龙血素A对大鼠肝星状细胞增殖及Frizzled-4受体蛋白表达的影响

Effect of Loureirin A on Proliferation and Frizzled-4Expression of Rat Hepatic Stellate Cells in vitro

目的研究WNT信号通路中卷曲蛋白-低密度脂蛋白相关蛋白复合受体(Frizzled/LRP)在体外培养活化大鼠肝星状细胞(a HSCs)中的表达及龙血素A(Loureirin A)干预对大鼠肝星状细胞(a HSC)增殖及α-平滑肌肌动蛋白、转化生长因子β1分泌的影响,探讨龙血素A抗肝纤维化的细胞分子机制.方法分离SD大鼠肝星状细胞,体外传代培养诱导细胞活化,倒置相差显微镜下观察肝星状细胞活化前后形态学变化,用不同浓度龙血素A处理活化的大鼠肝星状细胞(a HSCs).MTT法测定龙血素A对肝星状细胞的生长抑制率;Western Blot方法从蛋白水平检测肝星状细胞中Frizzled-4受体蛋白;Elisa测定药物干预后细胞培养上清液中α-SMA和TGFβ1的含量.结果与对照组相比,龙血素A抑制肝星状细胞增殖并有明显的时间-剂量效应关系,IC50=0.30μg/μL;龙血素A在蛋白水平显著下调受体蛋白Frizzled-4表达,同时抑制肝星状细胞分泌α-SMA、TGFβ1(P<0.05).结论 Frizzled-4受体蛋白在活化的大鼠肝星状细胞(a HSCs)中高表达,经龙血素A干预后,Frizzled-4表达量明显降低,细胞增殖受到抑制,α-SMA、TGFβ1合成和分泌量下降,这提示龙血素A可能与WNT信号通路调节肝纤维发生和血管新生的分子机制相关.

Objective To investigate the molecular mechanism of Loureirin A mediated anti-hepatic fibrosis by evaluting its effects on proliferation ,secretion of α-smooth muscle actin (α-SMA) and transforming growth factor-beta1 (TGF-β1),and expression of rat hepatic stellate cells in vitro . Methods Primary hepatic stellate cells were isolated and cultured fromSprague-Dawley rats. After activating and inducing primary hepatic stellate cells from qHSC to aHSC,the activated hepatic stellate cells model in vitro was established. Then we observed the morphological changes of static hepatic stellate cells and activated hepatic stellate cells with inverted phase contrast microscope. Cultured hepatic stellate cells were treated with different concentrations of loureirin A and the inhibitory rate of HSCs proliferation was measured by MTT assay. The expression of Frizzled-4 was measured by western blot analysis. The content of α-SMA and TGF-β1 in the cultured HSCs'supernatant were measured by enzyme-linked immunosorbent assay(ELISA) . Results Loureirin A the proliferation of inhibited activated hepatic stellate cells in a time-dose-dependent manner compared with the control group,IC50=0.30 滋g/滋L. After loureirinA treatment of the HSCs,western blot analysis showed that Frizzled-4 expression level was obviously lower than control group.Loureirin A also inhibited α-SMA and TGFβ1( <0.05)secretion in the cultured HSCs'supernatant in different degree by the assay of ELISA. Conclusions The molecular mechanism of Loureirin A and Wnt signaling pathway mediated anti-hepatic fibrosis and anti-angiogenesis may involve down-regulation the expression of Frizzled-4,inhibiting the synthesis and secretion ofα-SMA,TGF-β1and the proliferation of HSCs.