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  

Hydrogen inhibits C/EBP homologous protein-mediated macrophage apoptosis by activating autophagy

作　　者：田华[1] 高聪聪 郑冠琳 姚来彬 焦鹏[1] 杨娜娜[1] 孔雅茹 姚树桐[1,4] 秦树存[1] TIAN Hua;GAO Cong-cong;ZHENG Ouan-lin;YAO Lai-bin;JIAO Peng;YANG Na-na;KONG Ya-ru;YAO Shu-tong;QIN Shu-cun(Institute of Atherosclerosis,Key Laboratory of Atherosclerosis in Universities of Shandong;College of Life Sciences,4 College of Basic Medical Sciences,Taishan Medical University,Taian 271016,China;Taishan Vocational College of Nursing,Taian 271000,China.)

机构地区：[1]泰山医学院动脉粥样硬化研究所,山东省高校动脉粥样硬化重点实验室,山东泰安271016 [2]泰山医学院生命科学学院,山东泰安271016 [3]泰山护理职业学院,山东泰安271000 [4]泰山医学院基础医学院,山东泰安271016

出　　处：《中国病理生理杂志》2018年第8期1368-1375,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81570410;No.81370381);山东省泰山学者岗专项基金资助项目(No.ts201511057);山东省高等学校科技计划项目(No.J16LK55);泰山医学院高层次培养项目(No.2014GCC06;No.2015GCC05)

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.

关 键 词：氢分子 C/EBP同源蛋白 细胞自噬 氧化型低密度脂蛋白 巨噬细胞 细胞凋亡 

分 类 号：R543.5[医药卫生—心血管疾病] R363.2[医药卫生—内科学]