垂盆草总黄酮调控DNMTs抑制肝星状细胞活化的抗肝纤维化机制研究

Hepatic fibrosis (HF) is an inevitable part of a chronic liver injury that will develop into cirrhosis,and it is a common disease which is harmful to human health. Regulation of DNA methylation is an important epigenetic regulation mode.When DNMTs in liver tissue abnormally express, it will resulte in TGF-β receptors of DNA hypomethylation, overexpression of sensitive genes (such as TGF-β1,Smad3),induction the activation of HSC, all of which will lead to the occurrence and development of HF. Previous studies have demonstrated that Sedum Sarmentosum Total Flavanones can resist hepatic fibrosis, but its specific mechanism of action remains unclear. Our recent study has found that the drug can increase the levels of liver tissue DNMT3b and TβRⅡDNA methylation, and inhibit HSC activation.Therefore, we hypothesized that its mechanism of prevention and treatment of HF was to regulate DNMTs to inhibit the activation of HSC. The project intends to use HF model in vitro and vivo. By using MSP-PCR,WB and other technologies, we will observe whether the drug can increase DNMTs expression, promote TβR DNA hypermethylation, downstream sensitive gene expression of downstream pathways, and thereby inhibite the activation of HSC; by using 5-azadC to inhibite DNMTs active,it will clearly find out the relevance between regulating DNMTs and inhibiting the activation of HSC. The research will improve the drug's mechanism of HF's treatment, and provide a theoretical basis for the prevention and treatment of HF.

肝纤维化（HF）是各种慢性肝损伤发展成为肝硬化的必经环节，危害人类健康的常见病。DNA甲基化调控是表观遗传学的重要调节方式，肝组织DNMTs表达异常，导致TβR DNA低甲基化，TGF-β1、Smad3等过表达，诱导HSC活化，从而导致HF发生和发展。前期研究证实垂盆草总黄酮具有抗肝纤维化作用，但其具体作用机制仍不清楚。我们最近研究发现该药能提高肝组织DNMT3b表达和TβRⅡDNA甲基化水平，抑制HSC活化。因此，我们推测其防治HF作用机制是调控DNMTs抑制HF过程中HSC活化。本项目拟采用体内外HF模型，通过MSP-PCR、WB等技术，观察该药可否提高DNMTs表达，促进TβR DNA高甲基化，抑制下游通路敏感基因表达，从而抑制HSC活化；利用5-azadC抑制DNMTs活性，明确其调控DNMTs与抑制HSC活化相关性。研究将完善该药治疗HF作用机制，为其应用于HF治疗提供理论依据。

肝纤维化(liver fibrosis)是多种原因引起慢性肝损害所致的病理改变，其形成的核心环节是肝星状细胞（HSC）的活化。近年来的研究发现表观遗传调控在肝纤维化发病过程中起到重要作用，而DNA甲基转移酶(DNMTs)是催化基因发生甲基化必需的酶，同样与肝纤维化活动程度密切相关。前期研究垂盆草总黄酮具有抗CCL4诱导的大鼠肝纤维化作用。项目研究内容包含：1.观察垂盆草总黄酮对大鼠肝纤维化的药物疗效；2.垂盆草总黄酮对肝纤维化大鼠肝组织DNMTs表达、TGF-β/TβR/Smad通路和肝星状细胞活化指标的影响；3.垂盆草总黄酮对HSC-T6细胞活化增殖和凋亡的影响及相关作用机制。研究结果表明垂盆草总黄酮具有改善CCL4诱导的大鼠肝纤维化，抑制HSC-T6的活化增殖并诱导其凋亡，该作用与调控DNMTs表达和TGF-β/TβR/Smad通路，及改变TβRⅡDNA甲基化水平有关。通过本项目研究，将完善垂盆草总黄酮治疗肝纤维化作用机制，为该中药有效成分应用于肝纤维化治疗提供理论依据和肝纤维化的防治提供新的策略和靶点。