阿昔莫司对oxLDL诱导泡沫细胞自噬及AMPK/mTOR通路的影响

Effects of acipimox on oxLDL-induced foam cells autophagy and AMPK/mTOR pathway

目的基于AMPK/mTOR通路探讨阿昔莫司对氧化低密度脂蛋白(oxLDL)诱导泡沫细胞自噬的影响。方法体外培养THP-1细胞株(人单核细胞株),使用佛波醇酯促进其分化形成巨噬细胞,将巨噬细胞分为空白组、对照组、阿昔莫司组、氯喹组、阿昔莫司+氯喹组、Compound C(AMPK抑制剂)组、阿昔莫司+Compound C组。各组均添加对应药物作用于细胞0.5 h,除空白组外,其余组再添加oxLDL培养48 h。使用油红O染色检测细胞内脂滴含量;使用透射电子显微镜观察细胞中自噬体变化;采用免疫印迹法(Western Blot)检测细胞中自噬相关蛋白(LC3Ⅱ/LC3Ⅰ、P62)及AMPK/mTOR通路相关蛋白(p-AMPK/AMPK、p-mTOR/mTOR)水平。结果与空白组比较,对照组细胞红色脂滴含量升高(P<0.05),自噬体数量明显增多。与对照组比较,阿昔莫司组LC3Ⅱ/LC3Ⅰ水平升高,P62水平降低(P<0.05),而氯喹组LC3Ⅱ/LC3Ⅰ水平降低,P62水平升高(P<0.05);与阿昔莫司组比较,阿昔莫司+氯喹组LC3Ⅱ/LC3Ⅰ水平降低,P62水平升高(P<0.05)。与对照组相比,阿昔莫司组p-AMPK/AMPK、LC3Ⅱ/LC3Ⅰ水平升高,p-mTOR/mTOR、P62水平降低(P<0.05),而Compound C组p-AMPK/AMPK、LC3Ⅱ/LC3Ⅰ水平降低,p-mTOR/mTOR、P62水平升高(P<0.05);与阿昔莫司组比较,阿昔莫司+Compound C组p-AMPK/AMPK、LC3Ⅱ/LC3Ⅰ水平降低,p-mTOR/mTOR、P62水平升高(P<0.05)。结论阿昔莫司可以促进oxLDL诱导泡沫细胞自噬过程,且此过程可能与调控AMPK/mTOR通路有关。

Objective To investigate the effects of acipimox on oxLDL-induced foam cells autophagy and AMPK/mTOR pathway.Methods THP-1 cell line(human monocyte cell line)were cultured in vitro,which were promoted to differentiate into macrophages by using phorbol ester.The macrophages were divided into blank group,control group,acipimox group,and chloroquine group,acipimox+chloroquine group,compound C(AMPK inhibitor)group,acipimox+compound C group.The corresponding drugs were added to the cells for 0.5h.Then except for blank group,the other groups were added with oxidized low-density lipoprotein(oxLDL)and cultured for 48h,then oil red O staining was used to detect the content of lipid droplets in cells;transmission electron microscope was used to observe the changes of autophagosomes in cells;Western Blot was used to detect the levels of autophagy-related proteins(LC3Ⅱ/LC3Ⅰ,P62)and AMPK/mTOR pathway related proteins(p-AMPK/AMPK,p-mTOR/mTOR)in cells.Results Compared with those in blank group,the levels of red lipid droplets and the number of autophagosomes in control group were significantly increased(P<0.05).Compared with those in control group,the levels of LC3Ⅱ/LC3Ⅰin acipimox group were significantly increased,however,P62 levels were significantly decreased(P<0.05),and the levels of LC3Ⅱ/LC3Ⅰin chloroquine group were significantly decreased,but P62 levels were significantly increased(P<0.05).Compared with those in acipimox group,the levels of LC3Ⅱ/LC3Ⅰin the acipolimus+chloroquine group were significantly decreased,however,P62 levels were significantly increased(P<0.05).Compared with those in control group,the levels of p-AMPK/AMPK and LC3Ⅱ/LC3Ⅰin acipimox group were significantly increased,while,the levels of p-mTOR/mTOR and P62 were significantly decreased(P<0.05).Moreover the levels of p-AMPK/AMPK and LC3Ⅱ/LC3Ⅰin Compound C group were significantly decreased,however,the levels of p-mTOR/mTOR and P62 were significantly increased(P<0.05).Compared with those in acipolimus group,the levels of p-AMPK/AMPK and LC3Ⅱ/LC3Ⅰin acipolimus+compound C group were significantly decreased,but,the levels of p-mTOR/mTOR and P62 were significantly increased(P<0.05).Conclusion Acipimox can promote the autophagy process of foam cells induced by oxLDL,and its action mechanism may be related to the regulation of AMPK/mTOR pathway.