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丹酚酸B盐对转化生长因子-β1刺激肝星状细胞活化与胞内信号转导的作用
作者姓名:Liu C  Liu P  Hu Y  Zhu D
作者单位:1. 200032,上海中医药大学肝病研究所
2. 中国科学院上海药物研究所
基金项目:国家自然科学杰出青年基金资助项目 (3 982 5 168),国家自然科学基金资助项目 (3 9970 899)
摘    要:目的 探讨丹酚酸B盐(SA-B)抗肝纤维化的作用机理。方法 分离大鼠肝星状细胞(HSC),于无包被的塑料培养皿上原代培1、4、7d,细胞分别处于静止,中间活化与完全活化3种活化状态,予以100pmol/L转化生长因子β1(TGF-β1)刺激,观察细胞α-肌动蛋白表达与胶原分泌的变化。选择对TGF-β1刺激最为敏感的中间活化状态HSC为细胞模型,^3H]胸腺嘧啶掺入法观察0.1μmol/L-1mmol/LSA-B对该细胞增殖的作用,倒置显微镜观察药物对细胞形态的影响;Western印迹与Northern印迹等方法观察SA-B对TGF-β1刺激活化的HSC胞外基质基因与蛋白表达,α-肌动蛋白表达的影响;提取细胞质与细胞核蛋白,Western印迹方法观察SA-B对TGF-β1刺激的HSCSmad2/3蛋白表达,磷酸化与核转位的影响。结果 TGF-β1促进各活化状态HSC的胶原分泌,促进率分别为128.6%,207.3%与188.2%,中间活化状态HSC对TGF-β1的促胶原分泌最为敏感,除0.1mmol/L-1mmol/LSA-B引起部分细胞死亡外,0.1μmol/L-10μmol/LSA-B对细胞形态无影响,但可浓度依赖性地抑制细胞增殖,细胞内^3H]胸腺嘧啶掺入量分别为对照组的76%,60.1%与47.8%,1μmol/L-100644mol/LSA-B明显抑制TGF-β1刺激的HSC胶原分泌量(分别为对照组的68.6%与56.1%)。抑制α-肌动蛋白与纤维蛋白酶原激活物抑制子蛋白表达,下调I型前胶原基因表达,0.1μmol/L-10μmol/LSA-B不同程度地抑制Smad2/3的蛋白表达量,明显抑制Smad2蛋白胞内磷酸化与核转位。结论 SA-B抑制TGF-β1的HSC胞内信号转导,从而拮抗TGF-β1的促HSC活化,以上作用是SA-B抗肝纤维化的主要机理。

关 键 词:丹酚酸B盐  肝硬化  丹参  转化生长因子-β  信号传递  肝星状细胞活化
修稿时间:2002年4月19日

Effects of salvianolic acid-B on TGF-beta 1 stimulated hepatic stellate cell activation and its intracellular signaling
Liu C,Liu P,Hu Y,Zhu D.Effects of salvianolic acid-B on TGF-beta 1 stimulated hepatic stellate cell activation and its intracellular signaling[J].National Medical Journal of China,2002,82(18):1267-1272.
Authors:Liu Chenghai  Liu Ping  Hu Yiyang  Zhu Dayuan
Institution:Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China.
Abstract:OBJECTIVE: To investigate hepatic stellate cells (HSC) responses at different differentiation stages on transforming growth factor-beta 1, and to elucidate the mechanisms of salvianolic acid - B (SA-B), a water soluble compound from Salvia miltiorrhiza, against hepatic fibrosis, relating to interference with TGF-beta 1 stimulated HSC activation and intracellular signal transduction via Smads. METHODS: HSC was isolated from rat by in situ perfusion of liver and 8.2% nycondenz gradient centrifugation, and primarily cultured on uncoated plastic for 1 d, 4 d and 7 d respectively, which represented quiescent, intermediate and activated phenotypes. The cells were stimulated with 100 pmol/L TGF-beta 1, cell phenotypes were observed under inverted microscope, alpha-actin expression was checked by Western blot, and collagen secretion was measured with (3)H] proline incorporation and collangenase digestion, then HSC at one definite differentiation stage that responded most sensitively to TGF-beta 1 was selected as the cell model for the following study. 0.1 micromol/L - 1 mmol/L SA-B was incubated with HSC and the cell proliferation was measured by intracellular (3)H] thymidine pulse. SA-B was also incubated with TGF-beta 1 stimulated HSC, the collagen secretion was measured as above, alpha-actin and plasmin activator inhibitor-1 (PAI-1) were checked with Western blot, and alpha1 (I) procollagen mRNA levels were analyzed with Northern blot. The cytoplasmic and nuclear proteins were extracted, and cytoplasmic and nuclear Smad2, 3 expression and phosphorylation levels were measured with Western blot. RESULTS: As culture duration prolonged, HSC phenotypes underwent activation gradually, accompanied by the increase of alpha-actin expression and collagen secretion. TGF-beta 1 increased the basal collagen levels at d1, d4 and d7 by 128.6%, 207.7% and 188.2% of the control respectively, while d4 HSC had the most sensitive response, and this intermediate HSC was used as cell model for the following study. Except 0.1 mmol/L-1 mmol/L SA-B caused parts of HSC death, 0.1 micromol/L-10 micromol/L SA-B had no influence on cell shape, but decreased HSC proliferation in a dose depend manner, by 76%, 60.1% and 47.8% of the control respectively. 1 micromol/L-10 micromol/L SA-B remarkably inhibited the collagen secretion of TGF-beta 1 stimulated HSC by 68.6% and 56.1% of the control, PAI-1 and alpha-actin expression, and down-regulated alpha 1 (I) pro-collagen gene expression. 0.1 micro mol/L approximately 10 micro mol/L SA-B decreased the cytoplasmic and nuclear Smad2, 3 protein expression, especially inhibited Smad2 phosphorylation and nuclear translocation. CONCLUSIONS: SA-B obviously inhibits intermediate HSC proliferation, decreases TGF-beta 1 stimulated HSC activation and matrix protein and gene expression, and inhibited stimulated HSC Smad2, 3 protein expression, phosphorylation and nuclear translocation. The inhibition of TGF-beta 1 signaling in HSC and its biological responses is the important mechanism of SA-B against hepatic fibrosis.
Keywords:Liver cirrhosis  Salvia miltiorrhiza  Receptors  transforming growth factor-beta  Signal transduction
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