PM2.5通过激活巨噬细胞内质网应激进而影响ApoE^(-/-)小鼠动脉粥样硬化斑块

PM2.5 affects atherosclerotic plaques in ApoE^(-/-)mice by activating macrophage ER stress

目的探讨细颗粒物(PM2.5)通过激活巨噬细胞内质网应激促进巨噬细胞细胞质内Ca^(2+)的积累,进而影响ApoE^(-/-)小鼠动脉粥样硬化(AS)斑块的形成。方法体内实验:8周龄ApoE^(-/-)小鼠共计24只,随机分为对照组和PM2.5组,每组12只。采用高脂饲料喂养8周后,对照组小鼠气管内滴注生理盐水,PM2.5组小鼠气管内滴注PM2.5的生理盐水混悬液(30 mg/kg/d)。8周后主动脉根部石蜡切片行movat染色、DHE法检测氧自由基(ROS)水平;采用TUNEL法对斑块内细胞凋亡情况进行检测;Western Blot法检测内质网应激下游炎症因子IL-6、TNF-α、MCP-1蛋白的表达量。体外实验:分离培养小鼠原代腹腔巨噬细胞后,将细胞分为对照组(无刺激)、LPS组(模型组,LPS 100ng/mL,24 h)、LPS+PM2.5组(24 h)。采用钙离子荧光探针Fura-2 AM检测巨噬细胞内Ca^(2+)的积累量,Western Blot法检测巨噬细胞内IL-6、TNF-α、MCP-1蛋白的表达量。结果体内实验PM2.5组小鼠AS斑块面积明显增大,ROS水平显著增高(P<0.001);斑块内坏死凋亡细胞数量明显增多,内质网应激下游炎症因子IL-6、TNF-α、MCP-1蛋白表达量升高(P<0.001);体外实验LPS+PM2.5组与LPS组比较,细胞内Ca^(2+)积累量明显增多(P<0.001);IL-6、TNF-α、MCP-1蛋白的表达量均升高(P<0.001)。结论经PM2.5处理的高脂饲养的ApoE^(-/-)小鼠,PM2.5可作用于巨噬细胞,激活其内质网应激,导致巨噬细胞细胞质中Ca^(2+)积累增加,使氧自由基的产生与斑块内细胞坏死凋亡数量增加,对动脉粥样硬化斑块的发生发展起到促进作用。

Objective Particulate matter 2.5(PM2.5)promotes the accumulation of Ca^(2+)in the cytoplasm of macrophages by activating endoplasmic reticulum stress in macrophages,which in turn affects the study of atherosclerotic plaques in ApoE^(-/-)mice.Methods In vivo experiments Twenty-four 8-week-old ApoE^(-/-)mice were randomly divided into control group(n=12)and PM2.5 group(n=12)and fed with high-fat diet for 8 weeks,in which the control group was given normal saline by tracheal instillation and the PM2.5 group was given PM2.5 normal saline suspension by tracheal instillation(30 mg/kg/d).After 8 weeks,paraffin sections of aortic roots were stained with movat,and the levels of oxygen free radicals(ROS)were detected by DHE.The apoptosis of cells in plaques was detected by TUNEL.The expression of inflammatory factors IL-6,TNF-α and MCP-1 downstream of endoplasmic reticulum stress was detected by Western Blot.After isolation and culture of mouse primary peritoneal macrophages in vitro,the cells were divided into:1.Control group:no stimulation,2.Model group:LPS 100ng/mL(24h),3.LPS+PM2.5(24h).Results In vivo experiments showed that the area of atherosclerotic plaque was significantly increased and ROS level was significantly increased in PM2.5 group(P<0.001).The number of necrotic and apoptotic cells in the plaque was significantly increased,and the expression of inflammatory factors IL-6,TNF-α,MCP-1 protein downstream of endoplasmic reticulum stress was increased(P<0.001).In vitro experiment,compared with LPS group,the accumulation of intracellular Ca^(2+)was significantly increased in LPS+PM2.5 group(P<0.001),and the expression of IL-6,TNF-α,MCP-1 protein was increased(P<0.001).Conclusion In PM2.5-treated ApoE^(-/-)mice fed with high-fat diet,PM2.5 can act on macrophages and activate their endoplasmic reticulum stress,leading to increased accumulation of Ca^(2+)in the cytoplasm of macrophages,increasing the production of oxygen free radicals and the number of cell necrosis and apoptosis in plaques,thus promoting the progression of atherosclerotic plaques.