GPR48调控巨噬细胞炎症免疫反应参与动脉粥样硬化的作用及其机制研究

Mounting evidence suggested that inflammation and immune response play an important role in atherosclerosis. Our previous studies have shown that: 1) By using the exome sequencing, GPR48 active mutation (rs149204548) is independently associated with premature coronary artery disease; 2) GPR48, mainly expressed on macrophages, contributes to atherosclerosis progression; 3) GPR48 induces the expression and release of S100A8/S100A9 via cAMP-PKA-CREB signaling pathway; 4) Previous studies demonstrated that S100A8/S100A9 promotes the differentiation of proinflammatory M1 macrophages through TLR4/RAGE. Thus, based on these findings, we proposed that high cholesterol or shear stress activates GPR48 and promotes the expression and release of S100A8/S100A9 through cAMP-PKA-CREB signaling pathway, which induces the differentiation of M1 macrophages and participates in atherosclerosis progression. To this end, the present study aimed to further explore: 1) the functional role of GPR48 in atherosclerosis and the underlying molecular mechanism, by using GPR48 blocking antibody and macrophage specific GPR48 knockout mice on the background of ApoE knockout; 2) the relationship between GPR48-S100a8/S100a9 expression in circulating monocytes and severity of coronary artery disease. Our findings will provide new therapeutic target for ischemic heart disease.

炎症免疫反应在动脉粥样硬化中起到重要作用。我们研究发现：1）GPR48激活突变（rs149204548）与早发冠心病独立相关；2）GPR48主要表达于巨噬细胞，参与动脉粥样硬化；3）GPR48可能通过cAMP-PKA-CREB信号通路调控S100a8/S100a9表达和释放；4）S100a8/S100a9通过TLR4/RAGE促进促炎性M1巨噬细胞分化。基于以上结果，提出假设：高脂和血流剪切力激活巨噬细胞GPR48，通过cAMP-PKA-CREB信号促进S100a8/S100a9表达和释放，进而诱导M1巨噬细胞分化，参与动脉粥样硬化。为此，本课题将利用GPR48阻断抗体和巨噬细胞特异性敲除GPR48等技术明确GPR48参与动脉粥样硬化的作用和分子免疫机制，并从临床角度阐明GPR48-S100Aa8/S100a9与冠心病严重程度的相关性。为防治缺血性心脏病提供新的治疗策略和干预靶点。

GPR48可影响巨噬细胞极化和中性粒细胞聚集，加重冠状动脉粥样硬化进展。我们通过靶点预测和验证发现，GPR48激活突变与早发冠心病独立相关，然而其具体作用尚不明确。本研究旨在探索GPR48在动脉粥样硬化进展过程中的表达和作用机制。. 研究建立小鼠动脉粥样硬化模型，通过免疫荧光共染色、免疫组化技术和siRNA手段，明确GPR48的表达和调控机制。而后运用GPR48条件性敲除小鼠、RNA-Seq、流式细胞术，慢病毒敲减和重组蛋白过表达等手段，通过细胞培养、流式细胞学技术、qPCR及免疫印迹等方法探究GPR48靶向巨噬细胞S100a8/a9对机体炎症状态、巨噬细胞极化和斑块进展的影响。最后通过外显子测序等方法探究GPR48在早发冠心病人群中的表达水平。. 研究结果显示，GPR48主要表达于巨噬细胞，动脉粥样硬化斑块产生后主动脉组织中GPR48表达水平升高，GPR48敲除可通过抑制S100a8/a9表达，影响巨噬细胞向M1极化和中性粒细胞聚集，减少促炎细胞因子和趋化因子的产生，从而使动脉粥样硬化面积减小、改善斑块进展。同样，人动脉粥样硬化斑块样本也显示GPR48表达升高。外显子测序结果显示GPR48在早发冠心病人群中的激活突变率为3.51%，无冠心病人群为2.46%，差异具有统计学意义。. GPR48靶向S100a8/a9，在动脉粥样硬化进展中发挥了重要的功能，为动脉粥样硬化和冠心病提供了新的防治策略，具有重要的转化医学价值。