Effects Of Hydrogen On Mitochondrial Homeostasis And Mitophagy In OGD/R Injured Rat Hippocampal Neurons

Objective:To investigate the effects of hydrogen on mitochondrial homeostasis and mitophagy in oxygen-glucose deprivation/reperfusion injured primary cultured rat hippocampal neurons.Methods:The primary cultured hippocampal neurons,which are stable and mature until about the seventh day,are divided into :1.Control Group(Control Group,cultured under normal environment)2.Oxygen Glucose Deprivation/Reperfusion Group(OGD/R Group,put in oxygen glucose deprivation environment for 2 hours and at the stage of reperfusion cultured in normal environment for 24 hours)3.Oxygen Glucose Deprivation+ Hydrogen Group(OGD/R+H2 Group,put in oxygen glucose deprivation environment for 2 hours and at the stage of reperfusion cultured in a hydrogen-rich incubator for 24 hours)4.Oxygen Glucose Deprivation+ Rapamycin Group(OGD/R+RAP Group,put in oxygen glucose deprivation environment for 2 hours and at the stage of reperfusion,RAP was added into the culture media to a concentration of 200 nmol/L and than cultured in normal environment for 24 hours)5.Oxygen Glucose Deprivation + 3-methyladenine Group(OGD/R+3-MA Group,put in oxygen glucose deprivation environment for 2 hours and at the stage of reperfusion,3-MA was added into the culture media to a concentration of 10 mmol/L and than cultured in normal environment for 24 hours)6.Oxygen Glucose Deprivation + Rapamycin + 3-methyladenine Group(OGD/R+RAP+3MA Group,put in oxygen glucose deprivation environment for 2 hours and at the stage of reperfusion,RAP and 3-MA was added into the culture media to a concentration of 200 nmol/L and 10 mmol/L and than cultured in normal environment for 24 hours),7.Oxygen Glucose Deprivation+Hydrogen + 3-methyladenine Group(OGD/R+H2+3MA Group,put in oxygen glucose deprivation environment for 2 hours,at the stage of reperfusion,media was added 3-MA to a concentration of 10 mmol/L and than cultured in a hydrogen-rich incubator for 24 hour),8.Oxygen Glucose Deprivation+Hydrogen+ Rapamycin Group(OGD/R+H2+RAP Group,put in oxygen glucose deprivation environment for 2 hours,at the stage of reperfusion,media was added RAP to a concentration of 200 nmol/ and than cultured in a hydrogen-rich incubator for 24 hour).MTT assay was used to detect cell viability;flow cytometry was used to detect cell apoptosis;fluorescence microscopy was used to observe the level of reactive oxygen species in each group;JC-10 kit was used to detect the level of MMP in each group;plasmid-transfected cells were constructed to detect the level of mitophagy in each group;The expression of mitophagy-related factors LC3,PINK1 and Parkin were detected by Western blot and RT-PCR.Results: Compared with the Control Group,the survival rate of neurons and the level of MMP in OGD/R group were significantly lower(p < 0.01),the levels of reactive oxygen species,apoptotic cells and mitophagy were significantly higher(p < 0.01),and the expressions of mitophagy-related factors LC3,PINK1 and Parkin were also significantly higher than Control group;Compared with OGD/R group,OGD/R+H2 group and OGD/R+RAP group could increase cell survival rate and MMP level after OGD/R injury(p < 0.01),reduce the level of reactive oxygen species and apoptosis(p< 0.01),and further increase the level of mitophagy and expression of mitophagy-related factors(p < 0.01);On the contrary,the OGD/R+3-MA group further reduced the cell survival rate and MMP level(P<0.01),increased the level of reactive oxygen species and apoptosis(P<0.01),reduced mitophagy level and mitophagy related foctors expression level(P<0.01);In the OGD/R+H2+3-MA group and the OGD/R+RAP+3-MA group,compared with OGD/R+H2 group and OGD/R+RAP group,the addition of 3-MA decreased the cell survival rate and MMP level(P<0.01),increased the level of reactive oxygen species and cell apoptosis(P<0.01),reduced mitophagy level and mitophagy-related factors expression level(P<0.01).Conclusion: Both hydrogen and rapamycin can improve the cell survival rate and MMP level,reduce apoptosis and reactive oxygen levels,induce mitophagy and the expression of mitophagy-related factors,eventually have protective effects on oxygen glucose deprivation/reperfusion injury in hippocampal neurons.And their protective effects can be inhibited by 3-methyladenine.Taken together,our findings demonstrated that H2 had a neuroprotective effect on OGD/R damaged neurons by protecting mitochondrial function and the potential protection mechanism may closely related to enhancement of mitophagy mediated by PINK1/Parkin signaling pathway.