-PI3K, p-Akt and p-mTOR in tissues were detected by immunohistochemistry. The changes in vascular endothelial cells were observed by transmission electron microscopy. The apoptotic cells from granulation tissues were analyzed by fluorescence TUNEL assay. RESULTS: The local areas of granulation tissues in model group exhibited different degrees of thickening, necrosis and structural loss in the epidermal layer, and internal collagen fiber hyperplasia and necrosis were seen in the dermis. The collagen fiber structure disappeared, accompanied by infiltration of a large number of inflammatory cells. The blood vessels of granulation tissue exhibited congestion and dilatation, and the endothelial cells exhibited disordered structure with severe swelling mitochondria(ridge fracture and dissolution) and more autophagy in the cytoplasm. Compared with model group, the ulcer wound area of rats in XZM group was significantly reduced, with numerous scabs formed on the surface of the granulation tissue. The epidermis showed varying degrees of thickening. The collagen fibers in the dermis were interlaced, without inflammatory cell infiltration, vascular congestion and dilatation in the dermis, or granulation tissue hemorrhage. A very small amount of autophagy was observed in the cells, and the levels of p-PI3K, p-Akt and p-mTOR proteins increased(P<0. 05). Apoptosis rates in the granulation tissue of XZM, XZM+PI3Ki and XZM+mTORi groups were significantly decreased(P<0. 05). The mitochondria of endothelial cells in XZM+PI3Ki and XZM+mTORi groups showed more swollen and had more autophagosomes compared with XZM group. The apoptosis rate of endothelial cells was higher than that in XZM group. The fibrous tissue in the dermis was uneven and exhibited a similar degree of hyperplasia. The stratified squamous epithelium was thin or missing. There was still a large amount of inflammatory cell infiltration in the dermal tissues of rats in XZM+mTORi group. In addition, the protein levels of p-Akt and p-mTOR in PI3K group and XZM+mTORi group were significantly higher than those in model group(P<0. 05), and those in PI3K group and XZM+mTORi group were significantly lower than those in XZM group(P<0. 05). CONCLUSION: Xiaozhong-Zhitong mixture could repair wounds in a rat model of diabetic ulcer, alleviate the inflammatory response, and promote the healing of diabetic ulcer wound, which may be related to the regulation of PI3K/Akt/mTOR signaling pathway, as well as the autophagy and apoptosis of vascular endothelial cells.更多还原