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探讨雷公藤甲素(triptolide,TP)改善高剂量葡萄糖糖(高糖)诱导的足细胞上皮-间充质转分化(epithelial-mesenchymal transition,EMT)的作用和分子机制。将体外培养的小鼠永生化足细胞分为正常组(normal group,N)、高剂量葡萄糖组(high dose of glucose group,HG)、低剂量TP组(low dose of TP group,L-TP)、高剂量TP组(high dose of glucose group,HG)以及甘露醇组(mannitol group,MNT),分别进行不同的干预:N组加入D-葡萄糖(D-glucose,DG,5 mmol·L-1);HG组加入HG(25 mmol·L-1);L-TP组加入HG(25 mmol·L-1)+TP(3 ng·mL-1);H-TP组加入HG(25 mmol·L-1)+TP(10 ng·mL-1);MNT组加入DG(5 mmol·L-1)+MNT(24.5 mmol·L-1)。在干预后的各时间点(24 h,48 h,72 h),首先,观察TP对足细胞增殖活性的影响;其次,检测足细胞上皮细胞标志性分子(nephrin和podocin)、间充质细胞标志性分子(desmin和I型胶原)以及EMT相关介质snail的蛋白表达水平;最后,检测足细胞Wnt3α/β-catenin信号通路关键信号分子Wnt3α和β-catenin蛋白表达水平。结果表明,HG不仅能引起足细胞nephrin和podocin蛋白低表达,desmin,I型胶原和snail蛋白高表达,诱导足细胞EMT,而且,能促使足细胞Wnt3α和β-catenin蛋白高表达,激活Wnt3α/β-catenin信号通路。此外,L-TP对足细胞增殖能力没有影响,与HG联合干预不仅能明显恢复足细胞nephrin和podocin蛋白表达水平,抑制其desmin,I型胶原和snail蛋白表达水平,改善足细胞EMT,而且,能明显降低HG诱导的足细胞Wnt3α和β-catenin蛋白高表达,抑制Wnt3α/β-catenin信号通路活性。总之,HG在体外能激活Wnt3α/β-catenin信号通路而诱导足细胞发生EMT;L-TP在体外能明显抑制Wnt3α/β-catenin信号通路活性而改善足细胞EMT,这可能是其干预足细胞EMT的作用和分子机制之一。
To investigate the effect and molecular mechanism of triptolide (TP) on the epithelial-mesenchymal transition (EMT) induced by high dose glucose (high glucose). Immortalized podocytes cultured in vitro were divided into normal group (N), high dose of glucose group (HG), low dose of TP group (L-TP) , High dose of glucose group (HG) and mannitol group (MNT) were given different interventions: group D was given D-glucose (DG, 5 mmol·L-1 ); HG (25 mmol·L-1); HG (25 mmol·L-1) + TP (3 ng · mL-1) in L-TP group; L-1) + TP (10 ng · mL-1); MNT group (5 mmol·L-1) + MNT (24.5 mmol·L-1). At each time point (24 h, 48 h, 72 h) after intervention, the effect of TP on the proliferation of podocytes was observed. Second, the expressions of nephrin and podocin, mesenchymal cells The expression of Wnt3α / β-catenin signaling molecules and the expression of Wnt3α / β-catenin in podocytes. The results showed that HG not only induced low expression of nephrin and podocin protein in podocytes, but also induced high expression of desmin, type I collagen and snail protein, induced podocyte EMT, and also induced high expression of Wnt3α and β-catenin proteins in podocytes. Activation of Wnt3α / β-catenin signaling pathway. In addition, L-TP had no effect on the proliferation of podocytes. Combined with HG, the expression of nephrin and podocin protein, the expression of desmin, type I collagen and snail proteins, and the podocyte EMT were all improved, Can significantly reduce HG-induced podocyte Wnt3α and β-catenin protein expression, inhibit Wnt3α / β-catenin signaling pathway activity. In conclusion, HG can activate Wnt3α / β-catenin signaling pathway in vitro and induce EMT in podocytes. L-TP can significantly inhibit the activity of Wnt3α / β-catenin signaling pathway and improve podocyte EMT in vitro, which may be due to its intervention in podocytes One of the roles and molecular mechanisms of EMT.