Effect Of Exogenous H₂S On Neuronal Mitochondrial Damage After Ischemia-reperfusion And Its Related Signaling Pathway

Objective:Oxygen glucose deprivation and reperfusion（OGD/R）model constructed from human neuroblastoma cell line（SH-SY5Y）and Vascular dementia model by two-vessel method（2-VO）,to investigate the changes of neuronal oxidative stress,mitophagy and mitochondrial membrane potential.the effects of exogenous H₂S on mitochondrial function and mitophagy and its related signalling pathways in hippocampal neurons of vascular dementia rats.Methods:In vitro experiment:construct Oxygen and glucose deprivation/Reoxygenation（OGD/R）model with SH-SY5Y cells,and control,3/12（glucose deprivation 3 h/Reoxygenation12h）,3/24（glucose deprivation 3 h/Reoxygenation 24h）,6/12（glucose deprivation 6h/Reoxygenation 12h）,6/24（glucose deprivation 6 h/Reoxygenation 24h）,9/12（glucose deprivation 9 h/Reoxygenation 12h）,9/24（glucose deprivation 9h/Reoxygenation 24h）,12/12（glucose deprivation 12 h/Reoxygenation 12h）,12/24（glucose deprivation 12 h/Reoxygenation 24h）nine groups were set up.The autophagosomes in each group were observed by transmission electron microscopy.The control,3/12,3/24,6/12,6/24 groups were selected,the mitochondrial membrane potential and intracellular ROS content were detected by flow cytometry.Western blotting to determine the expression of Beclin1 and P62 in mitochondrial proteins.In vivo experiments:were construced by using 2-vessel occlusion（2-VO）methed,according to the postoperative survival time were set 1 day,7 days,30 days three time periods,each time period has sham Group（Sham）,model group（VaD）,positive control group（Nimodipine）,NaSH low dose group（Low-NaSH）,NaSH high dose group（High-NaSH）five groups,In order to maintain consistent drug treatment time,1 day and 7 days of Nimodipine group nimodipine was administered nimodipine by intragastric administration for 30 days,NaSH low-dose group and NaSH high-dose group were administered with NaSH for 30 days before operation,VaD group and Sham group received normal saline for 30 days before operation;the 30-day group was treated with nimodipine for 30 days,The NaSH low-dose group and the NaSH high-dose group were intragastrically administered with NaSH for 30 days.VaD group and sham group were given normal saline for 30 days.The water maze was used to assess the learning and memory ability of rats in each group for 30 days.The MDA content of brain tissue was detected by TBA method at 1 day,7 days and 30days.The SOD activity of brain tissue was detected by WST-8 method.The expression of Beclin1 and P62 in the mitochondria protein of hippocampal neurons and the expression of Akt,P-Akt,Mtor and P-mTOR in the cytoplasm protein of hippocampal neurons were detected by Western blotting.Results:1.After OGD/R,the mitochondrial membrane potential of SH-SY5Y cells was depolarized,and the intracellular ROS abandance increased.The MDA content in the brain tissue of VaD rats increased and the SOD activity increased in each day.The MDA content and SOD after H₂S treatment.The activity decreased.2.Observable autophagosomes were observed in SH-SY5Y cells of OGD/R group.The expression of Beclin1 was increased in mitochondrial proteins in 3/24,6/12 and 6/24 groups,and the expression of P62 was increased in 3/12 and 3/24 groups.Compared with the OGD/R group,the mitochondrial ROS content was higher in the 24h reperfusion group than in the corresponding 12h group,and the Beclin1 expression was also higher,and the P62expression in the 6/24 group was higher than that in the other groups.The expression of Beclin1 in hippocampal neurons mitochondrial protein of VaD rats in 1 day group decreased,but the expression of P62 did not change significantly.The expression of Beclin1 in hippocampal neurons mitochondrial protein of VaD rats in 7-day group and 30-day group was increased,and the expression level of P62 was consistent with that of Beclin1.3.The learning and memory abilities of VaD rats in 30-day rats was significantly higher than that in Sham group.Lossed,learning and memory ability and navigation ability improved after NaSH treatment.The expression of Beclin1 was decreased in different days after NaSH treatment.There was no significant change in P62 in the 1 day group.The expression of P62 in the 7-day group increased after H2S treatment,and decreased in the 30-day group.The expression of P-Akt and P-mTOR in the cytoplasm of hippocampal neurons of VaD rats decreased in 1 day,7 days and30 days.After NaSH treatment,the expression levels of P-Akt and P-mTOR in rats in1 day group continued to decrease.The NaSH high-dose group was less active than the low-dose group;However,the expression of P-Akt and P-mTOR was increased in the 7-day and 30-day groups.The expression levels of P-Akt and P-mTOR in the high-dose NaSH group were higher than those in the low-dose group.Conclusion:I/R can cause damage to mitochondrial function of nerve cells and induce mitochondrial autophagy.Exogenous H₂S can attenuate the degree of mitochondrial autophagy in hippocampal neurons of VaD rats and protect mitochondrial function,thereby improving learning and memory abilities in VaD rats.Protection might achieved by up-regulating the Akt/mTOR signalling pathway.