S1PR2通过GSK3β介导PI3K/Akt与Wnt/β-catenin信号通路交互作用促肾小球内皮细胞间质转化机制研究

Chronic vascular complications constitute the major cause of morbidity and mortality in patients with diabetes, and diabetic nephropathy (DN) is a glomerular lesion characterized mainly by microvascular damage. As the key target of hyperglycemia and other abnormal stimuli, endothelial mesenchymal transition（EndMT） in glomerular endothelial cells (GECs) is an early and critical step leading to DN, which has already become a hot topic in the field of DN. However, the underlying mechanisms remain poorly understood. Our previous studies have demonstrated that EndMT in hyperglycemia-treated GECs is intimately related to the high expression of S1PR2, which regulates the expression and function of several key molecules in the EndMT signaling pathway of GECs, including ROCK1, AKT, GSK-3β, and β-catenin. Therefore, the purpose of the present study is to evaluate the role of high glucose in the process of EndMT in hyperglycemia-treated GECs in vitro and DN mouse model in vivo. We will further determine the molecular mechanisms by assessing the effect of S1PR2 in the interaction of PI3K/Akt with Wnt/β-catenin signal pathway mediated by GSK3β under diabetic conditions, thus providing a new intervention target and the mechanistic basis for preventing and treating DN.

血管病变是糖尿病最重要的并发症，糖尿病肾病（DN）是一种以微血管损害为主的肾小球病变。肾小球内皮细胞(GECs)作为高血糖等异常刺激首当其冲的靶细胞，发生内皮间质转化（EndMT），是导致DN的早期关键步骤，已成为研究的热点；然而，其机制目前尚未明确。我们前期研究中新发现了高糖处理的GECs发生EndMT与1-磷酸鞘氨醇受体2（S1PR2）的高表达密切相关；且S1PR2可调控GECs的EndMT信号通路中ROCK1、AKT、GSK-3β、β-catenin表达量或活性的变化。因此，本项目拟采用体外高糖刺激的GECs细胞模型和DN小鼠模型，通过调控EndMT信号通路中关键靶蛋白，阐明高糖状态下S1PR2通过GSK3β介导PI3K/Akt与Wnt/β-catenin信号通路交互作用，促使肾小球内皮细胞的内皮间质转化过程及其具体机制，为DN的预防和治疗提供新的干预靶点及理论依据。

血管病变是糖尿病最重要的并发症，糖尿病肾病（DN）是一种以微血管损害为主的肾小球病变。肾小球内皮细胞(GECs)作为高血糖等异常刺激首当其冲的靶细胞，发生内皮间质转化（EndMT），是导致DN的早期关键步骤。本项目采用高糖和棕榈酸(HGPA)处理人脐静脉内皮细胞建立糖尿病细胞损伤模型，腹腔注射链脲佐菌素(STZ)联合高脂饮食(HFD)建立小鼠糖尿病肾病(DN)模型。采用 CCK-8、划痕实验和 FITC- 葡聚糖通透性实验研究了 S1PR2对 HGPA 处理的 HUVEC 细胞活力、迁移和单层细胞屏障功能的影响。采用组织病理学染色、免疫组织化学、免疫荧光和伊文思蓝染色方法，检测糖尿病肾病小鼠肾小球内皮细胞的内皮间质转化和屏障功能损伤，评价 S1PR2抑制剂对 DN 小鼠肾小球 EndMT 的疗效。Western blot 检测细胞和组织水平的wnt3a/RhoA/ROCK1/β-catenin/snail 信号通路的蛋白变化。研究结果显示， 在HGPA处理的人脐静脉内皮细胞损伤模型中，HGPA可能通过上调内皮细胞 S1PR2的表达，激活wnt3a/RhoA/ROCK1/β-catenin/snail 信号通路，导致内皮细胞发生间质转化。采用腹腔注射链脲佐菌素(STZ)联合高脂饮食(HFD)建立的小鼠糖尿病肾病(DN)模型中， 发现DN 小鼠出现了严重的肾损伤和肾纤维化，DN小鼠的肾小球 S1PR2表达增加，激活wnt3a/RhoA/ROCK1/β-catenin/snail 信号通路；而抑制 S1PR2的表达可以逆转小鼠肾小球内皮细胞的 EndMT 和内皮屏障功能损伤。本项目研究表明，HGPA可能通过S1PR2/wnt3a/RhoA/ROCK1/β-catenin/snail 信号通路介导肾小球内皮细胞发生内皮间质转化(EndMT)和内皮屏障功能的损伤，最终导致糖尿病肾病的发生。