Liu C H, Liu P, Hu Y Y, Xu L M, Tan Y Z, Wang Z N, Liu C
Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China.
Acta Pharmacol Sin. 2000 Aug;21(8):721-6.
To investigate the effects of salvionolic acid-A (SA-A), one of main effective components of Salvia miltiorrhiza for its antifibrotic action, on the cell proliferation and collagen production in cultured hepatic stellate cells (HSC).
HSC were isolated through in situ perfusion of liver with pronase E and collagenase, and gradient centrifugation with Nycodenz. The cultured HSC were incubated with SA-A 0.1-100 mumol/L for 24 h. MTT spectrometric assay and intercellular incorporation of methyl-[3H]thymidine ([3H]TdR) was used to assess the cell proliferation. The amount of collagen was semi-quantified by ponceau staining and image analysis, the amount of type I collagen secretion was measured with ELISA and normalized by the total protein of cell layer. The total RNA was prepared from the control cells and the drug treated cells respectively, and the expression of pro-collagen alpha 2 (I) mRNA was semi-quantitatively analyzed with RT-PCR.
SA-A 100 mumol/L showed a little cytotoxity, SA-A 0.1-10 mumol/L did not influence cell morphology, and SA-A 1-100 mumol/L decreased the cell proliferation significantly in a concentration-dependent manner (P < 0.05). SA-A 1, 10, 100 mumol/L decreased the cell collagen deposition by 78.6%, 71.8%, and 61.3% of the control respectively (P < 0.05), and decreased type I collagen secretion to 53.1%, 52.6%, and 49.5% (P < 0.01 or P < 0.05). Both SA-A 1 and 10 mumol/L downregulated procollagen alpha 2 (I) mRNA expression remarkably (P < 0.05).
SA-A inhibited HSC proliferation and collagen expression. The inhibitory effect on HSC activation is the main mechanism of SA-A action against liver fibrosis.
研究丹参主要有效成分之一的丹酚酸A（SA-A）对培养的肝星状细胞（HSC）增殖及胶原生成的抗纤维化作用。
通过用链霉蛋白酶E和胶原酶原位灌注肝脏，并用Nycodenz进行梯度离心分离HSC。将培养的HSC与0.1 - 100μmol/L的SA-A孵育24小时。采用MTT比色法和甲基 - [3H]胸腺嘧啶核苷（[3H]TdR）掺入法评估细胞增殖。通过丽春红染色和图像分析对胶原量进行半定量，用ELISA法测定Ⅰ型胶原分泌量并用细胞层总蛋白进行标准化。分别从对照细胞和药物处理细胞中提取总RNA，并用RT-PCR法对前胶原α2（Ⅰ）mRNA的表达进行半定量分析。
100μmol/L的SA-A显示出轻微的细胞毒性，0.1 - 10μmol/L的SA-A不影响细胞形态，1 - 100μmol/L的SA-A以浓度依赖方式显著降低细胞增殖（P < 0.05）。1、10、100μmol/L的SA-A分别使细胞胶原沉积量比对照降低78.6%、71.8%和61.3%（P < 0.05），并使Ⅰ型胶原分泌量降至53.1%、52.6%和49.5%（P < 0.01或P < 0.05）。1和10μmol/L的SA-A均显著下调前胶原α2（Ⅰ）mRNA表达（P < 0.05）。
SA-A抑制HSC增殖和胶原表达。对HSC激活的抑制作用是SA-A抗肝纤维化作用的主要机制。
