自噬相关基因5在脑缺血再灌注损伤性别差异中的作用及炎性机制研究

Males and females respond differently to stroke, but the mechanism about how stroke affects the 2 sexes in terms of survival and severity remains uncertainly. Our previous studies showed macrophages may be involved in sex differences in experimental stroke, macrophages can exert pro-inflammatory effects after cerebral ischemia reperfusion injury (CIRI) depending on the funtional phenotypes, M1 or M2, to which they are polarized. We previously found interleykin-4 knockout (IL-4 KO) resulted in severe outcome compared to wild type mice, and there is no sex differences in infarct volume and infiltrating immune cells in IL-4 KO mice which may be related with mcrophages polarization and M1/M2 ratio, but the function and the related mechanisms post stroke of M1 and M2 in sex difference still have not been clarified. Recent studies showed autophagy and NLRP3 inflammasome may contribute macrophages polarization and CIRI, our preliminary experiments showed infarction volume was larger and neurological score was greater in macrophage-specific autophagy-related gene 5 knockout mice （Atg5 KO） compared with WT in both sexes, and there is no sex difference in Atg5 KO mice, Atg5 KO mice also showed more M1 polarization tendency in bone marrow derived monocytes (BMDM) stimulated with lipopolysaccharide (LPS) compared with WT mice. Altogether, there should be some internal connecting links between autophagy，NLRP3 inflammasome and macrophages polarizaton in CIRI, but the underlying mechanisms of M1 and M2 regulated by autophagy/NLRP3 inflammasome in sex differences with CIRI still need further clarification. By using flow cytometry, confocal microscopy and Biomarker system, we try to identify the role of autophagy/NLRP3 inflammasome on macrophage polarization in sex diferences with cerebral ischemic injury, our results would provide new methods and potential treatment for the basic research and ischemic cerebrovascular diseases.

免疫炎症反应及巨噬细胞（mФ）在脑缺血再灌注损伤（CIRI）及其性别差异中的作用日益受到重视，脑缺血损伤区mФ来源于中枢小胶质细胞及外周单核细胞，并在不同微环境下极化为经典激活mФ(M1)或替代激活mФ(M2)。我们前期研究显示，CIRI性别差异可能与炎性细胞浸润、巨噬细胞极化及M1/M2比值密切相关，但其机制尚未完全阐明。最近研究显示自噬可调控CIRI后巨噬细胞NLRP3炎症小体活化，我们预实验采用mФ条件性自噬相关基因5（Atg5）敲除小鼠，CIRI模型结果显示Atg5敲除小鼠脑梗塞面积增大、性别差异消失，其可能与调控NLRP3炎症小体活化及mФ极化相关，但Atg5如何调控mФ极化及其在CIRI性别差异中的作用及机制尚未阐明。本项目拟应用流式分选、单细胞多基因分析系统和透射电镜等技术，阐明雌激素、自噬及巨噬细胞极化三者在CIRI性别差异中的相互作用及其信号机制，为脑卒中防治提供新思路。

背景: 自噬在脑缺血/再灌注（I/R）损伤中既具有保护又具有致病作用。在缺血性脑卒中中，自噬和铁死亡之间的内在联系还没有得到明确的研究。本研究的目的是探索自噬相关基因5（ATG5）在实验性缺血性中风中的作用以及在性别差异中的作用。.主要研究内容：注射ATG5 shRNA慢病毒后或者利用ATG5巨噬细胞条件性敲除小鼠，小鼠接受了短暂性大脑中动脉闭塞（MCAO）手术引起的局灶性脑缺血。评估了不同性别小鼠的梗塞体积、神经功能、细胞凋亡、活性氧（ROS）、自噬和铁水平。.重要结果：在MCAO之后，与野生型小鼠相比，ATG5敲低的小鼠的梗塞体积更小，神经功能障碍更少。通过ATG5敲除，缺血小鼠大脑中的细胞凋亡和ROS水平得到了缓解。在MCAO后，通过ATG5敲除，LC3 I/II的表达减少。此外，I/R后beclin1和LC3 II的表达增加，但这种增加被ATG5敲除的预处理抵消了。缺血性中风后，Fe2+和丙二醛（MDA）的水平增加，但它们被ATG5敲除后减少。同样，谷胱甘肽过氧化物酶4（GPX4）和谷胱甘肽（GSH）的表达因I/R而减少，但因ATG5敲除而升高。与雄性 ATG5f/f 相比，雌性小鼠梗塞面积较小。相比同源野生型 ATG5f/f，ATG5f/fLysMcre+小鼠缺血区表达更高的炎症因子 IL-1β、 IL-6、TNFα。动情期雌性小鼠及雌激素替代小鼠梗塞面积缩小，而雌性卵巢切除对照组梗塞 同野生雄性小鼠无差异。.科学意义：本研究表明，ATG5基因敲除可减弱自噬诱导的铁死亡并且在缺血性脑卒中性别差异中具有重要作用，这可能为缺血性中风的治疗提供一种新的潜在方法。