土茯苓总黄酮通过p38/ERK MAPK通路调控膜性肾病大鼠足细胞上皮-间充质转化的作用机制研究

Effect of Total Flavonoids of Tufuling(Smooth Greenbrier)on Podocyte Epithelial Mesenchymal Transformation in Rats with Membranous Nephropathy Through p38/ERK MAPK Pathway

目的:探讨土茯苓总黄酮(SGF)对膜性肾病大鼠足细胞上皮-间充质转化(EMT)的影响及对p38/细胞外调节蛋白激酶(ERK)-丝裂原活化蛋白激酶(MAPK)通路的调节作用。方法:将76只造模成功的膜性肾病SD大鼠随机分为模型组、SGF低剂量组(15 mg/kg)、SGF高剂量组(30 mg/kg)和阳性药物组(环孢素,10 mg/kg),每组19只,另取20只正常SD大鼠作为对照组。各组采用相应药物干预6周。HE染色和TUNEL法观察大鼠肾皮质组织病变和PC凋亡。检测24 h尿蛋白(24 h UTP)、血清血肌酐(Scr)、血尿素氮(BUN)水平,血清白细胞介素-4(IL-4)、转化生长因子-β1(TGF-β1)、肿瘤坏死因子-α(TNF-α)水平,肾皮质组织细胞外调节蛋白激酶(ERK)mKNA、p38MAPK mKNA、Nephrin mKNA、α-平滑肌肌动蛋白(α-SMA)mRNA,以及ERK、p-ERK、p38MAPK、p-p38MAPK、Nephrin、α-SMA蛋白水平。结果:模型组大鼠肾小球体积增大,细胞萎缩,系膜与基质出现明显增生,肾小管扩张;SGF低剂量组、SGF高剂量组及阳性药物组大鼠肾脏病理损伤均有不同程度减轻。与模型组比较,SGF低剂量组、SGF高剂量组及阳性药物组大鼠IL-4、TGF-β1水平,Nephrin mRNA和蛋白水平均明显升高(P<0.05),24h UTP、Scr、BUN、TNF-α、肾小球足细胞凋亡指数(AI)、α-SMA mRNA、p-ERK蛋白、p-p38MAPK蛋白、α-SMA蛋白水平均明显降低(P<0.05),其中以阳性药物组大鼠各指标变化最显著,其次是SGF高、低剂量组(P<0.05)。结论:SGF可有效改善膜性肾病大鼠炎性反应和细胞凋亡,抑制EMT过程,可能是通过调控p38/ERK MAPK通路发挥作用。

Objective:To investigate the effects of total flavonoids of Tufuling(SGF)on epithelial mesenchymal transformation(EMT)of podocyte(PC)in rats with membranous nephropathy(MN)and the regulation of p38/extracellular regulated protein kinase(ERK)-mitogen-activated protein kinase(MAPK)pathway.Methods:76 SD rats with membranous nephropathy were randomly divided into control group,model group,SGF low-dose group(15 mg/kg),SGF high-dose group(30 mg/kg)and positive drug group(cyclosporine,10 mg/kg).19 rats in each group,and 20normal SD rats were taken as the control group.Each group was intervened with corresponding drugs for 6weeks.HE staining and TUNEL method were used to observe the pathological changes of renal cortex and PC apoptosis in rats.The levels of 24-hour urinary protein(24 h UTP),serum creatinine(Scr),the level of blood urea nitrogen(BUN),serum interleukin 4(IL-4),transforming growth factorβ1(TGF-β1),the level of tumor necrosis factorα(TNF-α),extracellular regulated protein kinase in renal cortex(ERK)mKNA,p38MAPK mKNA,Nephrin mKNA,α-smooth muscle actin(α-SMA)mRNA and ERK,p-ERK,p38MAPK,p-p38MAPK,Nephrin,α-SMA protein levels in renal cortex were measured.Results:In the model group,the glomerular volume increased,the cells shrank,the mesangium and matrix proliferated significantly,and the renal tubules expanded.The pathological damage of kidney in SGF low-dose group,SGF high-dose group and positive drug group decreased in varying degrees.Compared with the model group,the levels of IL-4,TGF-β1,nephrin mRNA and protein in SGF low-dose group,SGF high-dose group and positive drug group increased significantly(P<0.05),and the levels of UTP,Scr,BUN,TNF-α,glomerular podocyte apoptosis index(AI),α-SMA m RNA,p-ERK protein,p-p38MAPK protein andα-SMA protein decreased significantly at 24 hours(P<0.05).Among them,the changes of various indexes in positive drug group were the most significant,followed by SGF high-dose and low-dose group(P<0.05).Conclusion:SGF can effectively improve the inflammatory response and apoptosis in rats with membranous nephropathy and inhibit the process of EMT,which may play a role by regulating p38/ERK MAPK pathway.