基于DJ-1/GPX4通路探讨加味二至丸对动脉粥样硬化巨噬细胞铁死亡的影响  

Modified Erzhi pill inhibits ferroptosis of macrophages in atherosclerosis via DJ-1/GPX4 pathway

作　　者：马贵萍 陈冉 肖稳康 洪创雄[3] MA Guiping;CHEN Ran;XIAO Wenkang;HONG Chuangxiong(Beijing University of Chinese Medicine Affiliated Shenzhen Hospital,Shenzhen 518172,China;Shanghai University of Traditional Chinese Medicine Affiliated Longhua Hospital,Shanghai 200032,China;The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine,Guangzhou 510405,China)

机构地区：[1]北京中医药大学深圳医院(龙岗),广东深圳518172 [2]上海中医药大学附属龙华医院,上海200032 [3]广州中医药大学第一附属医院,广东广州510405

出　　处：《中国病理生理杂志》2024年第4期627-636,共10页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81373799);广东省中医药强省建设指标体系构建研究(No.20173004)。

摘　　要：目的:探讨加味二至丸(MEP)对动脉粥样硬化(AS)巨噬细胞铁死亡的作用及机制。方法:(1)50只载脂蛋白E基因敲除(ApoE^(-/-))小鼠构建AS模型,随机分为模型组,辛伐他汀组,以及低、中、高剂量MEP组,每组10只,以10只同背景的C57BL/6J为空白对照组。按照分组干预10周后,HE染色观察小鼠主动脉窦病理学改变,RT-qPCR法检测各组小鼠主动脉组织DJ-1和谷胱甘肽过氧化物酶4(GPX4)的mRNA表达。(2)将40只SD大鼠随机分为空白对照组和MEP组,制备MEP含药血清。体外培养RAW264.7小鼠巨噬细胞,将细胞分为空白对照血清组、氧化型低密度脂蛋白(ox-LDL)诱导的模型组、铁死亡抑制剂(ferrostatin-1)组、MEP含药血清组和MEP含药血清+铁死亡诱导剂(erastin)组共5组。MTT实验检测不同浓度MEP含药血清对细胞活力的影响,油红O染色观察各组细胞脂质沉积,ELISA检测各组细胞活性氧(ROS)、丙二醛(MDA)、总超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)及铁离子水平,透射电镜观察各组细胞线粒体结构和形态,RT-qPCR法和Western blot法分别检测检测各组细胞DJ-1、GPX4、溶质载体家族7成员11(SLC7A11)、铁蛋白重链1(FTH1)、p53、前列腺素-内过氧化物合酶2/环加氧酶2(PTGS2/COX2)和NADPH氧化酶1(NOX1)的mRNA和蛋白表达。结果:(1)动物实验结果提示,MEP可以减轻小鼠主动脉窦根部斑块形成,显著升高主动脉DJ-1和GPX4的mRNA表达。(2)细胞实验结果提示,ox-LDL诱导下的RAW264.7细胞脂质沉积显著增多,ROS、MDA和总SOD水平显著升高,GSH水平显著降低,p53、PTGS2/COX2和NOX1的mRNA及蛋白表达显著上调,DJ-1、GPX4、SLC7A11和FTH1的mRNA及蛋白表达显著下调;与ox-LDL组比较,MEP含药血清组细胞脂质沉积显著减少,ROS、MDA和总SOD水平显著降低,GSH水平显著升高,p53、PTGS2/COX2和NOX1的mRNA及蛋白表达显著下调,DJ-1、GPX4、SLC7A11和FTH1的mRNA及蛋白表达显著上调。结论:MEP可以抑制AS巨噬细�AIM:To investigate the effect of modified Erzhi pill(MEP)on macrophage ferroptosis in atherosclerosis(AS),and to explore its mechanisms.METHODS:(1)Fifty apolipoprotein E gene knockout(ApoE^(-/-))mice were used to construct AS model and then randomly divided into model group,simvastatin group,and low-,medium-and high-dose MEP groups,with 10 mice in each group.Ten C57BL/6J mice with the same background were used as blank controls.After 10 weeks of intervention according to the grouping,HE staining was performed to observe the pathological changes of mouse aortic sinus tissues,and RT-qPCR was used to detect DJ-1 and glutathione peroxidase 4(GPX4)mRNA expression.(2)Forty SD rats were randomly divided into blank control group and MEP group to prepare MEP-containing serum.Mouse RAW264.7 macrophages were cultured,and divided into blank control serum group,oxidized low-density lipoprotein(ox-LDL)group,ferroptosis inhibitor(ferrostatin-1)group,MEP-containing serum group and MEP-containing serum+ferroptosis inducer(erastin)group.The effect of MEP-containing serum on cell viability was detected by MTT assay.Oil red O staining was performed to observe lipid deposition in the cells.The levels of reactive oxygen species(ROS),malondialdehyde(MDA),total superoxide dismutase(SOD),glutathione(GSH)and iron ions in the cells were measured by ELISA.Transmission electron microscopy was used to observe the mitochondrial structure and morphology of the cells,and RT-qPCR and Western blot were used to detect the mRNA and protein expression of DJ-1,GPX4,solute carrier family 7 member 11(SLC7A11),ferritin heavy chain 1(FTH1),p53,prostaglandin-endoperoxide synthase 2/cyclooxygenase 2(PTGS2/COX2)and NADPH oxidase 1(NOX1).RESULTS:(1)The MEP alleviated the formation of plaques at the root of the aortic sinus in mice and significantly increased the mRNA expression of DJ-1 and GPX4 in plaques.(2)Cellular lipid deposition was significantly increased by ox-LDL induction.The ROS,MDA and total SOD levels were significantly elevated,whereas the GSH le

关 键 词：加味二至丸 动脉粥样硬化 巨噬细胞 铁死亡 

分 类 号：R543[医药卫生—心血管疾病] R289[医药卫生—内科学] R363.2[医药卫生—临床医学]