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.