补肾活血汤抑制糖尿病肾病引起的足细胞损伤及EMT发生机制研究

Mechanism of Bushen Huoxue decoction in inhibiting podocyte injury and EMT induced by diabetic nephropathy

[目的]观察补肾活血汤对高糖诱导的小鼠足细胞损伤及上皮间充质转化(EMT)的影响,并探讨其对足细胞损伤及发生EMT的作用机制。[方法]体外培养永生化的小鼠足细胞系MPC5,分为对照组、高糖组及补肾活血汤组,经相应处理48h后,采用噻唑蓝(MTT)法检测各组足细胞增殖情况,采用流式细胞术检测各组足细胞凋亡情况,采用Transwell和划痕实验检测各组足细胞体外迁移能力,采用反转录聚合酶链式反应(RT-PCR)检测各组足细胞标志P-cadherin、ZO-1及EMT发生过程标志desmin、FSP-1的相对表达量。在高糖诱导的足细胞中分别加入Rac1抑制剂NSC23766或Rac1激活剂PMA,采用Westernblot检测不同处理组足细胞中GTP-Rac1、p-PAK1、p-p38、p-β-catenin、p65蛋白表达水平。[结果]与对照组相比,高糖处理可抑制足细胞增殖,诱导足细胞凋亡,促进细胞的迁移及EMT的发生,差异具有统计学意义(P<0.05)。补肾活血汤可缓解由高糖诱导的足细胞的增殖抑制作用,降低高糖诱导的细胞损伤、凋亡、迁移,逆转EMT的发生,差异具有统计学意义(P<0.05)。补肾活血汤通过调控GTP-Rac1、p-PAK1、p-p38、p-β-catenin、p65蛋白表达水平,影响Rac1及其下游信号通路的活化。[结论]高糖可导致小鼠足细胞损伤及EMT的发生,补肾活血汤可抑制高糖诱导的足细胞损伤及EMT的发生,其作用机制是补肾活血汤通过调控Rac1及下游信号通路,起到保护足细胞损伤的作用。

[Objective]To observe the effect of Bushen Huoxue Decoction on high glucose-induced podocyte injury and epithelial mesenchymal transition(EMT)in mice,and to explore its mechanism of podocyte injury and EMT.[Methods]The immortalized mouse podocyte line MPC5 was cultured in vitro.The cells were randomly divided into control group,high glucose group and Bushen Huoxue Decoction+high glucose group.After corresponding treatment for 48 h,the groups were detected by MTT assay.The proliferation of podocytes was detected by flow cytometry.The migration ability of podocytes in each group was detected by Transwell and scratch test.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect each.The relative expression levels of des-min and FSP-1 in the process of P-cadherin,ZO-1 and EMT were detected.Rac1 inhibitor NSC23766 or Rac1 activator PMA was added to high glucose-induced podocytes,and GTP-Rac1,p-PAK1,p-p38,p-β-catenin and p65 proteins were detected by Western blot in podocytes of different treatment groups.The expression level.[Results]Compared with the control group,high glucose treatment inhibited podocyte proliferation,induced podocyte apoptosis,and promoted cell migration and EMT.The difference is statistically significant(P<0.05).Bushen Huoxue Decoction can alleviate the proliferation inhibition of podocytes induced by high glucose,reduce the damage,apoptosis and migration induced by high glucose,and reverse the occurrence of EMT.The difference is statistically significant(P<0.05).Bushen Huoxue Decoction can affect the activation of Rac1 and its downstream signaling pathway by regulating the expression levels of GTP-Rac1,p-PAK1,p-p38,p-β-catenin and p65.[Conclusion]High glucose can cause podocyte injury and EMT in mice.Bushen Huoxue Decoction can inhibit podocyte injury and EMT induced by high glucose.The mechanism of action is that Bushen Huoxue Decoction protects Rac1 and downstream signaling pathways.The role of podocyte injury.