基于外泌体miR-126调控PIK3R2/SPRED1探讨黄芪甲苷促糖尿病皮肤溃疡血管新生的机制研究

Angiogenesis is the key of diabetic skin ulcers heal. The current research finds that astragaloside IV can promote angiogenesis, but the mechanism is unknown. Research group early found that exosomes secreted by endothelial progenitor cells(EPC-Exos)induced by astragaloside IV could be absorbed by endothelial cells to enhance its tubulogenic function; further in the preliminary experiment, it was found that the secreted EPC-Exos were rich in miR-126, and PIK3R2 and SPRED1 were found to be potential target genes regulating angiogenesis of miR-126 through target gene prediction. In the comprehensive preliminary study, the research group speculated that astragalosis-mediated EPC-Exos loaded miR-126 and regulates the PIK3R2/SPRED1 signaling axis to play a role in angiogenesis. This study will use "EPC-Exos miR-126-PIK3R2/SPRED1 signal axis angiogenesis" as the axis, disscuss the effect on EPC-Exos mediated by astragaloside IV to the expression of angiogenic proteins and genes in hyperglycemia damaged endothelial cells and diabetic skin ulcers, endothelial cell proliferation and destroyed surface vascularization from molecular biology, cell biology and experimental animal level, to elucidate the specific signal pathway and mechanism of its action, and provide theoretical basis for astragaloside IV used for destroyed surface repair.

血管新生是糖尿病皮肤溃疡愈合的关键。目前研究发现黄芪甲苷能促进血管新生，但作用机制不详。课题组前期研究发现黄芪甲苷促内皮祖细胞分泌的外泌体（EPC-Exos）可被内皮细胞摄取以增强其成管功能；进一步在预实验中研究发现所分泌的EPC-Exos富含miR-126，且经靶基因预测发现PIK3R2和SPRED1为miR-126调控血管新生潜在靶基因。综合前期研究结果，课题组推测黄芪甲苷介导的EPC-Exos负载miR-126调控PIK3R2/SPRED1发挥血管新生作用。本研究将以“EPC-Exos→miR-126-PIK3R2/SPRED1信号轴→血管新生”为主线，从分子生物学、细胞生物学及实验动物水平探讨黄芪甲苷介导的EPC-Exos对高糖受损内皮细胞和糖尿病皮肤溃疡血管新生相关蛋白基因表达、内皮细胞增殖和创面血管化的影响，以阐明其作用的具体信号通路及机制，为黄芪甲苷用于创面修复提供理论依据。

糖尿病皮肤溃疡创面是糖尿病中常见的，迁延难愈且反复发作的并发症，处理不当可能导致截肢，甚至危及生命。目前研究发现高糖毒性导致的微小血管病变和内皮细胞功能障碍，从而引发的血管新生困难是糖尿病皮肤溃疡创面不能顺利愈合的主要原因。本项目课题组前期研究发现黄芪甲苷能促进内皮祖细胞分泌负载miR-126的外泌体。本项目在前期研究的基础上进一步检测了100mg/L的黄芪甲苷对内皮祖细胞分泌的外泌体中miR-126-3P和miR-126-5P含量的影响，鉴定了外泌体的表征。鉴于miR-126-3P更高的表达量及生信预测miR-126-3P对PI3KR2和SPRED1均有调节作用，本项目选择miR-126-3P进行实验。实验中分别构建过表达和沉默表达的miR-126-3P外泌体，对比研究黄芪甲苷介导的内皮祖细胞外泌体对高糖诱导损伤内皮细胞增殖、迁移、成管、凋亡和焦亡，血管生长因子（VEGFa、VEGFb、VEGFc、FGF、Ang-1），PI3KR2和SPRED1及其下游信号通路的影响。另通过敲除内皮细胞中的miR-126、PI3KR2和 SPRED1，研究PI3KR2和 SPRED1参与miR-126-3P介导的内皮细胞成血管化。同时在动物实验水平验证黄芪甲苷刺激的内皮祖细胞外泌体和未刺激内皮祖细胞外泌体对伤口愈合的影响，以及miR-126-3P对PI3KR2/SPRED1信号轴的调控作用。实验研究发现miR-126-3P对PI3KR2和SPRED1均具有调控作用，双荧光素酶实验验证了上述结论。同时发现PIK3R2 介导的PI3K/AKT 信号通路和SPRED1 介导的MAPK/ ERK信号通路可能在血管新生中发挥作用。细胞实验中的成管实验和动物实验中的新生内皮细胞检测充分说明了结果的可靠性。本研究的意义在于为糖尿病皮肤溃疡血管新生寻找新的细胞药物治疗靶点及方法，进一步阐释黄芪甲苷促血管新生的作用机制，为黄芪甲苷（黄芪）用于糖尿病皮肤溃疡的治疗提供理论依据。