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

关键词:  黄连素  糖尿病肾病  足细胞  微小RNA-29b  上皮-间充质转化

Key words:  berberine  diabetic nephropathy  podocyte  micro RNA-29b  epithelial-mesenchymal transition

摘要:

[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+50 μmol/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。

Abstract:

[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 + 50 μmol/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 proteins α-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 of α-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 of α-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 of α-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.