腺苷A1和A2A受体信号通路介导酒精性肝纤维化中HSC活化增殖的作用机制及咖啡因的影响

腺苷A1（A1R）和A2A受体（A2AR）是腺苷受体（ARs）最主要的2种亚型。在酒精性肝纤维化形成过程中，由A1R和A2AR介导的环磷酸腺苷（cAMP）和磷脂酰肌醇（PI）信号通路均可能调控HSC的活化增殖。由于对cAMP依赖途径的相反调节作用，A1R和A2AR调控通路可能具有不同的效应。ARs介导HSC活化增殖的作用是A1R或A2AR某一亚型的直接作用还是两者共同参与?何种信号通路发挥主导作用？是否存在受体后信号交联？尚不清楚。为此，我们拟建立酒精性肝纤维化大鼠模型，分离、培养HSC，采用基因转染、RNAi以及免疫共沉淀等技术以及不同ARs激动剂和拮抗剂，观察A1R和A2AR在HSC中的表达及作用，以及其介导的cAMP和PI信号通路对HSC活化增殖的影响，并探讨两者相互联系以及咖啡因的作用，为进一步阐明乙醇致肝纤维化机制，以及如何合理利用ARs调节剂防治酒精性肝纤维化提供新的思路。

Adenosine receptor A1 (A1R) and receptor A2A (A2AR) are two major receptors of adenosine receptors (ARs). cAMP and PI signaling pathway mediated by A1R and A2AR, respectively, are thought to regulate the activation and proliferation of hepatic stellate cells (HSC) in the progress of alcoholic liver fibrosis. As A1R and A2AR mediated signal has opposite effects on cAMP dependent pathway, we reason that A1R and A2AR mediated signal may has different role during alcoholic liver fibrosis. Until now we are unclear which receptor, A1R, A2AR, or both participates the activation and proliferation of HSC mediated by ARs. What the major downstream signal pathway mediated these effects? cAMP or PI signaling pathway? Is there cross-talking between different pathways after ARs activation? Whether caffeine can improve the natural history of alcoholic liver fibrosis? What's its underlying mechanism? These questions are important issues to address. To answer these questions, we will establish alcoholic liver fibrosis animal model, isolate and culture HSC, use Real Time PCR, Western blot, flow cytometry, gene transfection, RNAi, and co-immunoprecipitation methods and different ARs agonist or antagonist. We will first observe the expression and role of A1R and A2AR in HSC. Then we will investigate the effects of ARs mediated cAMP and PI signal pathway on the activation and proliferation of HSC and confirm which pathway play major role in this progress. We also will investigate the cross-talk between cAMP and PI pathway and the protective role of caffeine. Further, we will elucidate how caffeine inhibit HSC activation and proliferation via A1/A2AR signal. The overall of this project will further precisely demonstrate the mechanisms of alcoholic liver firbrosis. This project will provide new idea for how to use ARs regulators more reasonable for application of alcoholic liver fibrosis therapy.