基于TGF-β/Smad和Fas/FADD信号通路探讨白芍总苷减轻肝纤维化的作用机制

The mechanism of action of total glycosides of paeony in attenuating liver fibrosis based on TGF-β/Smad and Fas/FADD signaling pathways

关键词:  白芍  白芍总苷  肝纤维化  凋亡  肝星状细胞

Key words:  Paeoniae Radix Alba  total glycosides of paeony  hepatic fibrosis  apoptosis  hepatic stellate cells

摘要:

目的 基于转化生长因子β（TGF-β）/Smad和肿瘤坏死因子受体超家族成员6（Fas）/Fas相关死亡域蛋白（FADD）信号通路，探讨白芍总苷（TGP）对TGF-β1诱导的活化HSC-LX2细胞纤维化的作用机制。方法 采用10 ng/mL TGF-β1造模药工作液诱导HSC-LX2细胞活化建立肝细胞纤维化模型。用不同浓度的TGP（125、250、500 μg/mL）干预48 h，通过细胞活力检测试剂盒（CCK-8）检测各组细胞存活率，流式细胞仪检测各组细胞凋亡率，酶联免疫吸附法（ELISA）检测炎性因子环氧合酶2（COX2）、白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）含量，蛋白免疫印迹法（Western blot）检测Ⅰ型胶原（COL-Ⅰ）、Ⅲ型胶原（COL-Ⅲ）、α-平滑肌肌动蛋白（α-SMA）的蛋白表达，以此判断模型是否成立及TGP药效。其次，检测金属蛋白酶抑制剂1（TIMP1）和基质金属蛋白酶1（MMP1）蛋白表达情况，更进一步判断细胞外基质沉积程度。随后检测TGF-β1、Smad2、Smad3及Fas、FADD、半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）、半胱氨酸天冬氨酸蛋白酶-8（Caspase-8）mRNA和蛋白表达情况，揭示TGP通过TGF-β/Smad和Fas/FADD信号通路调控肝细胞纤维化的可能机制。结果 TGP干预48 h后，与空白组比较，模型组HSC-LX2细胞活化增殖，COL-Ⅰ、COL-Ⅲ、α-SMA和TIMP1蛋白表达升高，MMP1蛋白表达下降（P＜0.05），表明肝细胞纤维化模型建立。与模型组相比，125、250和500 μg/mL TGP显著抑制活化HSC-LX2细胞活性（P＜0.05），增强细胞凋亡率（P＜0.001），降低COX2、IL-1β和TNF-α含量（P＜0.01或P＜0.001）；250、500 μg/mL TGP降低COL-Ⅰ、TIMP1蛋白表达（P＜0.05），提高MMP1蛋白表达水平（P＜0.05）；500 μg/mL TGP降低COL-Ⅲ、α-SMA蛋白表达（P＜0.05）；各浓度TGP不同程度抑制TGFβ/Smad信号通路中TGF-β1、Smad2、Smad3 mRNA和蛋白表达，刺激Fas/FADD信号通路中Fas、FADD、Caspase-3、Caspase-8 mRNA和蛋白表达（P＜0.05）。结论 TGP诱导活化HSC-LX2细胞纤维化作用机制可能通过抑制TGF-β/Smad信号通路来缓解肝细胞纤维化过程中的胶原纤维积累及细胞外基质沉积，而促使活化HSC-LX2细胞凋亡的机制则可能是通过上调Fas/FADD信号通路来实现的。

Abstract:

Objective To investigate the mechanism of action of total glucosides of paeony(TGP) on TGF-β1-induced fibrosis in activated HSC-LX2 cells based on transforming growth factor-β(TGF-β)/Smad and Fas/Fas-associated with death domain protein(FADD) signaling pathways.Methods Hepatic fibrosis model was established using 10 ng/mL TGF-β1 modeling drug working solution to induce HSC-LX2 cell activation. Cells were intervened with different concentrations of TGP(125, 250, 500 μg/mL) for 48 h. The cell survival rate of each group was detected by cell viability assay kit(CCK-8), the apoptosis rate of each group was detected by flow cytometry, and the inflammatory factors COX2, IL-1β, and TNF-α content were detected by enzyme-linked immunosorbent assay(ELISA), and Collagen I(COL-Ⅰ), Collagen Ⅲ(COL-Ⅲ), and α-smooth muscle actin(α-SMA) protein expressions were detected by protein immunoblotting(Western blot). These are to determine whether the model is valid and TGP efficacy. Secondly, protein expression of tissue inhibitor of metal protease 1(TIMP1) and matrix metallopeptidase 1(MMP1) was detected to determine the degree of extracellular matrix deposition even further. Subsequently, TGF-β1, Smad2, Smad3, Fas, FADD, Caspase-3, and Caspase-8 mRNA and protein expression were detected to reveal the possible mechanisms by which TGP regulates hepatocyte fibrosis through TGF-β/Smad and Fas/FADD signaling pathways.Results After 48 h of TGP intervention, compared with the blank group, HSC-LX2 cells in the model group showed activation and proliferation, elevated COL-Ⅰ, COL-Ⅲ, α-SMA, and TIMP1 protein expression, and decreased MMP1 protein expression(P < 0.05), indicating that the hepatocyte fibrosis model was established. Compared with the model group, 125, 250, and 500 μg/mL TGP significantly inhibited activated HSC-LX2 cell activity(P < 0.05), enhanced apoptosis rate(P < 0.001), and decreased COX2, IL-1β, and TNF-α content(P < 0.01 or P < 0.001); 250 μg/mL and 500 μg/mL TGP decreased COL-Ⅰ and TIMP1 protein expression(P < 0.05) and increased MMP1 protein expression level(P < 0.05); 500 μg/mL TGP decreased COL-Ⅲ and α-SMA protein expression(P < 0.05);various concentrations of TGP inhibited TGF-β1, Smad2, and Smad3 mRNA and protein expression in the TGFβ/Smad signaling pathway and stimulated Fas, FADD, Caspase-3, and Caspase-8 mRNA and protein expression in the Fas/FADD signaling pathway(P < 0.05) to varying degrees.Conclusion The mechanism of TGP-induced fibrotic action in activated HSC-LX2 cells may be through the inhibition of the TGF-β/Smad signaling pathway to alleviate collagen fiber accumulation and extracellular matrix deposition in the process of hepatocyte fibrosis, whereas the mechanism of prompting apoptosis in activated HSC-LX2 cells may be through the up-regulation of the Fas/FADD signaling pathway.