丹酚酸B对大鼠肝星状细胞胶原生成与丝裂原激活蛋白激酶活性的影响

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.

Effect of salvianolic acid B on collagen production and mitogen-activated protein kinase activity in rat hepatic stellate cells

AIM: To investigate the mechanism of salvianolic acid-B (SA-B) action against liver fibrosis relating to mediating hepatic stellate cell (HSC) activation and transforming growth factor-beta1(TGF-beta1) intracellular signal transduction. METHODS: HSC was isolated from normal rat through in situ perfusion of liver with pronase E and density-gradient centrifugation with 11 % nycondenz, then cells were subcultured. Cell proliferation was observed by 3H]TdR uptake. Cellular collagen deposition was measured with Ponceau S stain and semi-quantified with image analytic system. Type I collagen secretion in the supernatant was detected with ELISA. The gene expression of type I pro-collagen was analyzed by RT-PCR. The supernatant was acidified and active TGF-beta1 contents were assayed with ELISA. Mitogen-activated protein kinase (MAPK) activity was analyzed with immunoprecipitation and Western blot. RESULTS: SA-B 0.1, 1, 10, and 100 micromol/L suppressed HSC proliferation concentration-dependently as determined by 3H]TdR uptake by 94.1 %, 82.4 %, 62.7 %, and 4 % of the control respectively (P<0.05 or P<0.01). SA-B 1, 10, and 100 micromol/L inhibited soluble type I collagen secretion by 75.3 %, 69.8 %, and 63.5 % of the control and decreased the matrix collagen deposition to 86.2 %, 75.4 %, and 73.4 % (P<0.05 or P<0.01). SA-B 1 and 10 micromol/L decreased the cell active TGF-beta1 secretion by 63.3 % and 15.6 % of the control, down-regulated pro-collgen alpha1(I) mRNA expression to 77.0 % and 51.8 % respectively (P<0.05). SA-B 1 and 10 micromol/L also inhibited MAPK activity by 1 to 2 fold respectively. CONCLUSION: SA-B inhibited HSC proliferation and collagen production as well as decreased the cells' TGF-beta1 autocrine and MAPK activity, which might contribute to the mechanism of SA-B action against hepatic fibrosis.