PCSK9介导巨噬细胞凋亡在稳定动脉粥样硬化斑块中的作用和机制研究

Low-density lipoprotein (LDL) plays a major role in the formation of atherosclerosis(AS) and plaque instability.Statins is known to decrease the circulating LDL, but the concentration- dependent incrase of Proprotein Convertase Subtilisin/Kexin type 9(PCSK9) caused by statins can directly reduce the effect of LDL reduction. Overexpression of PCSK9 is found to inversely downregulate the low-density lipoprotein receptor (LDLR) and reduce the clearance of plasma cholesterol, so to inhibit PCSK9 overexpression is significant in the preventation and treatment of AS. In pre-study, we find PCSK9 is significantly increased in atherosclerotic plaques, so that we presume PCSK9 could influence the stability of atherosclerotic plaques, but the exact mechanisms by which PCSK9 effects the stability of atherosclerotic plaques are still unclear. In this study, we firstly establish a APOE-/- mouse atherosclerotic model by high-cholesterol diet to observe the influence of PCSK9 inhibitor on atherosclerotic plaques, macrophages, LDLR and Fas/FasL. Then we use conditioned media from human aortic smooth muscle cells to intervent macrophages, combind with PCSK9 siRNA and LDLR siRNA, to make sure the exact mechanisms in PCSK9 affecting plaque's stability. This study result would richen the theory of PCSK9 in atherosclerotic plaques stability and provide a theoretical basis to the anti- atherosclerosis therapy of PCSK9 inhibitor combined with statins lowering lipid.

低密度脂蛋白（LDL）是动脉粥样硬化（AS）形成和斑块不稳定的关键。他汀类药物引起的前蛋白转化酶枯草溶菌酶9（PCSK9）浓度依赖性上调直接减弱了其降LDL的效应，PCSK9表达上调后能负相关的使LDL受体（LDLR）表达下调，减弱对血浆胆固醇的清除，因此抑制PCSK9表达对AS防治有重要意义。在前期工作中，申请者发现PCSK9在AS斑块内表达显著升高，推测PCSK9可能影响斑块稳定性，但具体机制尚待研究。我们采用高脂饲喂APOE-/-小鼠AS模型，观察PCSK9抑制剂对斑块，斑块巨噬细胞、LDLR、Fas/FasL的影响；然后采用人主动脉平滑肌细胞和巨噬细胞，结合PCSK9 siRNA和LDLR siRNA，运用平滑肌细胞培养液干预巨噬细胞，明确PCSK9在影响斑块稳定性的具体机制。本项目完成将丰富PCSK9在AS斑块稳定性方面的理论，为PCSK9抑制剂联合他汀降脂、抗AS提供理论基础。

研究背景：低密度脂蛋白（LDL）是动脉粥样硬化（AS）形成和斑块不稳定的关键。他汀类药物引起的前蛋白转化酶枯草溶菌酶9（PCSK9）浓度依赖性上调直接减弱了其降LDL的效应，PCSK9表达上调后能负相关的使LDL受体（LDLR）表达下调，减弱对血浆胆固醇的清除，因此抑制PCSK9表达对AS防治有重要意义。在前期工作中，申请者发现PCSK9在AS斑块内表达显著升高，推测PCSK9可能影响斑块稳定性，但具体机制尚待研究。研究内容：我们采用高脂饲喂APOE-/-小鼠AS模型，观察PCSK9抑制剂对斑块，斑块巨噬细胞、LDLR、Fas/FasL的影响；然后采用THP-1巨噬细胞进行体外实验，研究PCSK9抑制剂对巨噬细胞增殖、凋亡以及LDLR、Fas/FasL表达的影响。重要结果：动物实验：①GW4064降低APOE-/-小鼠血清TC、LDL-C水平；②GW4064可以延缓AS斑块并减少斑块内巨噬细胞表达；③GW4064可以降低斑块内Fas、Fasl表达；④GW4064抑制AS斑块内PCSK9基因的表达，提高LDLR表达水平。细胞实验：①通过CCK8方法发现GW4064可以增强THP-1源性巨噬细胞活性；②流式细胞仪检测方法发现GW4064能抑制THP-1源性巨噬细胞凋亡；③荧光定量PCR和Western Blot 方法发现GW4064能抑制PCSK9表达，升高LDLR表达，降低Fas、Fasl表达水平。研究结论及科学意义：PCSK9抑制剂具有积极的抗AS及稳定斑块的作用，这一作用与其能抑制斑块内巨噬细胞凋亡有关。本项目的完成明确了PCSK9对AS斑块巨噬细胞凋亡和对斑块稳定性的积极作用，阐明PCSK9通过Fas/FasL凋亡途径促使巨噬细胞凋亡的机制，丰富了PCSK9在影响AS斑块稳定性方面的理论，为PCSK9抑制剂联合他汀降脂、抗AS提供了理论基础。