基于mTOR信号片仔癀调控肝细胞自噬的肝保护作用机制研究

The mTOR signaling pathway is a key link to initiate autophagy, while the both facts play an important role in many liver diseases. It may be an effective therapeutic strategy treated liver diseases via regulating autophagy by mTOR signaling pathway. Pien Tze Huang (PTH) effectively treated many live diseases. However, its mechanism is unclear. So we propose a scientific hypothesis that the effect of PTH on protecting liver is realized by regulating autophagy via mTOR signaling pathway. in In the present study, we will treat chronic immunological liver injury mice model with PTH, and evaluate the protecting liver via pathological observation and the analysis of liver function. While we will observe the formation of autophagosome via transmission electron microscope and labeling regulated proteins including LC3-B、Beclin-1 by immunohistochemistry. And then, we will analyze the expressions of the upstream and downstream proteins of mTOR signaling pathway, screen and verify autophagy-related gene by PCR array analysis. It is to explore the mechanism of PTH treated chronic immunological liver injury via regulating autophagy by mTOR signaling pathway. Our projects are closely related with the hotspot on researching new drugs to regulate autophagy, and pay attention on the common and refractory disease related with wide people livelihood to study the national protected traditional medicines, furthermore are to expand the research category of pharmacological action of PTH, and supply the scientific mentality and direction to explore and exploit the traditional Chinese medicine and the new drugs treated liver diseases.

mTOR信号是启动细胞自噬的关键环节，二者在各类肝病发病过程中发挥了关键作用，干预mTOR信号调控细胞自噬可能是治疗肝病的有效策略之一。片仔癀(Pien Tze Huang，PTH)可有效治疗各类肝脏疾病，但机制并不明确。故我们提出PTH保肝作用可能是通过干预mTOR信号调控细胞自噬实现的科学假设。本课题采用PTH治疗刀豆蛋白A诱导的小鼠慢性免疫性肝损伤，评价PTH保肝护肝的疗效，同时借透射电镜、免疫组织化学法标记LC3-B、Beclin-1等相关蛋白等方法观察自噬体的形成，并检测mTOR信号上、下游蛋白表达，并通过PCRarray分析法筛选自噬相关基因并验证其表达，旨在从干预mTOR调控细胞自噬探明PTH治疗慢性免疫性肝损伤的作用机制，该课题紧扣研发调控细胞自噬的新药的热点，密切关注影响广大民生的常见病和难治病，研究国家中药保护品种，有助于进一步拓展和完善PTH的药理作用研究范畴。

mTOR信号是启动细胞自噬的关键环节，在各类肝病发病过程中发挥了关键作用，也是其药物作用机制研究热点，提示干预mTOR信号调控细胞自噬可能是治疗肝病的有效策略之一。在本课题中，我们采用刀豆蛋白A（ConA）尾静脉注射诱导的小鼠免疫性肝损伤，经片仔癀治疗后，发现小鼠体重变化率下降明显减缓，肝重及肝重指数明显降低，肝脏组织病理损伤明显缓解；肝功能中ALT,AST,ALP,A/G水平明显好转；同时降低了CAT,MDA,NO和i-NOS的表达水平，升高了SOD和GSH-Pх表达水平，缓解了ConA所诱导的氧化应激损伤，Elisa检测发现肝脏组织中IL-6、IL-12、IL-17和INF-γ的表达水平显著下调，而IL-6、IL-12和TGF-β的表达水平明显上升；透射电镜显示肝脏组织中自噬小体和自噬溶酶体明显增多；同时免疫组化显示Beclin-1的表达明显恢复，表明片仔癀可促进免疫性肝损伤小鼠肝细胞自噬的水平。同时Western blotting显示 mTOR信号上游蛋白ERK1/2，Akt，p-Akt和PI3K表达水平明显下降，而 P53表达水平明显上升；mTOR信号核心蛋白mTOR,p-mTOR,Raptor,Rictor,Rheb水平明显下降，而TSC2,Atg13,p-Atg13水平明显上升；及mTOR信号下游蛋白ULK1，p-ULK1,FIP200,AMBRA1,VPS34,p-VPS34,Beclin-1,p-Beclin-1,LC3B水平显著升高，同时mTOR mRNA,Rictor mRNA表达水平明显下降，而Beclin-1 mRNA，LC3B mRNA和Atg13 mRNA表达水平明显上升，提示片仔癀可通过调控影响ConA诱导的免疫性肝损伤小鼠肝脏组织中mTOR信号活化的表观遗传学相关基因的表达从而抑制mTOR信号活化，进一步提示片仔癀可能通过调控mTOR信号活化的表观遗传学相关基因的表达进而抑制mTOR信号活化而调节细胞自噬而有效治疗免疫性肝损伤。