扯根菜浸膏对TGF-β1肝星状细胞胞内信号转导通路的影响

Effects of Penthorum chinense Pursh extractum on TGF-β1 activated signal transduction within hepatic stellate cells

目的观察扯根菜浸膏对TGF-β1肝星状细胞胞内信号转导通路的影响。方法HSC-T6常规培养后分为正常组、模型组和药物组,用含0.5%FBS的DMEM培养,模型组在相同培养条件下加入TGF-β1刺激,药物组分别用不同浓度药物培养液加TGF-β1刺激,采用Alamar Blue法检测细胞活力,四甲基偶氮唑蓝(MTT)法检测药物细胞毒性;根据Alamar Blue法及MTT的结果,选择药物组用含2mg/ml药物的0.5%FBS培养液加上TGF-β1刺激,24h后采用Western blotting法检测细胞I型胶原、α-肌动蛋白、ERK蛋白表达及磷酸化水平。结果不同浓度扯根菜浸膏均能抑制细胞增殖,在24h增殖抑制明显,以2mg/ml浓度最明显;MTT结果示2mg/ml药物组对细胞无明显的毒性,而10mg/ml、50mg/ml药物组对细胞有明显的毒性;扯根菜浸膏对I型胶原及α-肌动蛋白的表达均有明显抑制,与模型组比较,P<0.01;对ERK蛋白表达未见明显抑制,与模型组比较,P>0.05;但扯根菜浸膏对磷酸化ERK水平及表达均有明显的抑制,与模型组比较,P<0.01。结论扯根菜浸膏能明显抑制肝星状细胞分泌I型胶原及α-肌动蛋白,减少细胞外基质沉积,其部分作用机制可能与抑制TGF-β1的胞内ERK信号转导有关。

Objective To observe the effects of Penthorum chinense Pursh extractum on TGF-β1 activated signal transduction within hepatic stellate cells.Methods HSC-T6 cells were divided into 3 groups:the cells of the normal group incubated with DMEM including 0.5%NBS,the cells of the model group treated with DMEM including 0.5% NBS and TGF-β1,the cells of the drug group treated with DMEM including 0.5%NBS and different concentration of drug Penthorum chinense Pursh extractum and TGF-β1.The HSC-T6 viability was observed by Alamar Blue assay,while the toxicity of Penthorum chinense Pursh extractum was measured by MTT assay.According to the results of the Alamar Blue assay and MTT assay,the drug group treated with 2mg/ml of drug was chosen as the model of the later experiment.After 24 h,The expression of Col I,α-SMA,ERK protein and phosphorylation are analyzed by Western blotting assay.Results Different concentrations of the drug could all inhibit HSC-T6 proliferation,of which was the most significant at 24 h and in 2 mg/ml concentration of drug.MTT assay indicated that 2mg/ml concentration of drug had no obvious toxicity to HSC-T6(P>0.01),while 10mg/ml and 50mg/ml concentrations of drug had obvious toxicity to HSC-T6,and there were significant difference(P<0.01,respectively).Compared to the model group,the drug significantly down-regulated the protein expression of Col I,α-SMA and ERK phosphorylation,there were distinct difference(P<0.01,respectively).While the protein expression of ERK was obviously not restrained by the drug,and there was no significant difference(P>0.05).Conclusions Drug Penthorum chinense Pursh extractum can significantly decrease α-SMA and Col I secretion from activated hepatic stellate cells,inhibit the deposition of ECM in liver,and its part mechanism of action may be related to the inhibition of the ERK signal transduction of TGF-β1.