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

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(P0.01)。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(P0.01)。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。

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

AIM: To investigate the protective effect of hydrogen (H₂) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H₂-saturated medium was added to murine RAW264.7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine (3-MA) and 3 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 by 3-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 by 3-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.