Role of mitochondrial permeability transition pore in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration in rat neurons

Abstract

Objective To evaluate the role of mitochondrial permeability transition pore (mPTP) in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration (OGD/R) in rat neurons. Methods Cortical neurons isolated from neonatal Sprague-Dawley rats born within 24 h, were cultured primarily and seeded in 6-well plates (2 ml/well) at a density of 1×106 cells/ml.The neurons were randomly divided into 5 groups (n=72 each) using a random number table: control group (group C), OGD/R group (group O), mPTP opener group (group A), mPTP opener + sevoflurane group (group AS), and sevoflurane group (group Sev). The cells were cultured in normal culture medium in group C, and the cells were subjected to OGD for 90 min followed by restoration of O2-glucose supply for 24 h in the other groups.In A and AS groups, mPTP opener atractyloside 20 μmol/L was added immediately after OGD.In Sev and AS groups, the cells were post-conditioned with 2% sevoflurane for 1 h immediately after OGD.The neurons were collected at 24 h of OGD in O and A groups, or at 23 h of OGD in Sev and AS groups.Annexin Ⅴ-FITC/PI double staining was performed to count apoptotic neurons, apoptotic rate was calculated, and cell survival rate was measured using MTT assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescence probe as indicator.The opening of mPTP was determined through assessing the changes of mitochondrial optical density (ΔOD540 of mPTP). The expression of pro-apoptotic factors Bid, Bim, Puma protein and mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction and Western blot. Results Compared with group C, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in the other groups.Compared with group O, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in Sev group.Compared with group Sev, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in A and As groups.Compared with group A, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in group AS. Conclusion Sevoflurane mitigates OGD/R-induced apoptosis in rat neurons through inhibiting mPTP opening. Key words: Mitochondrial membrane transport proteins; Anesthetics, inhalation; Reperfusion injury; Neurons; Apoptosis