虎杖苷抑制肝星状细胞增殖活化的作用和机制研究

目的:研究虎杖苷 (Polydatin) 在体外对HSC-T6细胞的影响，探讨虎杖苷抑制肝纤维化发生的作用及相关机制。方法:体外培养大鼠肝星状细胞HSC-T6，分为对照组和虎杖苷低、中、高浓度（125、250、500 μmol/L）给药组。MTT法检测细胞增殖情况；比色法检测细胞培养上清液中乳酸脱氢酶（LDH）的活性；酶消化法检测细胞培养上清液中羟脯氨酸（Hyp）的含量；蛋白印迹法检测α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（CollagenⅠ）、核因子NF-E2相关因子2（Nrf2）和血红素加氧酶-1（HO-1）蛋白表达水平。多组间比较采用单因素方差分析，若方差齐则采用LSD检验，若方差不齐采用Dunnett T3检验，以P<0.05认为差异有统计学意义。结果:与对照组比较，虎杖苷125、250、500 μmol/L给药组细胞活力显著降低（F=252.01, P<0.001），Hyp含量明显减少（F=16.50，P<0.05），α-SMA和CollagenⅠ蛋白表达显著减少（F=19.89、182.91, 均P<0.05），且随着浓度的增加，抑制作用越强，呈一定的剂量依赖性；与对照组比较，虎杖苷125、250、500 μmol/L给药组Nrf2蛋白表达显著增加（F=23.70，P<0.05），虎杖苷250、500 μmol/L给药组HO-1蛋白表达显著增加（F=12.40，P<0.01），且随着浓度增加，上调作用越明显，呈一定的剂量依赖性。结论:虎杖苷通过抑制肝星状细胞增殖活化及胶原合成发挥抑制肝纤维化发生的作用，该作用可能与其上调Nrf2/HO-1通路有关。

Inhibitory effects and mechanism of Polydatin on the proliferation and activation of hepatic stellate cell

Objective: To examine the effect of Polydatin on HSC-T6 cells in vitro, and to investigate the inhibitory effects and related mechanism of Polydatin on liver fibrogenesis. Methods: Rat hepatic stellate cells HSC-T6 were cultured in vitro and were divided into control group and low, middle and high concentration(125, 250, 500 μmol/L)Polydatin groups. Cell proliferation was measured by MTT assay. LDH activity in cell supernatant was detected by colorimetryassay. Hypcontentin cell supernatant was examined by enzyme digestion assay.Protein expression levels of α-SMA(Alpha-smooth muscle actin), CollagenⅠ,Nrf2（NF-E2-related factor 2） and HO-1 (heme oxygenase-1) were detected by Western blot. One-Way ANOVA was used for comparison among multiple groups. If the variances were homogeneous, LSD test was used; if the variances were heterogeneous, Dunnett T3 test was used. A value of P<0.05 was considered different significantly. Results: Compared with the control group, for the Polydatin 125, 250, 500 μmol/L groups, the cell viability was significantly reduced(F=252.01, P<0.001), the Hyp content was significantly reduced(F=16.50, P<0.05), the protein expression of α-SMA and CollagenⅠwere decreased significantly(F=19.89, 182.91, all P<0.05). And with the increase of the concentration, the inhibitory effect was stronger and in a certain dose-dependent manner. Compared with control group, the protein expression of Nrf2 was increased significantly in Polydatin125, 250, 500 μmol/L groups (F=23.70, P<0.05) and the protein expression of HO-1 was increased significantly in Polydatin 250, 500 μmol/L groups(F=12.40, P<0.01), and with the increase of the concentration, the upregulative effect was stronger and in a certain dose-dependent manner. Conclusion:Polydatin can inhibit hepatic fibrogenesis by inhibiting the collagen synthesis and proliferation and activation of hepatic stellate cells, which may be associated with the upregulation of Nrf2/HO-1 pathway.