The Role Of Mitochondrial Permeability Transition Pore In Nerve Injury Induced By Sevoflurane Exposure And Its Mechanism

Objective To investigate the role and mechanism of mitochondrial permeability transition pore（MPTP）in sevoflurane-induced injury in hippocampal neurons of SD rat fetuses after 6 h exposure.Methods The fetal rat hippocampus tissue of SD rats on pregnancy d（18±1）was taken,digested and pipetted to make a cell suspension,and then planted in DMEM/F12medium and randomly divided into control group（Con group）and cyclosporine A group（Cs A group）.group),3%sevoflurane exposure group for 6 h（Sevo group）,and cyclosporine A pretreatment+3%sevoflurane group for 6 h（Sevo+Cs A group）.After12 h of cell culture in each group,the medium was changed to Neurobasal medium,and 5-Fluoro-2-deoxyuridine（FUDR）was added to inhibit the growth of glial cells on the third day of neuronal culture.By observing the growth state（d 1,d 3,d 5,d 7,d 9,d 13,d 17,d 21）of the hippocampal neurons under natural growth at different culture time points and detecting the neurons in the natural growth state Viability to determine the time to establish a sevoflurane cell injury model.The sevoflurane cell injury model was established by placing the cells into a control chamber filled with 3%sevoflurane when the cells were cultured to day 7,and then placing the control chamber containing3%sevoflurane into a CO₂cell incubator for treatment.During 6h,the concentration of sevoflurane in the chamber was detected intermittently,and the concentration in the control chamber was kept constant.Cells in Cs A group and Sevo+Cs A group were pretreated with corresponding concentrations of Cs A for 24 h on the sixth day.After 24hours,the Con group and Cs A group were placed in a control chamber filled with 95%空气+5%CO₂,and then placed in a CO₂cell incubator for 6 hours;the Sevo group and the Sevo+Cs A group were placed in a control chamber filled with 3%sevoflurane.The cell viability of neurons in different groups was detected by CCK-8 kit,neuronal apoptosis was detected by TUNEL staining,mitochondrial membrane potential（MMP）was detected by JC-1 kit,MPTP opening degree was detected by Calcein AM staining,and the brain was detected by Western blot.The expression of derived neurotrophic factor（BDNF）,postsynaptic compact protein 95（PSD-95）,Snaptophysin,Snapsin-1,CypD and other proteins.Results Hippocampal neurons grew rapidly from the adherence to the 7th day.On the7th day,the neuron cell body was full and there was a halo around it,and the cell protrusions formed a rich and dense network.The cell viability test showed that the cell viability of hippocampal neurons increased gradually from 1 to 7 days,the cell viability was the highest on the 7th day,and the cell viability gradually decreased from the 8th day.Compared with the Con group,the effect of pretreatment with different concentrations of Cs A（0.1,0.5,1μmol/L）for 24 h on the cell viability in the Cs A group was not statistically significant;compared with the Sevo group,the concentration of Cs A was 0.1μmol Cell protection was the best at/L（P<0.01）.Compared with the Con group,the Sevo group had an increased neuronal apoptosis rate（P<0.01）,decreased synaptic protein expression（P<0.05）,decreased mitochondrial membrane potential（P<0.01）,and increased MPTP opening（P<0.01）.),CypD protein expression increased（P<0.01）;compared with the Sevo group,0.1μmol/L Cs A pretreatment could reduce the apoptosis rate（P<0.01）,increase the synaptic protein expression（P<0.05）,prevent mitochondrial Membrane potential decreased（P<0.05）,decreased MPTP opening（P<0.01）,and decreased CypD protein expression（P<0.01）.Conclusion Sevoflurane exposure can induce neuronal damage in the hippocampus of SD rats,and the mechanism is related to the promotion of CypD-mediated MPTP opening.Cs A,a specific inhibitor of MPTP opening,can reduce sevoflurane-induced apoptosis and synaptic damage in hippocampal neurons,maintain mitochondrial membrane potential,inhibit MPTP opening and downregulate CypD protein expression.