汉防己甲素对TGF-β1诱导的MRC-5细胞中Col-I及FN表达的影响

Effect of Tetrandrine on Col-I and FN in TGF-β1-induced MRC-5 Cells

目的:探讨汉防己甲素(Tet)对人转化生长因子-β1(TGF-β1)刺激的人胚肺成纤维细胞MRC-5的影响。方法:不同质量浓度的TGF-β1(0,2.5,5,10,20,40μg·L^-1)作用于MRC-5细胞,应用细胞增殖与毒性检测法(CCK-8)检测TGF-β1对MRC-5细胞的增殖毒性,蛋白免疫印迹法(Western blot)检测MRC-5细胞中α-平滑肌肌动蛋白(α-SMA)及波形蛋白(Vimentin)表达水平的变化,酶联免疫吸附测定法(ELISA)检测MRC-5细胞上清液中I型胶原(Col-I)及纤维黏连蛋白(FN)表达水平的变化,以筛选出TGF-β1活化MRC-5细胞的适宜浓度。结合筛选出的TGF-β1的浓度条件,与不同浓度的Tet(0,2.5,5,10,20,40μmol·L^-1)共同作用于MRC-5细胞,再次应用CCK-8法筛选TGF-β1及Tet共培养对MRC-5细胞的安全浓度,Western blot检测MRC-5细胞中α-SMA及Vimentin表达水平的变化,ELISA检测MRC-5细胞上清液中Col-I及FN表达水平的变化。结果:与空白组比较,给药24 h时,20μg·L^-1及40μg·L^-1的TGF-β1对MRC-5细胞有毒性作用(P<0.05),给药48 h时,10,20,40μg·L^-1的TGF-β1对MRC-5细胞有毒性作用(P<0.05);加入Tet培养24 h时,对MRC-5细胞的半数抑制浓度(IC50)值为14.07μmol·L^-1,培养48 h时,IC50值为7.51μmol·L^-1;与空白组比较,5μg·L^-1的TGF-β1刺激MRC-5细胞时,细胞中α-SMA,FN及Col-I的相对含量明显升高(P<0.05),Vimentin相对含量显著升高(P<0.01),10μg·L^-1组细胞中α-SMA,Vimentin,FN及Col-I的相对含量显著升高(P<0.01);10μg·L^-1的TGF-β1与不同浓度的Tet共培养,与TGF-β1组比较,Tet 5μmol·L^-1组,细胞中α-SMA,Vimentin及FN相对含量显著降低(P<0.01),Col-I相对含量明显降低(P<0.05),Tet 10μmol·L^-1组,细胞中α-SMA,Vimentin,FN及Col-I的相对含量均显著降低(P<0.01)。结论:TGF-β1能够升高MRC-5细胞中Col-I,FN等细胞外基质的水平,而Tet可以有效抑制这种变化的发生,提示Tet可能对肺纤维化的形成中,成纤维细胞大量分泌细胞外基质的过程有抑制作用。

Objective:To investigate the effect of tetrandrine on transforming growth factor-β1(TGF-β1)stimulated MRC-5 cells.Method:Different concentrations of TGF-β1(0,2.5,5,10,20,40μg·L^-1)were applied to MRC-5 cells.Proliferation toxicity of TGF-β1 to MRC-5 was detected by cell counting kit-8(CCK-8)method.Detection of alpha smooth muscle actin(α-SMA)and Vimentin’s expression levels in MRC-5 by Western blot.Detection of changes of collagen I(Col-I)and fibronectin(FN)’s expression levels in MRC-5 supernatants by enzyme linked immunosorbent assay(ELISA)kit.And the appropriate concentration of TGF-β1 activated MRC-5 cells was screened.The appropriate concentration of TGF-β1 and different concentrations of Tet(0,2.5,5,10,20,40μmol·L^-1)were applied to MRC-5 cells,and CCK-8 method was used to screen safe concentration again.Western blot was used to detect changes inα-SMA and Vimentin expression levels in MRC-5 cells,and ELISA method to detect changes in Col-I and FN in MRC-5 cell supernatant.Result:Compared with the blank group,20,40μg·L^-1 of TGF-β1 had toxic effects on MRC-5 cells at 24 hours(P<0.05),and 10,20,40μg·L^-1 of TGF-β1 had toxic effects on MRC-5 cells at 48 h(P<0.05).When Tet is added for 24 h,the half inhibitory concentration(IC50)value was 14.07μmol·L^-1,and when cultured for 48 h,the IC50 value was7.51μmol·L^-1.Compared with the blank group,the relative contents ofα-SMA,FN and Col-I in the 5μg·L^-1 of TGF-β1 group were obviously increased(P<0.05),and the relative contents of Vimentin were significantly increased(P<0.01),and the relative contents of FN and Col-I,α-SMA and Vimentin in 10μg·L^-1 group were significantly increased(P<0.01).10μg·L^-1 of TGF-β1 was co-cultured with Tet at different concentrations.Compared with the TGF-β1 group,the relative levels ofα-SMA,Vimentin and FN in the 5μmol·L^-1 of Tet group were significantly reduced(P<0.01),and the relative levels of Col-I were obviously reduced(P<0.05).In the Tet 10μmol·L^-1 group,the relative contents of theα-SMA,Vimentin,FN and Col-I were significantly reduced(P<0.01).Conclusion:TGF-β1 can increase the levels of Col-I,FN and other extracellular matrices in MRC-5 cells,and Tet can effectively inhibit the occurrence of this change.It is suggested that Tet may inhibit secreting extracellular matrix of fibroblasts in the formation of pulmonary fibrosis.