丹参有效成分对TGF-β1诱导的肺上皮细胞间质转化的影响

Effect of Effective Components of Salvia Miltiorrhiza on TGF-β1 Induced Interstitial Transformation of Pulmonary Epithelial Cells

目的:通过建立肺上皮间质转化的体外模型,筛选出对肺纤维化有抑制作用的丹参活性成分。方法:设立丹酚酸A(10,20,40,80,160μmol·L^-1)组,丹酚酸B(10,20,40,80,160μmol·L^-1)组,丹参素(10,20,40,80,160μmol·L^-1)组,丹参酮ⅡA(10,20,40,80,160μmol·L^-1)组及空白组作用于A549细胞,用细胞增殖与毒性检测法(MTS)检测这些有效成分对A549细胞的毒性作用,以筛选出丹参有效成分的安全浓度。在安全浓度范围下,再将细胞分为空白组,模型组,丹酚酸A组,丹酚酸B组,丹参素组及丹参酮ⅡA组,用MTS法检测丹参有效成分对转化生长因子-β1(TGF-β1)诱导的A549细胞的增殖抑制作用;酶联免疫吸附测定(ELISA)检测丹参有效成分对纤连蛋白(FN),I型胶原(COL-Ⅰ)表达的影响。综合以上结果,筛选出抑制作用较好的丹参有效成分,用蛋白免疫印迹法(Western blot)检测对细胞E-钙粘(E-Cad)蛋白表达的影响。结果:与空白组比较,丹酚酸A 40μmol·L^-1,丹酚酸B 160μmol·L^-1,丹参素160μmol·L^-1对A549细胞具有毒性作用(P<0.05)。在无毒浓度范围下,与模型组比较,丹酚酸A 10,20μmol·L^-1,丹酚酸B 80μmol·L^-1在培养24 h后对TGF-β1诱导的肺上皮细胞增殖有抑制作用(P<0.05);培养72 h后,丹酚酸A 5,10,20μmol·L^-1,丹酚酸B 40,80μmol·L^-1的抑制作用非常显著(P<0.01)。ELISA结果显示,与空白组比较,模型组细胞FN,COL-Ⅰ的表达显著增加(P<0.01);与模型组比较,丹酚酸A20μmol·L^-1,丹酚酸B 40,80μmol·L^-1对FN,COL-Ⅰ的表达有明显抑制作用(P<0.05);Western blot结果显示,与空白组比较,模型组细胞E-Cad的表达显著降低(P<0.01);与模型组比较,丹酚酸A 20μmol·L^-1,丹酚酸B 80μmol·L^-1时,细胞E-Cad的表达显著增加(P<0.01)。结论:丹酚酸A,丹酚酸B对TGF-β1诱导的上皮间质转化具有抑制作用,可能是治疗肺纤维化的有效成分。

Objective:To screen out the effective components of Salvia miltiorrhiza by establishing an in vitro model of pulmonary epithelial mesenchymal transformation.Method:Different concentrations of salvianolic acid A(10,20,40,80,160μmol·L^-1),salvianolic acid B(10,20,40,80,160μmol·L^-1),tanshinol(10,20,40,80,160μmol·L^-1),tanshinoneⅡA(10,20,40,80,160μmol·L^-1)and the blank group were applied to A549 cell,cell proliferation and cytotoxicity assay(MTS)were used to detect the proliferation effect of menthol on A549 cells.After screening the safe concentration of the active ingredients of salvia miltiorrhiza by MTS,cells were divided into blank group,model group,salvianolic acid A group,salvianolic acid B group,tanshinol group and tanshinoneⅡA.Then,the inhibitory effect of the active ingredients of salvia miltiorrhiza on the proliferation of A549 cells induced by TGF-β1was detected by MTS.Enzyme linked immunosorbent assay(ELISA)method to detect salvia miltiorrhiza effective component of fiber protein(FN),collagen type I(COL-Ⅰ)expression.Based on the above results,the active components of salvia miltiorrhiza,which have best inhibition were screened out,and their effects on the expression of E-calcium-viscosity(E-Cad)protein were detected by Western blot.Result:Compared with blank group,salvianolic acid A 40μmol·L^-1,salvianolic acid B 160μmol·L^-1,tanshinol 160μmol·L^-1had toxic effects on A549 cells(P<0.05).In the non-toxic concentration range,compared with the model group,salvianolic acid A 10,20μmol·L^-1,salvianolic acid B 80μmol·L^-1showed inhibition effect after 24 h culture(P<0.05).After 72 h culture,salvianolic acid A5,10,20μmol·L^-1,salvianolic acid B 40,80μmol·L^-1inhibition effect was very significant(P<0.01).ELISA results showed that with the blank group,model group cells the expression of FN and COL-Ⅰincreased significantly(P<0.01).Compare with model group,salvianolic acid A 20μmol·L^-1,salvianolic acid B80μmol·L^-1inhibited FN and COL-Ⅰ(P<0.05).Western blot results showed that salicylic acid A and salicylic acid B had protective effects on E-Cad(P<0.01).Conclusion:Salvianolic acid A and salvianolic acid B have inhibitory effects on epithelial mesenchymal transformation by TGF-β1,which may be the main effective components of salvianolic acid in the treatment of pulmonary fibrosis.