GPR137B介导自噬参与动脉粥样硬化发病的机制研究

Atherosclerosis (AS) is an arterial disease associated with dyslipidemia and changes in the composition of the blood vessel wall. Despite the success of treating AS, there is a continuing need to find new therapeutic targets. There are many evidences that autophagy is involved in the formation of AS, providing new clues for the prevention, control, and reversal of AS. Autophagy occurs in endothelial cells, macrophages and smooth muscle cells in human AS plaques, but there is no report of how upstream signaling mediates autophagy in AS. GPR137B, a novel orphan GPCR, shows that it mainly exists in lysosomal membrane by immunofluorescence and Western blotting, suggesting that GPR137B participates in a number of biological functions of lysosome by regulating downstream signaling pathway. Microarray data show that AS mouse GPR137B expression is up-regulated. Our previous qPCR data showed that the expression of GPR137B was up-regulated in the liver and spleen of AS mice. The level of GPR137B gene and protein was significantly increased during inhibition of autophagy.The hypothesis that this gene is involved in the pathogenesis of AS may be directly related to the regulation of lysosomes and autophagosomes through the relevant signaling pathways of the integration, or indirectly affect the formation of autophagosomes to interfere with autophagy activity in order to participate in the pathogenesis of AS, this topic will be verified by experiments to verify the authenticity of the hypothesis.

动脉粥样硬化（AS）是与血脂异常及血管壁成分改变有关的动脉疾病。尽管治疗AS取得成效,还需不断寻找新的治疗靶点。有证据表明自噬参与AS形成，为预防、控制、逆转AS提供新线索。自噬发生在人类AS斑块中的内皮细胞、巨噬细胞和平滑肌细胞中，但上游如何介导自噬参与AS并无相关报道。GPR137B为一种新型孤儿GPCR，主要存在于溶酶体膜上，推测其通过调控下游信号通路，参与溶酶体的多项生物学功能，但无参与自噬报道，也无参与AS发病报道。有基因芯片数据显示，AS小鼠GPR137B表达上调。课题组前期qPCR数据表明，在AS小鼠肝与脾中，GPR137B表达均出现上调，抑制细胞自噬时GPR137B基因与蛋白水平均明显升高。本课题提出该基因参与AS发病的假说，其机制可能是直接通过相关信号通路调控溶酶体与自噬体的融合，或间接影响自噬体的形成来干预自噬活动从而参与AS发病，本课题将通过实验来验证该假说的真实性。

探讨溶酶体膜蛋白受体GPR137B与自噬以及动脉粥样硬化的相关性，进一步探索GPR137B通过参与自噬途径影响动脉粥样硬化发病的可能机制。构建动脉粥样硬化发病小鼠模型。与正常小鼠相比，动脉粥样硬化造模组小鼠肝脏与脾脏组织中的GPR137B基因表达水平均明显升高，GPR137B蛋白水平也呈现明显升高；利用Raw264.7细胞及骨髓原代巨噬细胞BMDM，给予不同浓度的自噬激动剂和抑制剂分别刺激3h、6h、12h。与对照组相比，RAPA处理组和BafA1处理组的GPR137B表达水平均无明显变化；说明改变自噬不影响GPR137B水平，此结果与在HUVEC中的不一致。在293T细胞中过表达GPR137B后，与对照组相比，GPR137B过表达组 LC3B-Ⅱ升高，P62显著降低，酸性溶酶体数量增多，说明自噬流通畅且自噬水平升高，提示GPR137B过表达可促进自噬。GPR137B过表达后给予自噬抑制剂BafA1和CQ，显示GPR137B可以明显解除BafA1和CQ对自噬的抑制作用。与对照组相比，GPR137B过表达组mTOR及其上游PI3K/Akt、Erk1/2信号通路蛋白表达水平均降低；BMDM源性泡沫细胞造模成功后，自噬相关蛋白LC3B-II和P62表达水平上升，同时在24h时GPR137B蛋白表达水平上升。提示GPR137B可通过促进自噬参与泡沫细胞的形成。.ApoE-/-雄鼠喂食高脂饲料8周后继续喂食高脂饲料同时给予OCE（500mg/kg/d）8周，其TC、TG、LDL-C水平呈明显下降趋势，与高脂对照组血脂水平的比较差异均具有统计学意义。.OCE减少主动脉粥样硬化斑块沉积，无论是斑块面积、浸润程度、血管狭窄程度都得到显著改善，与普食组小鼠主动脉相比也较为接近，且呈现明显量效关系。OCE组与高脂对照组比较：在基因水平上，mNr1h3表达增高，mFAS、mSREBP1、mSREBP2、mSR-B1、mHmgcr表达降低。 给予OCE后AMPK、PPAR-γ蛋白水平明显升高，而SREBP1、SREBP2和FAS水平则下降。取肝脏组织做WB检测，发现OCE能降低AS发病后升高的GPR137B的蛋白水平，提示OCE通过降低GPR137B的蛋白水平，抑制自噬从而抑制泡沫细胞的形成来缓解主动脉粥样硬化斑块沉积。