基于肝星状细胞自噬及调控通路探讨片仔癀抗肝纤维化的作用机制研究

Activation of hepatic stellate cells (HSC) is the key for the development of various types of chronic liver injury to liver fibrosis. In addition, autophagy can regulate HSC activation, so it becomes the important target for anti-fibrosis research. Pien Tze Huang has a protective effect on chronic liver injury. Our previous studies have shown that Pien Tze Huang can reduce hepatic fibrosis in rats induced by carbon tetrachloride, inhibit the proliferation of HSC and down-regulate the expression of miR-155 in vitro. This study intends to further from animal and cell experiment level, using transmission electron microscopy and immunofluorescence techniques to observe the effect of Pien Tze Huang on HSC activation and cell autophagy in vivo and in vitro, and using immunohistochemistry, Western blot, real-time PCR and other methods to observe the regulatory effect of Pien Tze Huang on the expression of miR-155 and key signaling molecules in downstream mTOR/autophagy signaling pathway. Then construction of miR-155 overexpression and knockdown lentiviral expression vector to establish HSC cell lines of high and low miR-155 expression is used to reverse verification, and then to validate the targets and regulation mechanism of Pien Tze Huang. The study will establish a theoretic and experimental foundations for using Pien Tze Huang against liver fibrosis.

肝星状细胞（HSC）活化是各种类型慢性肝损伤发展为肝纤维化的关键环节，而细胞自噬能够调控HSC活化，被认为是抗肝纤维化研究的重要靶点。片仔癀临床上治疗慢性肝损伤疗效明确。前期研究表明片仔癀可明显减轻CCl4肝纤维化模型大鼠肝组织的纤维化；在体外可抑制HSC的增殖活性，并下调miR-155的表达量。本项目拟进一步从动物和细胞实验水平，采用透射电镜、免疫荧光等技术观察片仔癀对体内外HSC活化、细胞自噬的影响，并用免疫组织化学染色、western blot、real-time PCR等方法观察片仔癀对miR-155及下游mTOR/自噬信号通路上关键信号分子表达的调控作用，并构建过表达和抑制表达miR-155的慢病毒载体，建立高表达和低表达miR-155的HSC细胞株进行反向验证，进而探讨片仔癀治疗肝纤维化的分子靶点及调控机制，为片仔癀临床用于抗肝纤维化治疗奠定理论和实验基础。

片仔癀为国家一级中药保护品种，主要由三七、麝香、牛黄、蛇胆等中药组成，在临床上消炎、保肝等疗效确切。前期研究表明，片仔癀可减轻四氯化碳诱导的大鼠肝纤维化，并可明显抑制体外培养的肝星状细胞（HSC）的增殖力，下调细胞内微小RNA-155（miR-155）的表达量。本项目在此基础上，以体内与体外实验研究相结合发现片仔癀能够减轻四氯化碳诱导的肝损伤，改善肝功能，减少肝内细胞外基质（ECM）及胶原的沉积，并且下调HSC活化标志物-SMA、Col-I以及自噬蛋白LC3、Beclin-1的表达，提示其抗纤维化的作用机制与调控HSC细胞自噬和活化相关，进一步从miRNA、mRNA、蛋白质等多个层次揭示片仔癀对细胞凋亡和自噬相关信号通路，包括miR-155/mTOR、TGF-β/Smads以及NF-kB信号通路的调控机制。结果显示片仔癀具有促进HSC细胞凋亡，其作用机制可能与调控Bcl-2、Bax、p-Bad、caspase-3的表达相关。片仔癀还具有调控miR-155、mTOR、RHEB、RICTOR和RPS6KB2、TGF-β、Smads、NF-kB、IkB-a和IkK-β等多个自噬相关信号通路上的关键分子靶点的作用，初步阐明了其抗纤维化的作用机制。本项目在中医“整体观念” 理论指导下采用现代生命科学的技术手段揭示了复方中药多成分、多途径、多靶点的特点。尽管研究仍有待进一步深入，但已获得的研究结果将为片仔癀在临床上更广泛和安全地应用提供科学证据。