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目的:探讨丹参酚酸B盐(salvianolic acid B,SA-B)对转化生长因子β1(transforming growthfactor-β1,TGF-β1)活化的大鼠肝星状细胞(hepatic stellate cell,HSC)内细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号转导通路的影响。方法:采用原位灌流酶消化及Nycodenz密度梯度离心的方法分离正常大鼠HSC,将TGF-β1和SA-B直接添加于原代HSC的无血清培养液中。蛋白印记法检测HSC内磷酸化和总ERK,以及有丝分裂原激活蛋白激酶激酶MEK、有丝分裂原活化蛋白激酶激酶激酶Raf、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和Ⅰ型胶原蛋白的表达。结果:SA-B可抑制正常原代HSC及TGF-β1刺激的HSC内ERK上游激酶MEK的磷酸化;而对正常原代HSC及TGF-β1刺激的HSC内MEK上游激酶Raf-1的磷酸化无明显抑制。SA-B对TGF-β1刺激的HSC内α-SMA表达有明显的抑制作用,SA-B与ERK阻断剂联用,几乎接近完全阻断α-SMA的表达。SA-B对正常培养的HSC和经TGF-β1刺激的HSCⅠ型胶原蛋白的合成都有明显的抑制作用。结论:SA-B抑制TGF-β1刺激的HSC内ERK信号转导通路的作用环节在于抑制了MEK的磷酸化。SA-B通过抑制TGF-β1的ERK信号转导通路,减少了因HSC活化导致的α-SMA表达和Ⅰ型胶原蛋白合成增加。
OBJECTIVE: To investigate the effect of salvianolic acid B (SA-B) on the extracellular domain of rat hepatic stellate cell (HSC) activated by transforming growth factor β1 (TGF-β1) Effects of extracellular signal-regulated kinase (ERK) signal transduction pathway. Methods: Normal rat HSCs were isolated by in situ enzymatic digestion and Nycodenz density gradient centrifugation. TGF-β1 and SA-B were directly added to serum-free medium of primary HSCs. HSC phosphorylation and total ERK were detected by Western blotting, and mitogen-activated protein kinase kinase MEK, mitogen-activated protein kinase kinase Raf, α-smooth muscle actin (α-SMA) and type Ⅰ collagen Protein expression. Results: SA-B inhibited the phosphorylation of MEK in normal primary HSC and TGF-β1-stimulated HSC, while phosphorylation of MEK upstream kinase Raf-1 in normal HSC and TGF-β1-stimulated HSCs No significant inhibition. SA-B significantly inhibited the expression of α-SMA in HSC stimulated by TGF-β1. SA-B combined with ERK block almost completely blocked the expression of α-SMA. SA-B had a significant inhibitory effect on the synthesis of both normal HSC and TGF-β1-stimulated HSC type I collagen. Conclusion: SA-B inhibits the phosphorylation of MEK by inhibiting the TGF-β1-stimulated ERK signal transduction pathway in HSC. SA-B reduced the expression of α-SMA and the increase of type I collagen synthesis by inhibiting the ERK signal transduction pathway of TGF-β1.