BMAL1通过影响胆汁酸稳态调控血小板活化和血栓形成的机制研究

Studies showed that circadian rhythm disorder can lead to the pathogenesis of thrombotic diseases, and the core clock genes Bmal1 plays a crucial role in maintaining circadian rhythm. But the mechanism by which BMAL1 regulates platelet activation and thrombosis remains to be elucidated. We constructed BMAL1 knockout mice and found that BMAL1 knockout can enhance platelet aggregation. Furthermore, BMAL1 knockout accelerated the iron chloride-induced carotid artery thrombosis in mice. Bile acid homeostasis can be affected by circadian rhythm. Further study found that bile acid can inhibit platelet aggregation and delay carotid artery thrombosis in mice. Accordingly, BMAL1 regulates platelet activation and thrombosis by affecting bile acid homeostasis. We intend to elucidate the mechanism by which BMAL1 regulates the homeostasis of bile acids via detecting the bile acid level and the molecular levels in BMAL1 knockout mice. We played platelet function experiments to clarify the mechanism of bile acid regulation of platelet activation. Carotid artery thrombosis experiments were performed in vivo by adjusting bile acid levels in BMAL1 knockout mice. This project will enhance our understanding of the etiology of thrombotic diseases, thereby providing us with a better understanding of the molecular targets for prevention and therapeutic approaches. Bile acid may serve as an important target molecule for the prevention of thrombotic diseases caused by circadian rhythm disruption.

研究表明，生物节律紊乱可以导致血栓性疾病的发生，核心生物钟基因Bmal1在维持生物节律中起着至关重要的作用。但BMAL1如何调控血小板活化和血栓形成尚有待阐明。为此，我们构建了BMAL1敲除小鼠；初步研究发现，BMAL1敲除可以增强血小板的聚集，并加快氯化铁诱导的小鼠颈动脉血栓形成。由于生物节律紊乱可以影响胆汁酸稳态；进一步研究发现，胆汁酸能抑制血小板聚集，并延缓小鼠颈动脉血栓形成。本项目据此提出，BMAL1通过影响胆汁酸稳态来调控血小板活化和血栓形成。我们拟通过检测BMAL1敲除鼠胆汁酸水平和肝脏中调控胆汁酸稳态的因子水平来阐明BMAL1调节胆汁酸稳态的机制；进而通过各项血小板功能实验重点阐明胆汁酸影响血小板活化的机制；在体内，通过调节BMAL1敲除鼠的胆汁酸水平，经颈动脉血栓实验检测异常血栓形成的改善情况。本项目有望为研发基于机制的抗血小板药物及生物时辰防治血栓性疾病提供科学思路。

血栓性疾病的发生涉及众多因素，其中，血小板的激活是导致血栓性疾病的核心始动环节，因此血小板活化机制的研究和抗血小板药物的应用对治疗血栓性疾病具有重要意义。胆汁酸作为人体的重要代谢物质近来被发现与代谢综合征相关，而通过我们的进一步研究发现胆汁酸对血小板的活化具有抑制作用，其中次级胆汁酸的抑制作用较强。此外通过流式细胞仪检测发现胆汁酸能够抑制胶原引起的血小板p-selectin和JON/A的表达，并且能够抑制胶原与凝血酶引起的血小板ATP释放。通过血小板的粘附实验我们发现胆汁酸可减少血小板在胶原表面的黏附和在固化纤维蛋白原表面的扩展。对于体内血栓形成研究，我们发现胆汁酸能够抑制氯化铁诱导的动脉血栓，而对静脉血栓形成未有影响。进一步深入探索胆汁酸对血小板的抑制作用的分子机制，我们通过蛋白印迹实验发现胆汁酸能够抑制GPVI下游通路分子的磷酸化水平，如PLCγ2, Akt，ERK1/2等蛋白的磷酸化水平。重要的是，通过磷酸化蛋白组学检测发现Syntaxin-11分子的磷酸化水平受到胆汁酸的抑制，且在血小板活化中具有重要作用，此外，蛋白激酶NCK在血小板活化也具有重要意义，这为新型抗血小板药物和血栓性疾病的治疗提供了新的治疗靶点。