下瘀血汤调控TRPV1表达抑制肝星状细胞活化的抗肝纤维化机制

Activation of hepatic stellate cell (HSC) is now well established as a central driver of liver fibrosis occurrence and progression. The transient receptor potential vanilloid type-1 (TRPV1) is closely associated with pathological changes in organ fibrosis. Our previous primary results showed that TRPV1 mainly located in HSC in human liver; meanwhile, the phosphorylation level of p65, one subunit of NF-κB, up-regulated significantly when TRPV1 were knock-down in HSC cells using siRNA transfection in response to LPS stimulation. We also found that TRPV1 was down-regulated and NF-κB p-p65 increased remarkably in CCl4- and BDL-induced fibrotic mice compared with that in the control group, whereas, these pathological changes were reversed significantly by xia-yu-xue decoction (XYXD). Based on these new findings, we could hypothesize that XYXD inhibits liver fibrosis by regulating on TRPV1/NF-κB signal transduction in HSC. For these purposes, we would detect the TRPV1 expression, regulation, physiological function, and interact with sterile alpha and TIR motif-containing protein 1(SARM1) on NF-κB signaling in HSC in response to LPS stimulation using co-immunoprecipitation (Co-IP) and calcium imaging analysis. At the same time, we investigate the regulation effects of XYXD on these signaling transduction. Furthermore, we would explore the role of TRPV1 in liver fibrosis progression in CCl4-and BDL-induced liver fibrosis using western blot and real-time PCR analysis. Simultaneously, we would unravel the therapeutic effect of XYXD on liver fibrosis progression. This study is expected to elucidate the new mechanism and signal pathway in HSC activation which has been rarely reported at present. This project will help us reveal the mechanism of XYXD on liver fibrosis, which provides a theoretical basis for its clinical application.

肝星状细胞（HSC）活化是肝纤维化发生发展的核心事件，瞬时受体电位香草酸亚型1 (transient receptor potential vanilloid 1,TRPV1) 与纤维化病理变化密切相关。本项目前期发现TRPV1在HSC有明确表达，而通过基因沉默TRPV1可诱导NF-κBp65上调；下瘀血汤对肝纤维化过程中TRPV1下调和NF-κBp65上调有显著调控作用；因此，提出下瘀血汤调控TRPV1/NF-κB信号抑制HSC活化的抗肝纤维化机制的假说。拟运用免疫共沉淀及钙成像等技术，研究HSC活化过程中TRPV1的表达以及与无菌α TIR基序包含蛋白1相互作用对NF-κB的调控机制，解析下瘀血汤的干预作用；运用免疫印迹等技术，探析TRPV1对肝纤维化进展的影响与下瘀血汤干预效应。该研究有望从新的视角发现HSC活化的通路，阐释下瘀血汤抗肝纤维化的效应机制，为其临床发展应用提供理论基础。

肝星状细胞（HSC）活化是肝纤维化发生发展的核心事件，下瘀血汤是抗肝纤维化常用方剂，项目负责人发现下瘀血汤调控TRPV1/NF-κB信号转导抑制HSC活化机制。通过临床肝活检石蜡切片样本和冻存组织样本，我们检测人临床肝纤维化基因芯片、肝纤维化过程中TRPV1的mRNA、蛋白及免疫组化表达，采用组化双染TRPV1与LRAT共定位；运用CCl4和BDL模型检测了肝纤维化模型TRPV1表达及下瘀血汤干预效应；人/小鼠原代HSC细胞，采用CoIP技术检测了TRPV1/SARM1结合效应。因此，我们从临床-小鼠-细胞-分子-氨基酸等五个层面上揭示了：1）肝纤维化过程中TRPV1的mRNA表达、蛋白表达显著下调，TRPV1主要分布在肝窦，并与LRAT共染。2）采用Trpv1-/-小鼠和CCl4和BDL模型，与WT小鼠相比，Trpv1-/-肝纤维化显著加重，炎症因子显著上调；过表达TRPV1则显著抑制肝纤维化进展。3）TPRV1主要表达在HSC，并具有生理学功能，在HSC活化后TRPV1表达和生理学功能均显著下调。与WT-HSC细胞相比，Trpv1-/--HSC在LPS刺激后炎症因子显著上调；静止的HSC中SARM1与TRPV1相结合，HSC活化后两者结合能力显著降低。4）下瘀血汤对肝纤维化有显著抑制作用，但对Trpv1-/-肝纤维化无抑制作用，下瘀血汤对WT-HSC炎症因子有显著抑制作用，但对Trpv1-/--HSC无作用；同时下瘀血汤对LPS刺激诱导的TRPV1/SARM1结合能力下降有抑制作用。证实下瘀血汤调控TRPV1表达抑制肝纤维化作用机制。我们还新增了：TRPV1与SARM1相互作用的具体方式：SARM1的TIR亚基的His583在与TRPV1结合发挥关键作用；同时，我们发现TRPV1分子N末端的ARD序列的Ank5-6-E312在与SARM1结合中发挥关键作用。

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