葫芦茶苷通过调控Nrf2/HO-1信号通路抑制肝星状细胞增殖活化的作用

Inhibition effects of tadehaginoside on proliferation and activation of hepatic stellate cell by regulating Nrf2/HO-1 signaling pathway

目的分析葫芦茶苷对肝星状细胞(HSC)增殖活化的抑制作用,并基于核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)信号通路探讨其可能的作用机制。方法将人肝星状细胞系LX-2细胞分为空白对照组、模型组和葫芦茶苷不同剂量组。除空白对照组外,其余各组加入含转化生长因子β1(TGF-β1)的培养液使细胞增殖活化;刺激24 h后,葫芦茶苷不同剂量组加入相应剂量的葫芦茶苷干预24 h。检测各组LX-2细胞的增殖情况,细胞上清液Ⅰ型胶原蛋白(ColⅠ)、Ⅲ型胶原蛋白(ColⅢ)和α-平滑肌肌动蛋白(α-SMA)表达水平,以及细胞中α-SMA、ColⅠ、Nrf2、HO-1 mRNA和蛋白的表达水平。结果(1)与空白对照组比较,模型组细胞增殖明显(P<0.05);与模型组比较,葫芦茶苷各剂量组LX-2细胞的增殖均受到抑制,且随着药物剂量的增加抑制效果越明显(均P<0.05)。选取抑制率适宜的剂量20、40、80μg/mL(葫芦茶苷低、中、高剂量组)进行后续实验。(2)与空白对照组比较,模型组细胞上清液ColⅠ、ColⅢ和α-SMA表达水平升高,α-SMA和ColⅠ的mRNA和蛋白表达水平上调,Nrf2和HO-1 mRNA和蛋白表达水平下调(均P<0.05);与模型组比较,葫芦茶苷各剂量组细胞上清液ColⅠ、ColⅢ和α-SMA表达水平降低,α-SMA和ColⅠ的mRNA和蛋白表达水平下调,Nrf2和HO-1 mRNA和蛋白表达水平上调(均P<0.05);葫芦茶苷低、中、高剂量组细胞上清液ColⅠ和α-SMA表达水平、细胞中ColⅠ的mRNA表达水平依次降低,Nrf2和HO-1蛋白表达水平依次升高(均P<0.05)。结论葫芦茶苷可显著抑制HSC的活化与增殖,具有抗肝纤维化的作用,其可能是通过调节Nrf2/HO-1信号途径的表达而发挥作用。

Objective To analyze the inhibition effects of tadehaginoside on proliferation and activation of hepatic stellate cell(HSC),and to explore its possible mechanism of action based on nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)signaling pathway.Methods Human hepatic stellate cell line LX-2 cells were assigned to blank control group,model group,or different dose tadehaginoside groups.Except for the blank control group,the remaining groups were added with culture solution containing transforming growth factorβ1(TGF-β1)for cell proliferation and activation.After stimulation for 24 hours,the different dose tadehaginoside groups were added with corresponding doses of tadehaginoside for 24 hours of intervention.The proliferation states of LX-2 cells,and expression levels of cell supernatant collagen typeⅠ(ColⅠ),collagen typeⅢ(ColⅢ),andα-smooth muscle actin(α-SMA),as well as mRNA and protein expression levels ofα-SMA,ColⅠ,Nrf2,and HO-1 were detected in each group.Results(1)Compared with the blank control group,the model group exhibited significant cell proliferation(P<0.05);compared with the model group,the proliferation of LX-2 cells in the different dose tadehaginoside groups was inhibited,and with the increase of drug dosage,the inhibition effect became more obvious(all P<0.05).The doses of 20,40,and 80μg/mL(in the low-,moderate-,and high-dose tadehaginoside groups)with appropriate inhibition rates were selected to conduct subsequent experiments.(2)Compared with the blank control group,the model group yielded elevated expression levels of cell supernatant ColⅠ,ColⅢ,andα-SMA,up-regulated mRNA and protein expression levels ofα-SMA and ColⅠ,and down-regulated mRNA and protein expression levels of Nrf2 and HO-1(all P<0.05);compared with the model group,different dose tadehaginoside groups interpreted declines in expression levels of cell supernatant ColⅠ,ColⅢ,andα-SMA,down-regulated mRNA and protein expression levels ofα-SMA and ColⅠ,and up-regulated mRNA and protein expression levels of Nrf2 and HO-1(all P<0.05);the expression levels of cell supernatant ColⅠandα-SMA and the mRNA expression level of ColⅠin the cells interpreted in a descending sequence,whereas the protein expression levels of Nrf2 and HO-1 interpreted in an ascending sequence in the low-,moderate-,and high-dose tadehaginoside groups(all P<0.05).Conclusion Tadehaginoside can significantly inhibit the proliferation and activation of HSC,and has an effect against liver fibrosis,which may exert the effects by regulating the expression of Nrf2/HO-1 signaling pathway.