P2Y14R对急性痛风性关节炎的调控作用及先导化合物的靶向干预机制研究

Monosodium urate crystal (MSU)-induced pyroptosis of macrophages and NETosis of neutrophils play key roles in the disease course of acute gouty arthritis (GA), nevertheless, the regulatory mechanisms are poorly understood. P2Y14R inhibits cAMP-dependent signaling pathways through coupling Gi/o protein, subsequently participate in immune stress regulation, which is highly expressed in peripheral blood mononuclear cell of GA patients. Previous studies suggested that P2Y14R antagonist could inhibit MSU induced pyroptosis of macrophages and NETosis of neutrophils, which prompted us that P2Y14R was associated with GA, but the mechanisms were not clear. Since cAMP is closey related to signaling pathways involved in pyroptosis of macrophages and NETosis/apoptosis of neutrophils, we put forward a hypothesis that P2Y14R regulates death patterns of inflammatory cells through Gi/o-cAMP pathways in the disease course of GA, which will be confirmed by three perspectives of cells, animals and clinical samples. BPBC is a lead compound inhibited actions first synthesized by our research group as a potent P2Y14R inhibitor, which can attenuate GA in rats. We plan to establish in vivo and in vitro models to reveal the molecular mechanisms by which the lead compound improves GA, that is the lead compound inhibits macrophages pyroptosis and induces switch neutrophils death from NETosis to apoptosis.

急性痛风性关节炎（GA）病程中，尿酸盐结晶（MSU）介导的巨噬细胞焦亡和中性粒细胞NETosis发挥着关键作用，但其调控机制不清。P2Y14R通过偶联Gi/o蛋白抑制cAMP依赖的信号通路参与免疫应激调控，在GA患者外周血单个核细胞中高表达。前期研究显示，P2Y14R拮抗剂可抑制MSU诱导的巨噬细胞焦亡和中性粒细胞NETosis，提示P2Y14R与GA相关，但确切机制不明。基于cAMP与巨噬细胞焦亡和中性粒细胞NETosis/凋亡相关信号通路的联系，申请人提出P2Y14R通过Gi/o-cAMP途径调控炎症细胞死亡方式参与GA病程的假说，拟从细胞、动物和临床样本三个层面进行确证。BPBC是本课题组首次合成的具有P2Y14R抑制活性的先导化合物，能显著缓解大鼠GA。本项目拟构建体内外模型，揭示其靶向P2Y14R抑制巨噬细胞焦亡、诱导中性粒细胞由NETosis向凋亡转换，从而治疗GA的分子机制。

急性痛风性关节炎是由于尿酸盐晶体（MSU）沉积，触发机体固有系统而导致的关节及周围组织的急性炎症反应，缺少理想的治疗药物，故探索急性痛风性关节炎治疗的原创靶标，寻求全新的治疗策略成为了重点。本研究聚焦巨噬细胞和中性粒细胞介导的固有免疫反应在急性痛风性关节炎中的关键作用，提出了通过抑制巨噬细胞焦亡，诱导中性粒细胞由NETosis向凋亡的切换，从而实现缓解炎症和促进炎症消退的思路。基于嘌呤受体家族成员P2Y14受体（P2Y14R）的异常表达与痛风急性发作的临床相关性，本研究重点围绕P2Y14R对固有免疫细胞炎症反应的调控作用，探讨其参与急性痛风性关节炎病程的分子机制。结果显示，痛风急性发作期患者外周血中P2Y14R的表达显著升高，P2Y14R的敲除能够显著抑制MSU导致的大鼠急性痛风性关节炎。具体机制方面，P2Y14R依赖cAMP调控NLRP3炎症小体的活化，从而参与Caspase-1/GSDMD介导的巨噬细胞焦亡；而在中性粒细胞中，P2Y14R依赖cAMP/PKA途径，一方面激活Raf-MEK-ERK通路活化NADPH氧化酶系统，促进中性粒细胞NETs的释放，诱导细胞NETosis，另一方面抑制PI3K-Akt通路，组织细胞凋亡。在证明了P2Y14R可能是急性痛风性关节炎的潜在治疗靶标基础上，本研究一方面对现有唯一的P2Y14R选择性拮抗剂PPTN进行结构修饰以提高成药性，另一方面采用多构象整合预测模型对小分子化合物及天然产物数据库进行筛选，得到了若干高活性、结构新颖的全新P2Y14R拮抗剂，并对其结构进行优化，通过体内外模型的验证，阐明了先导化合物治疗急性痛风性关节炎的分子机制。本研究为急性痛风性关节炎的治疗提供了原创的潜在治疗靶标，同时也为其他急性炎症性疾病的治疗开辟了新的思路；同时，先导化合物的发现也为急性痛风性关节炎及其他P2Y14R相关疾病治疗药物的研发奠定了基础。