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目的:研究五灵胶囊有效成分对转化生长因子(TGF-β1)诱导HSC-T6细胞表达Ras/ERK、TGF-β/Smad信号通路蛋白的作用,探寻产生药效的主要化学成分。方法:HSC-T6细胞分组:对照组、TGF-β1(10 ng·ml~(-1))组、PD98059组、PT组、五味子醇甲、五味子乙素、柴胡皂苷、柴胡多糖、丹参新醌乙、X1、二氢丹参酮Ⅰ(各100μg·ml~(-1))、丹参酚酸B(10 ng·ml~(-1))组,培养24 h(10 ng·ml~(-1) TGF-β1于最后12 h加入);ELISA检测培养上清中COL-Ⅰ,Western Blot检测COL-Ⅰ、α-SMA、Ras、ERK、PERK、TβRⅠ、TβRⅡ、Smad2/3蛋白表达。结果:与TGF-β1组相比,各有效成分组能明显降低COL-Ⅰ分泌,其中以X1、丹参新醌乙、二氢丹参酮Ⅰ、五味子醇甲、丹参酚酸B为显著(P<0.05),同时各药物组能显著下调COL-I、α-SMA、ERK、P-ERK、TβRⅠ、TβRⅡ蛋白表达(P<0.05);五味子醇甲、X1、二氢丹参酮Ⅰ能显著下调表达Ras蛋白(P<0.01);柴胡皂苷对TβRⅠ表达无作用;X1、醇甲、乙素、二氢丹参酮Ⅰ、柴胡多糖能显著下调Smad2/3蛋白表达(P<0.01)。结论:五灵胶囊有效成分对Ras/ERK和TGF-β1/Smad信号通路蛋白的作用不尽相同,强度也不尽一致。抗纤维化作用的最强的成分是X1、二氢丹参酮Ⅰ、柴胡多糖、五味子醇甲,丹参新醌乙的作用较弱。
OBJECTIVE: To study the effect of active ingredients of Wuling Capsule on the expression of Ras/ERK and TGF-β/Smad signaling pathway proteins induced by transforming growth factor (TGF-β1) in HSC-T6 cells, and to explore the main chemical components that produce drug efficacy. METHODS: HSC-T6 cells were divided into control group, TGF-β1 (10 ng·ml -1) group, PD98059 group, PT group, Schizandrol, Schizandrin B, Saikosaponin, Bupleurum chinense polysaccharide, Salvia miltiorrhiza Bge. Xinqi B, X1, dihydrotanshinone I (100 μg·ml -1 each), salvianolic acid B (10 ng·ml -1) group, cultured for 24 h (10 ng·ml~(- 1) TGF-β1 was added in the last 12 h); COL-I was detected by ELISA; Western blot was used to detect the expression of COL-I, α-SMA, Ras, ERK, PERK, TβRI, TβRII, and Smad2/3 proteins. Results:Compared with TGF-β1 group, each active ingredient group can significantly reduce the secretion of COL-I, among which X1, Salvia miltiorrhiza B, Dihydrotanshinone I, Schizandrol A, Salvia miltiorrhiza B were significant (P<0.05). ), At the same time, each drug group can significantly down-regulate the expression of COL-I, α-SMA, ERK, P-ERK, TβRI, and TβRII proteins (P<0.05); Schisandrin, X1, and Dihydrotanshinone I can significantly down-regulate Ras protein expression. (P<0.01); saikosaponin had no effect on the expression of TβRI; X1, alcohols A, B, dihydrotanshinone I, Bupleurum chinensis polysaccharides could significantly down-regulate Smad2/3 protein expression (P<0.01). Conclusion: The active ingredients of Wuling capsules have different effects on Ras/ERK and TGF-β1/Smad signaling pathway proteins, and their intensities are not consistent. The strongest components of antifibrotic effects were X1, dihydrotanshinone I, Bupleurum chinense polysaccharides, and schisandrin A. Salvia miltiorrhiza B was less effective.