过表达Atg5蛋白对巨噬细胞泡沫化程度的影响
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摘要
目的探讨过表达Atg5蛋白对巨噬细胞泡沫化程度的影响。方法采用Atg5慢病毒和对照组病毒感染Raw264.7巨噬细胞,建立对照细胞系(对照组)和Atg5过表达细胞系(观察组)。两组细胞分别加入50 mL/L ox-LDL诱导形成泡沫化细胞。采用Western blot分析两组细胞蛋白p62、LC3、CD36和SR-A的变化;采用高效液相法检测两组细胞内胆固醇脂(CE)、游离胆固醇(FC)和总胆固醇(TC)的变化;采用免疫荧光和电子显微镜分析两组细胞内脂滴的含量;采用ELISA检测两组细胞培养上清中炎症因子TNF-α、IL-6和IL-1β的水平。结果与对照组比较,观察组细胞自噬底物p62水平显著下调,LC3-II水平显著上调(P<0.05)。与对照组比较,观察组巨噬细胞CE、FC和TC含量均显著下调(P<0.05)。观察组细胞脂质水平和脂滴的数量较对照组明显下降(P<0.05)。与对照组比较,观察组细胞炎症因子TNF-α、IL-6和IL-1β的水平显著下调(P<0.05)。结论自噬核心蛋白Atg5过表达可显著提高巨噬细胞自噬水平,可能通过自噬降解胞内胆固醇等脂质,预防巨噬细胞泡沫化,进而降低动脉粥样硬化的发生。
Aim To investigate the effect of over-expression of Atg5 protein on the degree of macrophage foam.Methods Raw264.7 macrophages were infected with Atg5 lentivirus and control virus. The control cell line( control group) and the Atg5 over-expression cell line( observation group) were established. Two groups of cells were added 50 mL/L ox-LDL( oxidized low-density lipoprotein) respectively to induce the formation of frothy cells. The levels of protein p62,LC3,CD36 and SR-A were analyzed by western blot. The changes of intracellular cholesterol metabolism( cholesterol CE,free cholesterol FC and total cholesterol TC) were detected by HPLC( high performance liquid chromatography).The contents of lipid droplets in the two groups were analyzed by immunofluorescence and electron microscopy. The levels of inflammatory factors including TNF-α,IL-6 and IL-1 beta were detected by ELISA in cell culture supernatants of two groups. Results Compared with the control group,the level of the autophagic substrate p62 was significantly downregulated and the level of LC3-II was significantly up-regulated in the observation group( P< 0.05). Compared with the control group,the contents of CE,FC and TC of macrophages in the observation group significantly decreased( P<0. 05).The level of cell lipid and the number of lipid droplets in the observation group were significantly lower than those in the control group( P<0.05). Compared with the control group,the levels of inflammatory cytokines TNF-α,IL-6 and IL-1 beta in the observation group were significantly down-regulated( P<0.05). Conclusions The over-expression of Atg5,autophagy core protein,can significantly enhance the autophagy level of macrophages. The Atg5 may degrade intracellular cholesterol and other lipids to prevent macrophage froth,thus reducing the occurrence of atherosclerosis.
Aim To investigate the effect of over-expression of Atg5 protein on the degree of macrophage foam.Methods Raw264.7 macrophages were infected with Atg5 lentivirus and control virus. The control cell line( control group) and the Atg5 over-expression cell line( observation group) were established. Two groups of cells were added 50 mL/L ox-LDL( oxidized low-density lipoprotein) respectively to induce the formation of frothy cells. The levels of protein p62,LC3,CD36 and SR-A were analyzed by western blot. The changes of intracellular cholesterol metabolism( cholesterol CE,free cholesterol FC and total cholesterol TC) were detected by HPLC( high performance liquid chromatography).The contents of lipid droplets in the two groups were analyzed by immunofluorescence and electron microscopy. The levels of inflammatory factors including TNF-α,IL-6 and IL-1 beta were detected by ELISA in cell culture supernatants of two groups. Results Compared with the control group,the level of the autophagic substrate p62 was significantly downregulated and the level of LC3-II was significantly up-regulated in the observation group( P< 0.05). Compared with the control group,the contents of CE,FC and TC of macrophages in the observation group significantly decreased( P<0. 05).The level of cell lipid and the number of lipid droplets in the observation group were significantly lower than those in the control group( P<0.05). Compared with the control group,the levels of inflammatory cytokines TNF-α,IL-6 and IL-1 beta in the observation group were significantly down-regulated( P<0.05). Conclusions The over-expression of Atg5,autophagy core protein,can significantly enhance the autophagy level of macrophages. The Atg5 may degrade intracellular cholesterol and other lipids to prevent macrophage froth,thus reducing the occurrence of atherosclerosis.
引文
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[2] Jackson AO,Regine MA,Subrata C,et al.Molecular mechanisms and genetic regulation in atherosclerosis[J].Int J Cardiol Heart Vasc,2018,21(25):36-44.
[3] Sargolzaei J,Chamani E,Kazemi T,et al.The role of adiponectin and adipolin as anti-inflammatory adipokines in the formation of macrophage foam cells and their association with cardiovascular diseases[J].Clin Biochem,2018,54(13):1-10.
[4] Chistiakov DA,Melnichenko AA,Myasoedova VA,et al.Mechanisms of foam cell formation in atherosclerosis[J].J Mol Med (Berl),2017,95(11):1153-1165.
[5] Martinez Lopez N,Tarabra E,Toledo M,et at.System-wide benefits of intermeal fasting by autophagy[J].Cell Metab,2017,26(6):856-871.
[6] Dong S,Jia C,Zhang S,et al.The REGγ proteasome regulates hepatic lipid metabolism through inhibition of autophagy[J].Cell Metab,2013,18(3):380-391.
[7] Komatsu M,Waguri S,Ueno T,et al.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice[J].J Cell Biol,2005,169(3):425-434.
[8] Martinez LN,Singh R.Autophagy and lipid droplets in the liver[J].Annu Rev Nutr,2015,35(3):215-237.
[9] Mizushima N,Yoshimori T,Ohsumi Y.The role of Atg proteins in autophagosome formation[J].Annu Rev Cell Dev Biol,2011,27(11):107-132.
[10] Nakatogawa H,Suzuki K,Kamada Y,et al.Dynamics and diversity in autophagy mechanisms:lessons from yeast[J].Nat Rev Mol Cell Biol,2009,10(7):458-467.
[11] Suzuki K,Kirisako T,Kamada Y,et al.The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation[J].EMBO J,2001,20(21):5971-5981.
[12] Mizushima NL,Yamamoto A,Hatano M,et al.Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells[J].J Cell Biol,2001,152(4):657-668.
[13] Nguyen TB,Olzmann JA.Lipid droplets and lipotoxicity during autophagy[J].Autophagy,2017,13(11):2002-2003.
[14] Chun SK,Lee S,Yang MJ.Exercise-induced autophagy in fatty liver disease[J].Exerc Sport Sci Rev,2017,45(3):181-186.
[15] Singh R,Kaushik S,Wang Y,et al.Autophagy regulates lipid metabolism[J].Nature,2009,458(7242):1131-1135.
[16] Singh V,Rana M,Jain M,et al.Curcuma oil attenuates accelerated atherosclerosis and macrophage foam-cell formation by modulating genes involved in plaque stability,lipid homeostasis and inflammation[J].Br J Nutr,2015,113(1):100-113.