从TGF-β1/Smads、ERK信号途径探讨山芝麻酸甲酯抗肝纤维化的作用机制

TGF-β1/Smads and ERK pathways can synergistically regulate the processes of cellular biology and play important roles in hepatic fibrosis. The research on both the singnal pathways will be helpful not only to understand the pathogenesis of hepatic fibrosis but also to find the novel anti-fibrotic targets. In our pilot experiments, we have found that methyl helicterate could significantly alleviate hepatic fibrosis in rats. Moreover, the further research showed that methyl helicterate could decrease the levels of TGF-β1 and Smad3, and inhibit the expression and phosphorylation of ERK, indicating that the anti-hepatic fibrosis of methyl helicterate was probably related to regulating and controlling both the TGF-β1/Smads and ERK signal pathways. However, the exact mechanism of methyl helicterate against hepatic fibrosis is unclear. In this study, the effects of methyl helicterate on TGF-β1/Smads and ERK signal pathways in hepatic stellate cells (HSC) will be detected. Furthermore, the cross-talk between TGF-β1/Smads and ERK pathways will be analyzed, and the effect of methyl helicterate on them will also be assessed simultaneously. In addition, the effects of methyl helicterate on the expression of both the signal pathways related genes and proteins in CCl4-induced hapatic fibrosis in rats will be analysed. In short, this study will contribute to expounding the mechanism of methyl helicterate on TGF-β1/Smads and ERK pathways in hepatic fibrosis, and will provid the experimental evidence and theoretical support for the treatment of hepatic fibrosis.

TGF-β1/Smads和ERK通路可协同调节细胞生物学过程，在肝纤维化发生过程中发挥重要作用。针对这两条通路的研究不仅有助于深入了解肝纤维化发病的分子机制，也为肝纤维化的防治研究提供新的干预靶点。我们前期发现山芝麻酸甲酯能明显抑制肝纤维化，进一步研究表明它能降低TGF-β1和Smad3的水平，抑制ERK的表达和磷酸化水平，提示，其抗肝纤维化的作用可能与调控TGF-β1/Smads和ERK通路有关，但机制尚未完全明确。本项目拟用原代HSC，研究山芝麻酸甲酯对这两条信号通路的干预效应；同时，探讨TGF-β1/Smads和ERK 通路的交互作用及山芝麻酸甲酯对其的影响。另外，用CCl4致大鼠肝纤维化模型，进一步了解山芝麻酸甲酯对这两条信号通路相关基因/蛋白表达的影响。本研究将系统阐明山芝麻酸甲酯对TGF-β1/Smads和ERK信号通路的调控作用机制，为其治疗肝纤维化提供实验依据和理论支持。

肝纤维化是慢性肝病发展到一定程度的共同病理特征，其特点是细胞外基质（ECM）的过度沉积。TGF-β1/Smads和ERK信号通路在活化的HSCs中通过调节ECM的合成而在肝纤维化发生机制中发挥重要调控作用，因而是目前药物治疗肝纤维化比较理想的靶点。本项目将从体内、外较全面地阐明山芝麻酸甲酯（MH）对肝纤维化的保护作用及机制。首先，我们采用大鼠肝纤维化模型研究MH对肝纤维化大鼠的保护作用。实验发现，MH能显著减轻大鼠肝损伤和肝纤维化程度。进一步的机制研究表明，MH能减少胶原沉积、抑制NF-κB活化从而减轻炎症反应和氧化损伤、提高肝代谢酶活性、并能抑制miR-21表达继而抑制TGF-β1/Smads和ERK信号通路的活化最终减少ECM的生成和沉积。其次，我们开展了体外细胞实验，即检测了MH对肝星状细胞（大鼠HSC-T6和人LX-2细胞）活化、增殖的作用及其机制。结果表明，MH可明显抑制HSCs增殖、集落形成和细胞迁移，促进细胞凋亡，同时阻滞细胞于G2期。进一步的实验发现，MH可下调Smads和ERK1/2磷酸化水平，提示，MH抑制HSCs活化增殖可能与阻滞TGF-β1/Smads和ERK信号通路有关。另外，实验发现，MH能够激活JNK信号通路以及抑制PI3K/AKT信号通路继而诱导HSCs自噬，同时MH还能降低线粒体膜电位(MMP)，这些结果提示，MH可能通过诱导HSCs的过度自噬以及破坏线粒体功能从而诱导HSCs的大量凋亡。此外，我们采用慢病毒转染技术沉默和过表达miR-21，观察MH对miR-21沉默或过表达时TGF-β1/Smads和ERK信号通路的影响。我们发现，miR-21是TGF-β1/Smads和ERK信号通路相互作用中起重要作用的调节靶点。MH干预后能够显著抑制miR-21介导的TGF-β1/Smads和ERK信号通路，从而抑制HSCs活化增殖、并促进其凋亡。本项目初步证实山芝麻酸甲酯对大鼠肝纤维化有明显的改善作用，其机制可能与调节JNK和PI3K/AKT信号通路诱导细胞自噬继而引起HSCs凋亡、以及抑制miR-21介导的TGF-β1/Smads和ERK信号通路继而抑制ECM的生成和沉积有关。本研究为山芝麻酸甲酯治疗肝纤维化提供了实验依据和理论支持，在慢性肝病基础研究方面显示出较高的学术价值和潜在临床应用价值。