miR-200b-RhoA招募mTORC1-ULK复合物在内皮细胞自噬中的作用及机制

Previous studies have suggested that the inducing of oxidative stress injury in vascular endothelial cells by glycosylation end products (AGEs) was closely associated with miR-200b-RhoA signal axis. Considering the facts that the RhoA could crosstalk with mTORC1 complex, a classical signaling molecule of autophagy pathway, we supposed that recruiting of mTORC1-ULK complex by miR-200b-RhoA during the process of autophagy plays an important role in AGEs-induced pathogenesis in endothelial cells . To address these issues, the endothelial cells were infected with adenovirus mRFP-GFP-LC3 followed by being stimulated with AGEs. The outbreak of ROS and autophagy flow were observed when cells were treated with agonists or antagonists for RhoA signal axis and mTORC1 complexes. Furthermore, spontaneous type 2 diabetic KKAy mice were introduced, the level of oxidative stress and aggregation of autophagy molecules LC3 in endothelial cells, as well as the phosphorylation levels of components in RhoA signal axis and mTORC1 complexes were evaluated. This study will contribute to the better understanding of AGEs induced vascular endothelial injury and provide a useful references for effective prevention and treatment of diabetic vascular lesions.

前期研究表明糖基化终产物（AGEs）诱导的血管内皮氧化应激损伤与miR-200b-RhoA信号轴密切相关， 而RhoA可与自噬途径mTORC1通路相互“串话”。因此，本课题提出miR-200b-RhoA招募mTORC1-ULK复合物诱导内皮细胞自噬在AGEs致病中发挥重要作用。拟利用mRFP-GFP-LC3腺病毒感染内皮细胞株，AGE刺激后观察内皮细胞ROS爆发和自噬流变化以及利用信号轴与mTORC1复合物激动剂和阻断剂干预观察两者的“串话”关系；以自发性2型糖尿病KKAy小鼠为模型检测AGEs、血管内皮细胞的氧化应激水平及自噬分子LC3的聚集；检测miR-200b-RhoA信号轴和mTORC1-ULK复合物各组分蛋白的表达或磷酸化水平。本研究有助于深入了解AGEs诱导的血管内皮损伤机制，为更为有效地进行糖尿病性血管病变防治提供有益借鉴。

血管内皮氧化应激损伤是糖尿病并发血管病变的重要因素，本课题利用生物信息学软件预测分析以探究miR-200b在糖尿病血管内皮氧化应激损伤及自噬发生过程中的分子调控机制，以验证miR-200b在糖尿病血管内皮氧化应激损伤中的改变，并探究其与氧化应激损伤及自噬发生之间的关联。 运用AGEs处理Eahy内皮细胞构建糖尿病血管病理性改变的细胞模型，通过检测细胞药物敏感增殖性及细胞ROS、SOD及MDA等水平，获得AGEs干预的最佳剂量及最佳时间。在上述研究基础上，将实验分成空白组、AGEs组、AGEs+PBS组、AGEs+miR-200b抑制剂组、AGEs+ROS抑制剂组，利用流式细胞技术及Elisa法检测血管内皮ROS、SOD及MDA的含量。同时构建2型糖尿病动物模型，对体外模型及在体实验使用RT-qPCR检测miR-200b的相对表达量，Western-blot检测mTOR及自噬蛋白LC3-II、p62的表达。研究证实，AGEs对血管内皮细胞的干预存在时间效应及浓度效应。糖尿病环境下血管内皮氧化应激水平增高，miR-200b表达增加，mTOR表达下降，自噬相关蛋白表达增加，而抑制miR-200b的表达，可使血管内皮氧化应激水平降低，并且可促使mTOR的表达而抑制自噬的发生。为此，我们推测miR-200b可促进血管内皮氧化应激反应的发生，并可通过抑制mTOR的表达参与血管内皮自噬的调控，可能是治疗糖尿病血管并发症的新靶点。