氢分子通过激活自噬抑制C/EBP同源蛋白介导的巨噬细胞凋亡
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摘要
目的:研究氢分子对氧化型低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导巨噬细胞凋亡的影响,并探讨可能的分子机制。方法:体外培养鼠源RAW264.7巨噬细胞,处理前更换为饱和含氢培养基,分别给予3-甲基腺嘌呤(3-methyladenine,3-MA;5 mmol/L)和雷帕霉素(rapamycin,Rap;3μmol/L)预处理1 h,再加入ox-LDL(100 mg/L)继续培养24 h。分别采用MTT法和Annexin V-FITC双染法检测细胞活力和凋亡情况,试剂盒测定培养基中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,Western blot法检测自噬标志分子beclin-1和内质网应激相关促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein,CHOP)表达的变化,激光共聚焦显微镜观测细胞内微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达变化。结果:氢分子显著抑制ox-LDL诱导的RAW264.7巨噬细胞活力降低、LDH漏出增加、细胞凋亡及CHOP表达上调;ox-LDL诱导巨噬细胞自噬反应,表现为beclin-1表达上调,LC3颗粒化聚集,而氢分子可进一步促进ox-LDL对细胞自噬的诱导作用,且氢分子的这种促进作用可被自噬抑制剂3-MA拮抗,而被自噬诱导剂Rap增强(P<0.01)。另外,氢分子对ox-LDL所致的巨噬细胞凋亡、细胞活力降低及CHOP上调的抑制作用也可被3-MA拮抗,而被Rap促进。在体外培养的人源THP-1巨噬细胞中也观察到类似的结果,即氢分子不仅可抑制ox-LDL诱导的细胞凋亡和CHOP上调,也可使beclin-1表达进一步上调(P<0.01)。结论:氢分子可通过下调CHOP表达抑制ox-LDL诱导的巨噬细胞凋亡,其上游机制可能是通过激活自噬实现的。
AIM: To investigate the protective effect of hydrogen( H_2) on oxidized low-density lipoprotein( ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H_2-saturated medium was added to murine RAW264. 7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine( 3-MA) and3 μmol/L rapamycin( Rap) for 1 h,and then treated with ox-LDL( 100 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI staining,respectively. The activity of lactate dehydrogenase( LDH) in medium was detected. The protein levels of beclin-1( a molecular marker of autophagy) and C/EBP homologous protein( CHOP,a key signaling component of endoplasmic reticulum stress-associaed apoptosis pathway) were determined by Western blot. Microtubule-associated protein 1 light chain 3( LC3,another molecular marker of autophagy) was observed under laser scanning confocal microscope. RESULTS: Hydrogen attenuated the reduction of cell viability,LDH leakage,apoptosis and CHOP upregulation induced by ox-LDL. Hydrogen promoted ox-LDL-induced autophagy in macrophages as assessed by beclin-1 upregulation,and LC3 granulation,and this promotion effect of hydrogen was inhibited by3-MA( an autophagy inhibitor) and further enhanced by Rap( an autophagy inducer). Moreover,the inhibitory effect of hydrogen on ox-LDL-induced macrophage apoptosis,reduction of cell viability and CHOP upregulation were also blocked by3-MA and enhanced by Rap. Similar results were obtained in human THP-1-derived macrophages,as assessed by the inhibition of ox-LDL-induced apoptosis and CHOP upregulation,and the promotion of beclin-1 expression by hydrogen. CONCLUSION: Hydrogen may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression,and the upstream mechanism may partially involved in the activation of autophagy.
AIM: To investigate the protective effect of hydrogen( H_2) on oxidized low-density lipoprotein( ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H_2-saturated medium was added to murine RAW264. 7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine( 3-MA) and3 μmol/L rapamycin( Rap) for 1 h,and then treated with ox-LDL( 100 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI staining,respectively. The activity of lactate dehydrogenase( LDH) in medium was detected. The protein levels of beclin-1( a molecular marker of autophagy) and C/EBP homologous protein( CHOP,a key signaling component of endoplasmic reticulum stress-associaed apoptosis pathway) were determined by Western blot. Microtubule-associated protein 1 light chain 3( LC3,another molecular marker of autophagy) was observed under laser scanning confocal microscope. RESULTS: Hydrogen attenuated the reduction of cell viability,LDH leakage,apoptosis and CHOP upregulation induced by ox-LDL. Hydrogen promoted ox-LDL-induced autophagy in macrophages as assessed by beclin-1 upregulation,and LC3 granulation,and this promotion effect of hydrogen was inhibited by3-MA( an autophagy inhibitor) and further enhanced by Rap( an autophagy inducer). Moreover,the inhibitory effect of hydrogen on ox-LDL-induced macrophage apoptosis,reduction of cell viability and CHOP upregulation were also blocked by3-MA and enhanced by Rap. Similar results were obtained in human THP-1-derived macrophages,as assessed by the inhibition of ox-LDL-induced apoptosis and CHOP upregulation,and the promotion of beclin-1 expression by hydrogen. CONCLUSION: Hydrogen may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression,and the upstream mechanism may partially involved in the activation of autophagy.
引文
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[22]Duewell P,Kono H,Rayner KJ,et al.NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals[J].Nature,2010,464(7293):1357-1361.
[23]马洪涛,陈红光,董艾莉,等.富氢液激活自噬减轻带状疱疹后神经痛大鼠痛敏并降低炎症因子的水平[J].细胞与分子免疫学杂志,2017,33(2):155-158,163.
[24]Du H,Sheng M,Wu L,et al.Hydrogen-rich saline attenuates acute kidney injury after liver transplantation via activating p53-mediated autophagy[J].Transplantation,2016,100(3):563-570.
[25]陈婷,张燕,田振军,等.氢水对反复力竭运动大鼠海马组织氧化应激损伤及细胞自噬的影响[J].北京体育大学学报,2017,37(7):69-74.
[26]潘震华,赵悦,于宏颖,等.饱和氢盐水对心肌缺血再灌注损伤老年大鼠心肌细胞自噬的影响[J].中华医学杂志,2015,95(25):2022-2026.
[2]Yu X,Wang Y,Zhao W,et al.Toll-like receptor 7 promotes the apoptosis of THP-1-derived macrophages through the CHOP-dependent pathway[J].Int J Mol Med,2014,34(3):886-893.
[3]Thorp E,Li G,Seimon TA,et al.Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe-/-and Ldlr-/-mice lacking CHOP[J].Cell Metab,2009,9(5):474-481.
[4]Yao S,Miao C,Tian H,et al.Endoplasmic reticulum stress promotes macrophage-derived foam cell formation by upregulating cluster of differentiation 36(CD36)expression[J].J Biol Chem,2014,289(7):4032-4042.
[5]Yao S,Tian H,Miao C,et al.D4F alleviates macrophage-derived foam cell apoptosis by inhibiting CD36 expression and ER stress-CHOP pathway[J].J Lipid Res,2015,56(4):836-847.
[6]姚树桐,苗成,刘庆华,等.槲皮素预处理对衣霉素所致巨噬细胞凋亡的抑制作用及机制[J].生理学报,2013,65(1):47-54.
[7]Tian H,Sun HW,Zhang JJ,et al.Ethanol extract of propolis protects macrophages from oxidized low density lipoprotein-induced apoptosis by inhibiting CD36 expression and endoplasmic reticulum stress-C/EBP homologous protein pathway[J].BMC Complement Altern Med,2015,15:230.
[8]Ohno K,Ito M,Ichihara M,et al.Molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases[J].Oxid Med Cell Longev,2012,2012:353152.
[9]田华,姚树桐,高凤美,等.含氢水对实验性小鼠肺纤维化的抑制作用及机制[J].中国现代医学杂志,2012,22(2):29-33.
[10]Song G,Tian H,Liu J,et al.H2 inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factorκB activation in endothelial cells[J].Biotechnol Lett,2011,33(9):1715-1722.
[11]Song G,Tian H,Qin S,et al.Hydrogen decreases atherosusceptibility in apolipoprotein B-containing lipoproteins and aorta of apolipoprotein E knockout mice[J].Atherosclerosis,2012,221(1):55-65.
[12]Song G,Zong C,Zhang Z,et al.Molecular hydrogen stabilizes atherosclerotic plaque in low-density lipoprotein receptor-knockout mice[J].Free Radic Biol Med,2015,87:58-68.
[13]Liu Y,Shoji-Kawata S,Sumpter RM Jr,et al.Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides,starvation,and hypoxiaischemia[J].Proc Natl Acad Sci U S A,2013,110(51):20364-20371.
[14]Sergin I,Razani B.Self-eating in the plaque:what macrophage autophagy reveals about atherosclerosis[J].Trends Endocry Metab,2014,25(5):225-234.
[15]Liao X,Sluimer JC,Wang Y,et al.Macrophage autophagy plays a protective role in advanced atherosclerosis[J].Cell Metab,2012,15(4):545-553.
[16]田华,马守原,康攀攀,等.自噬通过抑制C/EBP同源蛋白表达减轻氧化低密度脂蛋白诱导的巨噬细胞凋亡[J].中国病理生理杂志,2016,32(12):2192-2198.
[17]Cai J,Kang Z,Liu K,et al.Neuroprotective effects of hydrogen saline in neonatal hypoxia-ischemia rat model[J].Brain Res,2009,1256:129-137.
[18]Tsukano H,Gotoh T,Endo M,et al.The endoplasmic reticulum stress-C/EBP homologous protein pathway-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques[J].Arterioscler Thromb Vasc Biol,2010,30(10):1925-1932.
[19]Yang Z,Klionsky DJ.Eaten alive:a history of macroautophagy[J].Nat Cell Biol,2010,12(9):814-822.
[20]Robinet P,Ritchey B,Smith JD.Physiological difference in autophagic flux in macrophages from 2 mouse strains regulates cholesterol ester metabolism[J].Arterioscler Thromb Vasc Biol,2013,33(5):903-910.
[21]Schrijvers DM,De Meyer GR,Martinet W.Autophagy in atherosclerosis:a potential drug target for plaque stabilization[J].Arterioscler Thromb Vasc Biol,2011,31(12):2787-2791.
[22]Duewell P,Kono H,Rayner KJ,et al.NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals[J].Nature,2010,464(7293):1357-1361.
[23]马洪涛,陈红光,董艾莉,等.富氢液激活自噬减轻带状疱疹后神经痛大鼠痛敏并降低炎症因子的水平[J].细胞与分子免疫学杂志,2017,33(2):155-158,163.
[24]Du H,Sheng M,Wu L,et al.Hydrogen-rich saline attenuates acute kidney injury after liver transplantation via activating p53-mediated autophagy[J].Transplantation,2016,100(3):563-570.
[25]陈婷,张燕,田振军,等.氢水对反复力竭运动大鼠海马组织氧化应激损伤及细胞自噬的影响[J].北京体育大学学报,2017,37(7):69-74.
[26]潘震华,赵悦,于宏颖,等.饱和氢盐水对心肌缺血再灌注损伤老年大鼠心肌细胞自噬的影响[J].中华医学杂志,2015,95(25):2022-2026.