Role of OGT in methane-induced alleviation of oxygen-glucose deprivation and restoration injury to rat spinal cord neurons: relationship with Nrf2 expression / 中华麻醉学杂志

ABSTRACT

Objective:To evaluate the role of O-linked-β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) in methane-induced alleviation of oxygen-glucose deprivation and restoration (OGD/R) injury to rat spinal cord neurons and the relationship with Nrf2 expression.Methods:The primarily cultured spinal cord neurons of rats were seeded in 6-well plates at a density of 1×10 5 cells/ml and divided into 6 groups ( n=60 each) using a random number table method: control group (group C), group OGD/R, methane group (group M), and methane plus OGT inhibitor alloxan group (group MA). The medium was replaced with glucose- and serum-free Earle′s salt solution, and the neurons were exposed to 37 ℃ and 5%CO 2-95%N 2 in an incubator for 2 h followed by routine culture to establish the model of OGD/R.In group M, 200 μl methane-saturated saline (final concentration of methane 1.8 mmol/L) was added at oxygen-glucose restoration.Alloxan 8 mmol/L was added at 10 min after oxygen-glucose restoration to inhibit OGT in MA group.At 12 h of oxygen-glucose restoration, the neuronal survival rate, leakage rate of lactic dehydrogenase (LDH) and apoptotic rate of neurons were measured.The expression of OGT and H3K4me3 in Nrf2 promoter was measured by chromatin immunoprecipitation and fluorescent quantitative real-time polymerase chain reaction.Nucleoprotein was extracted for determination of O-GlcNAc glycosylation of RBBP5 (a H3K4 methyltransferase subunit) by immunoprecipitation and Western blot.The spatial proximity of MLL1 subunit of MLL complex to histone H3 was detected by proximity ligation assay.The expression of Nrf2 protein and mRNA was detected by Western blot and quantitative real-time polymerase chain reaction, respectively.The activities of superoxide dismutase (SOD) and catalase (CAT) and malonaldehyde (MDA) concentration were measured by enzyme-linked immunosorbent assay. Results:Compared with group C, the survival rate of neurons was significantly decreased, and the leakage rate of LDH, apoptotic rate and MDA content in supernatant were increased in OGD/R and M groups ( P<0.01). Compared with OGD/R, the survival rate of neurons was significantly increased, the leakage rate of LDH, apoptotic rate and MDA content in supernatant were decreased, the expression of OGT and H3K4me3 in Nrf2 promoter was up-regulated, O-GlcNAc glycosylation and spatial proximity level of MLL1 to histone H3 were increased, the expression of Nrf2 protein and mRNA was up-regulated, and the activities of SOD and CAT were increased in group M ( P<0.01). Compared with group M, the survival rate of neurons was significantly decreased, the leakage rate of LDH, apoptotic rate and MDA concentration were increased, the expression of OGT and H3K4me3 in Nrf2 promoter was down-regulated, O-GlcNAc glycosylation and spatial proximity level of MLL1 to histone H3 were decreased, the expression of Nrf2 protein and mRNA was down-regulated, and activities of SOD and CAT were decreased in group MA ( P<0.05 or 0.01). Conclusion:OGT is involved in methane-induced alleviation of OGD/R injury to rat spinal cord neurons, which is related to up-regulating Nrf2 expression.