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

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.

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。

酒精性肝纤维化是酒精性肝病（ALD）的重要中间环节，本研究针对在酒精性肝纤维化形成过程中腺苷A1R和A2AR信号通路调控HSC活化增殖的假设作用机制，在建立酒精性肝纤维化大鼠整体模型及离体HSC模型的基础上，同时建立腺苷A1R和A2AR过表达和表达沉默HSC模型，进一步明确在酒精性肝纤维化大鼠HSC中A1R和A2AR的表达显著增高，且A2AR的表达高于A1R，乙醛可通过激活HSC腺苷A1R和A2AR促进HSC活化增殖。阐明了腺苷A1R和A2AR共同参与调控酒精性肝纤维化中HSC的活化增殖，腺苷A1R和A2AR拮抗剂对酒精性肝纤维化中HSC活化增殖均具有显著的抑制作用，但二者对cAMP-PKA-CREB信号通路具有相反的调节作用，腺苷A2AR介导的cAMP信号通路发挥主导作用。本研究发现在大鼠酒精性肝纤维化HSC中腺苷A1R和A2AR间存在相互作用，腺苷A1R介导的PI通路在乙醛诱导的HSC活化增殖中与cAMP信号通路具有交互作用，且互为正向调节。本研究同时发现非选择性ARs拮抗剂caffeine对酒精性肝纤维化大鼠具有一定的防治作用，并抑制HSC增殖和活化，其机制与其抑制HSC cAMP-PKA-CREB和PLC-DAG-PKC信号通路有关。此外，本研究还发现cAMP/EPAC/Rap1信号通路在乙醛诱导的HSC活化增殖中也具有一定的作用，且EPAC和PKA介导的信号通路可能相互独立。嘌呤P2X7R通过PKC-GSK3β途径促进乙醛诱导的HSC活化，P2X7R抑制剂可能具有防治酒精性肝纤维化的作用。本项目研究成果为酒精性肝纤维化新治疗靶位的筛选提供研究基础，也为如何合理应用ARs调节剂防治酒精性肝纤维化提供新的启示。