温阳消癥方调控LncRNA MEG3对TGF-β_(1)诱导的肾小管上皮细胞纤维化因子的影响

Effect of Wenyangxiaozheng Decoction on TGF-β_(1)-induced Fibrosis in Renal Tubular Epithelial Cells by Regulating LncRNA MEG3

目的:探讨温阳消癥方对TGF-β_(1)诱导的人近端肾小管上皮细胞LncRNA MEG3的调控作用及对纤维化因子的影响。方法:体外培养人近端肾小管上皮细胞(HK-2),分为对照组、TGF-β_(1)组、TGF-β_(1)+3.5 mg/ml温阳消癥组、TGF-β_(1)+14 mg/ml温阳消癥组,MTT法检测细胞增殖,实时荧光定量聚合酶链式反应(RT-qPCR)检测α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)及结缔组织生长因子(CTGF)mRNA及长链非编码RNA母系表达基因3(LncRNA MEG3)表达,蛋白质印迹法检测α-SMA、FN、CTGF蛋白表达。结果:3.5、7、14 mg/ml温阳消癥不影响细胞增殖,35 mg/ml组细胞增殖明显降低(P<0.01);与对照组比较,TGF-β_(1)组中α-SMA、FN、CTGF mRNA、LncRNA MEG3及FN、CTGF蛋白表达显著升高(P<0.01),α-SMA蛋白表达升高(P<0.05);与TGF-β_(1)组比较,两个温阳消癥方治疗组α-SMA、FN、CTGF mRNA、LncRNA MEG3、FN蛋白表达,及TGF-β_(1)+14 mg/ml温阳消癥组α-SMA、CTGF蛋白表达均显著降低(P<0.01),TGF-β_(1)+3.5 mg/ml温阳消癥组α-SMA、CTGF蛋白表达降低(P<0.05);与TGF-β_(1)+3.5 mg/ml温阳消癥组相比,TGF-β_(1)+14 mg/ml温阳消癥组α-SMA、CTGF mRNA及CTGF蛋白表达均显著降低(P<0.01),FN mRNA及LncRNA MEG3表达降低(P<0.05)。结论:温阳消癥方可下调LncRNA MEG3的表达,抑制TGF-β_(1)诱导的HK-2细胞纤维化,其抑制纤维化的作用可能和调控LncRNA MEG3的表达有关。

Objective:To explore the regulatory effect of Wenyangxiaozheng decoction on LncRNA MEG3 of human proximal tubular epithelial cells induced by TGF-β_(1) and its influence on fibrosis factors.Methods:The human proximal tubular epithelial cells(HK-2)were cultured in vitro and divided into control group,TGF-β_(1) group,TGF-β_(1)+3.5 mg/ml Wenyangxiaozheng group and a TGF-β_(1)+14 mg/ml Wenyangxiaozheng group.Cell proliferation was detected by MTT assay.RT-qPCR was used to detect the expression ofα-SMA、FN、CTGF mRNA and LncRNA MEG3.Western blot was used to detect the protein expression ofα-SMA,FN and CTGF.Results:3.5,7 and 14 mg/ml Wenyangxiaozheng group did not affect cell proliferation,but 35 mg/ml group significantly reduced cell proliferation(P<0.01).Compared with the control group,the expression ofα-SMA,FN,CTGF mRNA,LncRNA MEG3 and FN,CTGF protein in TGF-β_(1) group increased significantly(P<0.01),and the expression ofα-SMA protein increased(P<0.05).Compared with TGF-β_(1) group,the expression ofα-SMA,FN,CTGF mRNA,LncRNA MEG3,FN protein in the two Wenyangxiaozheng group,and the expression ofα-SMA,CTG protein in the TGF-β_(1)+14 mg/ml Wenyangxiaozheng group decreased significantly(P<0.01),while the expression ofα-SMA,CTGF protein in the TGF-β_(1)+3.5 mg/ml Wenyangxiaozheng group decreased(P<0.05).Compared with the TGF-β_(1)+3.5 mg/ml Wenyangxiaozheng group,the expression ofα-SMA,CTGF mRNA and CTGF protein in the TGF-β_(1)+14 mg/ml Wenyangxiaozheng group decreased significantly(P<0.01),and the expression of FN mRNA and LncRNA MEG3 decreased(P<0.05).Conclusion:Wenyangxiaozheng decoction can down-regulate the expression of LncRNA MEG3 and inhibit the fibrosis of HK-2 cells induced by TGF-β_(1),and its inhibitory effect on fibrosis may be related to regulating the expression of LncRNA MEG3.