Role of TET3-induced DNA demethylation in methane-induced up-regulation of Nrf2 expression in rat spinal cord neurons subjected to oxygen-glucose deprivation and restoration injury
10.3760∕cma.j.issn.0254-1416.2019.04.013
- VernacularTitle:大鼠脊髓神经元氧糖缺失-复氧复糖损伤时TET3诱导DNA去甲基化在甲烷上调Nrf2表达中的作用
- Author:
Liping WANG
1
;
Xiaoming GUO
;
Guozhong CHEN
;
Xiaozhi WU
;
Dongsheng CHEN
;
Hao XU
Author Information
1. 福建医科大学福总临床医学院 联勤保障部队第九〇〇医院 厦门大学附属东方医院 福建中医药大学福总教学医院 蚌埠医学院福总教学医院麻醉与围术期医学科
- Keywords:
Methane;
NF-E2-related factor 2;
Spinal cord;
Reperfusion injury;
Ten-elev-en translocation protein;
DNA demethylation
- From:
Chinese Journal of Anesthesiology
2019;39(4):430-435
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the role of TET3-induced DNA demethylation in methane-in-duced up-regulation of nuclear factor-erythroid 2-related factor 2 ( Nrf2) expression in rat spinal cord neu-rons subjected to oxygen-glucose deprivation and restoration ( OGD∕R) injury. Methods The primarily cultured spinal cord neurons of rats were seeded in 6-well plates at a density of 1×105 cells∕ml and divided into 5 groups ( n=48 each) using a random number table method: control group ( group C) , group OGD∕R, methane group (group M), methane plus TET3-siRNA group (group M+siTET3) and methane plus negative siRNA group (group M+siCon). The medium was replaced with glucose- and serum-free Earle's salt solution, and the neurons were exposed to 5％ CO2-95％N2 in an incubator for 2 h followed by routine culture to establish the model of OGD∕R. In group M, 200μl methane-saturated saline ( final concentration of methane 1. 8 mmol∕L) was added at oxygen-glucose restoration. TET3-siRNA 100 pmol∕L and negative siRNA 100 pmol∕L were added at 24 h before oxygen-glucose restoration to perform transfection in M+siTET3 and M+siCon groups, respectively. At 12 h of oxygen-glucose restoration, the neuronal survival rate, release rate of lactic dehydrogenase ( LDH) and apoptotic rate of neurons were measured, and the ex-pression of TET3 and Nrf2 protein and mRNA was detected by Western blot and fluorescent quantitative re-al-time polymerase chain reaction, respectively, and contents of superoxide dismutase (SOD), catalase ( CAT) and malonaldehyde ( MDA) were measured by enzyme-linked immunosorbent assay. Neuronal DNA was extracted for determination of methylation and hydroxymethylation rates of DNA ( by enzyme-linked im-munosorbent assay) and methylation of CpG island in Nrf2 gene promoter ( by fluorescent real-time methyla-tion specific polymerase chain reaction). Results Compared with group C, the survival rate of neurons was significantly decreased, the release rate of LDH and apoptotic rate were increased in group OGD∕R ( P<0. 01) . Compared with OGD∕R, the survival rate of neurons was significantly increased, the release rate of LDH and apoptotic rate were decreased, the expression of TET3 and Nrf2 protein and mRNA was up-regula-ted, DNA hydroxymethylation rate and contents of SOD and CAT were increased, and the DNA and Nrf2 promoter methylation rates and MDA content were decreased in group M ( P<0. 05 or 0. 01) . Compared with group M, the neuronal survival rate was significantly decreased, the release rate of LDH and apoptotic rate were increased, the expression of TET3 and Nrf2 protein and mRNA was down-regulated, the DNA hydroxymethylation rate and contents of SOD and CAT were decreased, and the DNA and Nrf2 promoter methylation rates and MDA content were increased in group M+siTET3 ( P<0. 05 or 0. 01) , and no signifi-cant change was found in the parameters mentioned above in group M+siCon ( P>0. 05) . Conclusion The mechanism by which methane up-regulates Nrf2 expression in rat spinal cord neurons subjected to OGD∕R injury is related to activating TET3 and promoting DNA demethylation in Nrf2 promoter.
