黄连素调节miR-29b抑制高糖诱导的MPC-5足细胞上皮-间质转化研究

Study on the berberine regulates miR-29b to inhibit epithelial-mesenchymal transition of MPC-5 podocytes induced by high glucose

[目的]探讨miR-29b在高糖诱导的MPC-5足细胞上皮-间质转化中的作用及黄连素的保护机制。[方法]将MPC-5小鼠足细胞分为正常组、高糖组和高糖+50μmol/L黄连素组,培养48 h后,采用酶联免疫吸附法(ELISA)测定培养液上清液中的重组人转化生长因子-β1(TGF-β1),实时定量聚合酶链反应(RT-qPCR)法测定miR-29b表达,Western blot法检测上皮向间充质细胞转分化(EMT)相关蛋白[α-平滑肌肌动蛋白(α-SMA)、肌间线蛋白(desmin)、podocin]的表达。将足细胞分别转染miR-29b mimic或miR-29b inhibitor后,再给予高糖刺激和/或黄连素培养,ELISA法测定培养液上清液中的TGF-β1;Western blot法检测EMT相关蛋白的表达。采用双荧光素酶报告基因实验检测miR-29b与TGF-β1是否能够直接结合。[结果]与正常组相比,高糖组miR-29b表达降低,TGF-β1浓度上升,EMT相关蛋白α-SMA、desmin表达上升,podocin表达下降。与高糖组相比,高糖+黄连素组的miR-29b表达增加,TGF-β1浓度,α-SMA、desmin表达下降,podocin表达上升(P<0.01)。与高糖组相比,高糖+miR-29b mimic组的TGF-β1浓度下降,α-SMA、desmin表达下降,podocin表达上升(P<0.01)。与高糖+黄连素组相比,高糖+黄连素+miR-29b inhibitor组TGF-β1浓度上升,α-SMA、desmin表达上升,podocin表达下降(P<0.01)。双荧光素酶报告基因实验发现miR-29b可直接作用于TGF-β1并抑制其表达。[结论]黄连素可以通过调节miR-29b水平,进而降低高糖诱导的足细胞表达TGF-β1、降低足细胞发生上皮间质转化的程度。

[Objective]To investigate the role of miR-29b in the high glucose induced epithelial-mesenchymal transition of MPC-5 podocytes and the protective mechanism of berberine.[Methods]The podocytes of MPC-5 mice were divided into normal group,high glucose group and high glucose+50μmol/L berberine group.After 48 hours of culture,TGF-β1 in the supernatant of culture medium was measured by ELISA,the expression of miR-29b was measured by RT-qPCR,and EMT related proteins[α-smooth muscle actin(α-SMA),desmin and podocin]were detected by Western blot.The podocytes were transfected into miR-29b mimic or miR-29b inhibitor respectively,and then cultured with high glucose stimulation and/or berberine.TGF-β1 in the supernatant of the culture solution was determined by ELISA.The expression of EMT-related proteins was detected by Western blot.The double luciferase reporter gene experiment was used to detect whether miR-29b and TGF-β1 can be directly combined.[Results]Compared with the normal group,the expression of miR-29b decreased,the concentration of TGF-β1 increased,the expressions of EMT-related proteinsα-SMA and desmin increased,and the expression of podocin decreased in the high glucose group.Compared with high glucose group,the expression of miR-29b in high glucose+berberine group increased,the concentration of TGF-β1,the expression ofα-SMA and desmin decreased,and the expression of podocin increased(P<0.01).Compared with the high glucose group,the concentration of TGF-β1 decreased,the expression ofα-SMA and desmin decreased and the expression of podocin increased in the high glucose miR-29b mimic group(P<0.01).Compared with high glucose+berberine group,the concentration of TGF-β1,the expression ofα-SMA and desmin increased and the expression of podocin decreased in high glucose+berberine miR-29b inhibitor group(P<0.01).The experiment of double luciferase reporter gene showed that miR-29b could directly act on TGF-β1 and inhibit its expression.[Conclusion]Berberine can reduce the expression of TGF-β1 in podocytes induced by high glucose and the degree of epithelial-mesenchymal transition in podocytes by regulating the level of miR-29b.