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目的:研究丹酚酸B影响肝星状细胞活化与转化生长因子β1胞内信号转导的抗肝纤维化作用机制.方法:正常大鼠肝脏链酶蛋白酶原位灌流消化与11％nycondenz密度梯度离心分离肝星状细胞,传一代培养.[~3H]TdR掺入法测定细胞增殖,丽春红染色、图像分析半定量细胞胶原沉积量,ELISA法测定细胞培养上清Ⅰ型胶原分泌量,培养上清酸化处理后,ELISA法测定活性TGF-β1含量.RT-PCR法分析细胞前胶原α_1(Ⅰ)基因的表达,免疫沉淀与蛋白印迹法分析丝裂原激活蛋白激酶(MAPK)活性.结果:丹酚酸B 0.1-100 μmol/L浓度依赖性抑制星状细胞增殖,丹酚酸B 1-100 μmol/L浓度依赖性抑制细胞的Ⅰ型胶原分泌量与总胶原的沉积.丹酚酸B 1-10 μmol/L抑制TGF-β1自分泌量,下调α_1(Ⅰ)前胶原的基因表达.而且丹酚酸B 1-PffiO卫几明显抑制TGF-β1刺激的MAPK活性.结论:丹酚酸B可抑制肝星状细胞的增殖与胶原生成,抑制TGF-β1的自分泌与MAKP活性,这些作用是丹酚酸B抗肝纤维化的主要作用机制.
Aims: To study the anti-hepatic fibrosis mechanism of salvianolic acid B on the activation of hepatic stellate cells and the intracellular signal transduction of transforming growth factor-β1.Methods: Normal rat hepatic chain enzyme digestion was compared with 11% nycondenz density gradient The hepatic stellate cells were isolated by centrifugation and then subcultured. [~ 3H] TdR incorporation assay was used to measure cell proliferation, Ponceau staining, image analysis of semiquantitative collagen deposition, ELISA to determine the amount of type I collagen secretion in cell culture supernatant, The culture supernatant was acidified and the content of active TGF-β1 was measured by ELISA.RT-PCR was used to analyze the expression of procollagen α_1 (Ⅰ) gene and the activity of mitogen-activated protein kinase (MAPK) was analyzed by immunoprecipitation and Western blot. Results: Salvianolic acid B 0.1-100 μmol / L inhibited the proliferation of stellate cells in a concentration-dependent manner, and the concentration of 1-100 μmol / L of salvianolic acid B inhibited the secretion of type Ⅰ collagen and the deposition of total collagen in a dose- Acid B 1-10 μmol / L inhibited TGF-β1 autocrine secretion and down-regulated the gene expression of α_1 (Ⅰ) procollagen, and inhibited the activity of MAPK stimulated by TGF-β1. Conclusion: Phenolic acid B inhibits hepatic stellate cell proliferation and collagen production and inhibits TG The autocrine and MAKP activities of F-β1 are the main mechanisms of action of salvianolic acid B against hepatic fibrosis.