从miR-126靶向VEGF调控PI3K/Akt信号通路动态激活血管内皮细胞功能视角探索“煨脓长肉”理论促进糖尿病溃疡愈合机制的研究

Vascular disease is the key mechanism of the occurrence of diabetic ulcers. The whole course of ulcer development is accompanied by vascular endothelial cell dysfunction and insufficient of proliferation or migration. Studies have shown that VEGF can participate in endothelial cell proliferation, differentiation and migration through PI3K/Akt signaling pathway. Besides, specific miR-216 can maintain endothelial cell function and promote wound healing. Traditional Chinese medicine " pus-retaining and granulation-generating" can promote wound angiogenesis during liquefaction and necrotic tissue. Our research group showed that Zizhu ointment with " pus-retaining and granulation-generating " can increase the expression of miR-216 and VEGF to promote wound healing in diabetic ulcer. The hypothesis is put forward that Zizhu ointment can promote the healing of diabetic ulcers, and its mechanism is to regulate the expression of PI3K/Akt through targeting VEGF to regulate the expression of miR-216, thereby improving the dysfunction of vascular endothelial cells.In this study, research platform as the diabetic ulcers rat model and vascular endothelial cells, with the use of PI3K/Akt inhibitors, miR-126 inhibitors, miR-126 analogs, through cell transfection, flow cytometry and immunofluorescence technique, to observe the expression of miR-216 mediated vascular endothelial cells in the ulcer healing. In order to solve the key problem of "pus-retaining and granulation-generating" Zizhu ointment can regulate the expression of miR-216 dynamically, coordinate the function of vascular endothelial cells and promote the ulcer healing. Therefore, it provides a new experimental basis for the treatment of diabetic ulcers by Chinese traditional Chinese medicine "pus-retaining and granulation-generating".

血管病变是糖尿病溃疡发生的关键机制，溃疡全程中伴随着血管内皮细胞功能障碍。研究显示miR-216及VEGF可通过PI3K/Akt信号通路维持内皮细胞功能、促进创面愈合。中医“煨脓长肉”能液化坏死组织促进血管再生，前期研究显示“煨脓长肉”中药紫朱软膏能上调糖尿病溃疡愈合过程中的miR-216及VEGF表达。由此提出假说：紫朱软膏促进糖尿病溃疡愈合的机制是通过靶向VEGF调控PI3K/Akt信号通路以调节miR-216表达，改善血管内皮细胞功能。本研究以大鼠模型及血管内皮细胞为研究平台，利用PI3K/Akt抑制剂、miR-126类似物/抑制物，通过细胞转染、流式细胞及免疫荧光等技术，观察miR-216介导的血管内皮细胞在溃疡愈合中的表达。以期解决“煨脓长肉”中药紫朱软膏通过调节miR-216动态表达，协调血管内皮细胞功能，促进溃疡愈合的关键问题，为“煨脓长肉”中药治疗糖尿病溃疡提供实验基础。

糖尿病溃疡发生发展过程中伴随着血管内皮细胞的功能障碍及增殖迁移不足状态，内皮细胞特异性调节因子miR-216在维持内皮细胞功能稳定、血管壁完整及促进创面愈合中作用重大。前期研究提示“煨脓长肉”中药紫朱软膏促进糖尿病溃疡愈合或通过miR-216实现。.本研究以动物模型及血管内皮细胞为研究平台，以宏基因组学技术为切入点，研究miRNA在血管内皮细胞相关血管生成过程中的作用，以期解决“煨脓长肉”中药紫朱软膏通过调节miR-216动态表达，协调血管内皮细胞功能，促进溃疡愈合的关键问题。.体内实验提示，相比非糖尿病溃疡组（NUC），糖尿病溃疡组（DUC）中血管生成受到抑制，表现为VEGFa/ANG2表达下降，而PI3KR2/SPRED1表达升高；紫朱软膏治疗组（DUZ）可加快血管生成，并促进溃疡愈合。测序结果表明，相比NUC组，DUC组溃疡组织种共筛选出1072种已知miRNAs，其中94种有差异明显；溃疡用药前后(DUZ/DUC)，共筛选出1056种已知miRNAs，其中57种有差异明显；其中miR-126a-5p在造模后表达下降，用药后有明显升高。体外利用BEND.3构建高糖高脂损伤模型，通过划痕实验、Annexin-V/PI双染实验，发现紫朱醇提物能促进细胞迁移并减少凋亡；紫朱醇提物通过提高miR-126a-5p促进血管生成，用药组成管实验中成管数量明显增加，血管生成相关基因蛋白表达也明显升高，PI3K/AKT信号通路呈激活状态。以293T细胞为对象，通过细胞转染、RNA干扰、双荧光素酶报告基因实验证实miR-126通过靶向PIK3R2参与血管生成。.结论：紫朱软膏能促进血管新生，可能是通过上调miR-126a-5p靶向PIK3R2激活PI3K/Akt信号通路实现的，这也为“煨脓长肉”中药治疗糖尿病溃疡提供了新的实验基础。