miRNAs和Hh信号通路在NASH相关肝纤维化中的交互作用及慈菇消脂丸的干预研究

The core of NASH related liver fibrosis is the abnormal activation of hepatic stellate cells (HSC). In recent years, numerous studies have demonstrated that miRNAs and Hh signaling pathways are involved in different stages of liver fibrosis, including HSC activation, proliferation and ECM generation and deposition. But there is less research on the interaction between pathways, the research group proposed "Sagittaria Xiao-Zhi Pill play anti hepatic fibrosis through interaction and regulation of miRNAs and Hh signaling pathways" scientific hypothesis, established animal model of hepatic fibrosis and HSC cell model, and assayed the expression of liver fibrosis related miRNAs, target genes and related factors using Immunohistochemistry method, Western-blot method, Real-time PCR, gene chip and bioinformatics methods. The research group will reveal the pathogenesis of NASH related liver fibrosis at the molecular level, and provide a theoretical basis for the effective components of traditional Chinese medicine in the treatment of NASH related liver fibrosis, and also provides an effective method for the prevention and treatment of NASH related hepatic fibrosis by traditional Chinese medicine, gives full play to the advantages and characteristics of traditional Chinese medicine.

NASH相关性肝纤维化的核心环节为肝星状细胞(HSC)的异常活化。近年来，大量研究已经证明，miRNAs和Hh信号通路在肝纤维化的不同进展阶段都有参与，包括HSC活化，增殖和ECM生成和沉积。但对于通路之间的交互作用研究较少，本课题组提出“慈菇消脂丸通过调控miRNAs和Hh信号通路的交互作用发挥抗肝纤维化”的科学假说，分别建立肝纤维化动物模型和HSC细胞模型，采用免疫组化法、Western-blot法、Real-time PCR法、基因芯片、生物信息学方法等技术对肝纤维化相关miRNAs及靶基因和相关因子进行测定，将从分子水平揭示NASH相关性肝纤维化的发病机制，并为中医药治疗NASH相关性肝纤维化提供理论依据，亦为应用中医药防治NASH相关性肝纤维化提供有效方法，充分发挥中医药的优势和特色。

NASH相关性肝纤维化的核心环节为肝星状细胞（HSC）的异常活化。近年来，大量研究已经证明，miRNAs和Hh信号通路在肝纤维化的不同进展阶段都有参与，包括HSC活化，增殖和ECM生成和沉积。但对于通路之间的交互作用研究较少，本课题组提出“慈菇消脂丸通过调控miRNAs和Hh信号通路的交互作用发挥抗肝纤维化”的科学假说，分别建立肝纤维化动物模型和LX2细胞活化模型，采用免疫组化法、Western-blot法、Real-time PCR法、基因芯片、生物信息学方法等技术对肝纤维化相关miRNAs及靶基因和相关因子进行测定。生物信息学筛选差异miRNAs靶基因，结果显示：与正常组比较，2%含药血清组筛选出63个差异miRNAs，34个基因上调和29个基因下调；4%含药血清组筛选出72个差异miRNAs，37个基因上调和35个基因下调；6%含药血清组筛选出82个差异miRNAs，41个基因上调和41个基因下调。通过GO和KEGG富集分析，发现 Hh信号通路中9个显著差异miRNAs：miR-149-5p、miR-199a-5p、miR-210-3p、miR-222-3p、miR-27b-3p、miR-34a-5p、miR-378a-3p、miR-497-5p、miR-671-5p。进一步通过实验验证miR-378a-3p、miR-199a-5p、miR-34a-5p在NASH相关性肝纤维化中的作用及重要的干预机制。研究结果表明：miR-199a-5p、miR-34a-5p过表达和miR-378a-3p抑制表达均可使LX2细胞活化，Hh信号通路相关蛋白表达上调。中药慈菇消脂丸可以使LX2细胞中miR-199a-5p、miR-34a-5p基因表达下调，miR-378a-3p基因表达上调，从而抑制Hh信号通路相关蛋白表达而发挥抗肝纤维化的作用。