Hypothermia regulates mitophagy and apoptosis via PINK1/Parkin-VDAC 3 signaling pathway during oxygen-glucose deprivation/recovery injury.

Abstract

Post-cardiac arrest brain injury (PCABI), as the main cause of high mortality and long-term disability in patients, induces mitochondrial damage and cell apoptosis. Hypothermia is well-known as an effective neuroprotective therapy, but its underlying mechanisms deserve further exploration. Previous study has demonstrated that hypothermia provides neuroprotection via increasing PINK1/Parkin-mediated mitophagy. However, whether hypothermia can regulate both apoptosis and mitophagy through the PINK1/Parkin-VDAC3 signaling pathway or not. In this study, BV2 mouse microglial cells were cultured under oxygen-glucose deprivation for 6 h following reperfusion with or without hypothermia for 2-4 h. Cell viability was examined by trypan blue stain. Mitophagy was observed by transmission electron microscope. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening were determined respectively by JC-1 staining and BBcellProbe M61 staining using a flow cytometer. Expression of mitophagy-related proteins (Cleaved PINK1, Parkin, SQSTM1/p62, Beclin-1, LC3B II/LC3B I), apoptosis-related proteins (Bcl-2, Cytochrome C, caspase-3, cleaved caspase3) and VDAC3 were assessed using western blot analysis and quantitative real-time PCR. The interaction between Parkin and VDAC3 was confirmed by immunofluorescence colocalization. The results showed that hypothermia alleviated MMP damage, inhibited mPTP opening, then decreased cell apoptosis and activated mitophagy at 2 h after temperature intervention, which might be mediated by the PINK1/Parkin-VDAC3 signaling pathway. Moreover, the effects of hypothermia were reduced or reversed at 4 h after temperature intervention. In conclusion, the potential mechanisms of hypothermia during oxygen-glucose deprivation/recovery could be summarized as follows:1) At 2 h after temperature intervention, hypothermia provided neuroprotective effects via promoting mitophagy and reducing apoptosis through activating the PINK1/Parkin-VDAC3 signaling pathway. 2) The curative effect of hypothermia was timeliness. At 4 h after temperature intervention, hypothermia aggravated apoptosis through inhibiting Parkin recruitment to mitochondria and aggravating the release of Cyt C through open mPTP.