高脂饮食对ApoE敲除小鼠炎症反应及外周血单核细胞斑块内迁徙的影响

Effects of high-fat diet on inflammatory responses of ApoE^-/-mice and migration of peripheral blood monocytes in atherosclerotic plaque

目的比较ApoE敲除小鼠高脂喂养不同时间后,小鼠全身及斑块炎症动态变化,建立荧光标记技术,探讨炎症微环境对外周血单核细胞向主动脉根部斑块迁徙的影响。方法高脂饮食(0周、8周和16周)诱导ApoE敲除小鼠动脉粥样硬化模型,并实验观察。①油红O染色检测斑块形成;②主动脉炎症情况:实时定量PCR检测主动脉相关炎症因子生成及细胞含量;③全身炎症情况:ELISA检测血浆相关炎症因子水平,流式细胞术检测外周血炎症细胞百分比变化;④尾静脉注射PKH26标记单核细胞,激光共聚焦显微镜观察主动脉根部单核细胞迁徙情况。结果随高脂饮食时间的延长,ApoE敲除小鼠主动脉及主动脉根部动脉粥样硬化斑块增多(P<0.01);主动脉炎症反应增高:CCL2、TNF-α及IL-1βmRNA相对表达量高(P<0.01),主动脉组织巨噬细胞增多(P<0.001);伴随全身炎症反应增高:血浆CCL2、TNF-α含量增高(P<0.01)及外周血单核细胞百分比增高(P<0.05),外周血单核细胞向斑块内迁徙增多(P<0.01)。结论高脂饮食推动ApoE敲除小鼠全身及斑块组织炎症反应加重,有助于外周血单核细胞向动脉粥样硬化斑块迁徙。

Objective To investigate the dynamic changes of local and systemic inflammatory responses after high-fat diet feeding of ApoE^-/-mice so as to establish the method to assess the recruitment of PKH26-labeled monocytes into atherosclerotic plaque.Methods ApoE^-/-mice that had received a high-fat diet(HFD)for 8 and 16 weeks were assessed for:①atherosclerotic plaque burden(oil red O staining),②aortic tissue inflammation(by RT-qPCR to detect aortic inflammatory factors and cells),③systemic inflammatory responses(by ELISA to measure plasma proinflammatory cytokine levels and flow cytometry to assess inflammatory cellular profile in the peripheral blood),and④recruitment of PKH26 labeled monocytes into atherosclerotic lesions(by confocal microscope).Results Compared to those in high-fat diet 8 weeks group,ApoE^-/-mice in high-fat diet 16 weeks group had more serious atherosclerosis in the whole aorta and aortic root(P<0.01);significantly up-regulated aortic mRNA levels of inflammatory factors CCL2,TNF-αand IL-1β(P<0.01);an increased number of aortic local macrophages(P<0.001);significantly increased protein expression of plasma CCL2 and TNF-α(P<0.01);and the increased percentage of peripheral blood monocytes(P<0.05).Moreover,the number of PKH26 labeledmonocytes that migrated to aortic lesions were significantly higher in high-fat diet 16 weeks group(P<0.01).Conclusion High-fat diet promotes the severity of systemic and local inflammatory responses in ApoE knockout mice,which contributes to the migration of peripheral blood monocytes to atherosclerotic plaque.