同型半胱氨酸经PI3K-AKT-mTOR信号通路对巨噬细胞自噬及脂质沉积的影响

The Effect and Mechanism of Homocysteine on Macrophage Autophagy and Lipid Deposition through the PI3K-AKT-mTOR Pathway

目的探讨同型半胱氨酸(Hcy)对巨噬细胞自噬及脂质沉积的影响。方法将18只雄性ApoE-/-小鼠分为对照组、Hcy组、Hcy+Rap组,通过蛋白质免疫印迹检测自噬相关蛋白LC3BⅡ、p62、PI3K-AKT-mTOR信号通路中总蛋白及磷酸化蛋白水平;采用mRFP-GFP-LC3腺病毒感染细胞,观察自噬流的改变;Dil-ox-LDL染色及油红O染色检测Hcy对巨噬细胞内脂质沉积的影响;组织油红O染色观察小鼠主动脉根部斑块的脂质沉积情况;免疫组化检测小鼠主动脉根部斑块中巨噬细胞自噬改变。结果Western blot检测和自噬流实验结果显示,与对照组相比较,给予Hcy干预后,巨噬细胞自噬被抑制,PI3K、AKT、mTOR表达差异均无统计学意义(P均>0.05),但PI3K、AKT、mTOR磷酸化水平均升高(P均<0.05);与Hcy组相比较,Hcy+Rap组LC3BⅡ表达增高(P<0.05),p62表达降低(P<0.01);Dil-ox-LDL染色与油红O染色结果显示,与对照组相比较,Hcy组巨噬细胞内脂质沉积增加(P<0.01),而给予Rap干预后,细胞内脂质沉积减少(P<0.01);与对照组相比较,HMD组小鼠主动脉根部斑块中脂质沉积增多(P<0.01),注射Rap后,脂质沉积减少(P<0.01);免疫组化结果显示,与对照组相比较,HMD组自噬被抑制,注射Rap后,自噬得到恢复。结论Hcy能够抑制巨噬细胞自噬并促进脂质沉积,与PI3K-AKT-mTOR信息通路有关。

Objective To investigate the effects of Hcy on autophagy and lipid deposition in macrophages.Methods The 18 male ApoE-/-mice were divided into control group,Hcy group,and Hcy+Rap group.The levels of autophagy associated proteins LC3BⅡ,p62 and PI3K-AKT-mTOR signaling pathways were detected by Western blot.The cells were infected with mRFP-GFP-LC3 adenovirus and the changes of autophagy flow were observed.The effect of Hcy on lipid deposition in macrophages was detected by Dil-ox-LDL staining and oil red O staining.Lipid deposition in aortic root plaques of mice was observed by oil red O staining.The autophagy changes of macrophages in aortic root plaques in mice were detected by immunohistochemistry.Results Western blot and autophagy flow experiment showed that compared with the control group,autophagy of macrophages was inhibited after Hcy intervention,and the expressions of PI3K,AKT and mTOR were not significantly different(P all>0.05),but the phosphorylation levels of PI3K,AKT and mTOR were significantly increased(P all<0.05).Compared with the Hcy group,LC3BⅡexpression in the Hcy+Rap group was increased(P<0.05)and p62 expression was decreased(P<0.01).Dil-ox-LDL staining and oil red O staining showed that compared with the control group,the intracellular lipid deposition increased in Hcy group(P<0.01),while the intracellular lipid deposition decreased after Rap intervention(P<0.01).Compared with the control group,the lipid deposition in aortic root plaques in HMD group was increased(P<0.01),and the lipid deposition was decreased after Rap injection(P<0.01).Immunohistochemical results showed that compared with the control group,autophagy was inhibited in the HMD group and recovered after Rap injection.Conclusion Hcy can inhibit macrophage autophagy and promote lipid deposition,which is related to PI3K-AKT-mTOR pathway.