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目的:研究丹酚酸A对培养的大鼠肝星状细胞增殖与胶原生成的影响。方法:用链酶蛋白酶与胶原酶对肝脏进行原位灌流消化,Nycodenz密度梯度离心分离大鼠肝星状细胞,传一代培养。MTT法与[~3H]TdR掺入法测定细胞增殖。丽春红染色、图象分析法半定量细胞胶原沉积量,ELISA法测定细胞培养上清中Ⅰ型胶原分泌量,检测细胞层总蛋白量校正细胞数。RT-PCR法分析前胶原α_2(Ⅰ)基因的表达。结果:丹酚酸A 100μmol/L引起部分细胞脱壁与死亡,有一定毒性反应。丹酚酸A 0.1-10μmol/L对细胞形态无明显影响。丹酚酸A1-100μmol/L浓度依赖性抑制细胞增殖,降低胶原沉积量与Ⅰ型胶原分泌量。丹酚酸A1-10μmol/L对前胶原α_2(Ⅰ)mRNA表达均有明显抑制作用。结论:丹酚酸A抑制肝星状细胞增殖与胶原表达,是丹参抗肝纤维化的主要有效成分之一,抑制肝星状细胞活化是其抗肝纤维化的主要作用机制。
Objective: To study the effect of salvianolic acid A on the proliferation and collagen production of cultured rat hepatic stellate cells. Methods: Liver was digested by streptavidin and collagenase in situ. Rat hepatic stellate cells were isolated by Nycodenz density gradient centrifugation and subcultured. Cell proliferation was measured by MTT and [~ 3H] TdR incorporation. Ponceau was stained with semi-quantitative method. The amount of collagen Ⅰ secreted by cell culture supernatant was measured by ELISA. The amount of total protein in cell layer was measured. The expression of procollagen α 2 (Ⅰ) gene was analyzed by RT-PCR. Results: Salvianolic acid A 100μmol / L caused part of the cell wall and death, there is a certain toxicity. Salvianolic acid A 0.1-10μmol / L had no significant effect on cell morphology. Salvianolic acid A1-100μmol / L inhibited cell proliferation in a concentration-dependent manner and decreased collagen deposition and collagen Ⅰ secretion. Salvianolic acid A1-10μmol / L significantly inhibited the mRNA expression of procollagen α_2 (Ⅰ). Conclusion: Salvianolic acid A inhibits hepatic stellate cell proliferation and collagen expression, is one of the main active ingredients of anti-fibrosis of Salvia miltiorrhiza. Inhibition of hepatic stellate cell activation is the main mechanism of anti-hepatic fibrosis.