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OGT在甲烷减轻大鼠脊髓神经元氧糖缺失-复氧复糖损伤中的作用:与Nrf2表达的关系 被引量:1

Role of OGT in methane-induced alleviation of oxygen-glucose deprivation and restoration injury to rat spinal cord neurons:relationship with Nrf2 expression
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摘要 目的评价氧连氮-乙酰葡萄糖胺(O-GlcNAc)转移酶(OGT)在甲烷减轻大鼠脊髓神经元氧糖缺失-复氧复糖损伤中的作用及其与Nrf2表达的关系。方法原代培养的大鼠脊髓神经元,以1×105个/ml密度接种于6孔板,采用随机数字表法分为4组(n=60):对照组(C组)、氧糖缺失-复氧复糖组(OGD/R组)、甲烷组(M组)和甲烷+OGT抑制剂四氧嘧啶组(MA组)。采用无糖-无血清Earle平衡盐液,在37℃、5%CO2-95%N2缺氧培养箱中孵育2 h,然后正常培养的方法制备脊髓神经元氧糖缺失-复氧复糖损伤模型。M组于复氧复糖时在培养液中加入200μl甲烷饱和生理盐水(甲烷终浓度1.8 mmol/L);MA组于M组复氧复糖后10 min在培养液中加入8 mmol/L四氧嘧啶。复氧复糖12 h时,测定神经元存活率、LDH漏出率和凋亡率;采用染色质免疫沉淀和实时荧光定量PCR(qPCR)法检测Nrf2基因启动子区OGT和H3K4me3表达水平;提取核蛋白,采用免疫沉淀和Western blot法测定H3K4甲基转移酶复合物调节亚基RBBP5的O-GlcNAc糖基化水平;采用邻位连接法检测H3K4甲基转移酶复合物催化亚基MLL1与组蛋白H3空间邻近水平;采用Western blot和qPCR法测定Nrf2及其mRNA的表达;采用ELISA法测定上清液SOD、过氧化氢酶(CAT)活性和MDA浓度。结果与C组比较,OGD/R组和M组神经元存活率降低,LDH漏出率、凋亡率和上清液MDA浓度升高(P<0.01);与OGD/R组比较,M组神经元存活率升高,LDH漏出率、凋亡率和上清液MDA浓度降低,Nrf2启动子区OGT和H3K4me3表达上调,RBBP5亚基O-GlcNAc糖基化、MLL1亚基与组蛋白H3空间邻近水平增高,Nrf2及其mRNA表达上调,上清液SOD和CAT活性升高(P<0.01);与M组比较,MA组神经元存活率降低,LDH漏出率、凋亡率和上清液MDA浓度升高,Nrf2启动子区OGT和H3K4me3表达下调,RBBP5亚基O-GlcNAc糖基化、MLL1亚基与组蛋白H3空间邻近水平降低,Nrf2及其mRNA表达下调,SOD和CAT活性下降(P<0.05或0.01)。结论OGT参与了甲烷减轻大鼠脊髓神经元氧糖缺失-复氧复糖损伤的过程,与上调Nrf2表达有关。 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 1×105 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%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.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.
作者 王丽萍 薛海燕 郭晓明 陈国忠 徐皓 Wang Liping;Xue Haiyan;Guo Xiaoming;Chen Guozhong;Xu Hao(Department of Anesthesia and Perioperative Medicine,Fuzhou General Clinical Medical College of Fujian Medical University 900th Hospital of Joint Logistics Support Force Dongfang Hospital Affiliated to Xiamen University Fuzhou General Teaching Hospital of Fujian University of Traditional Chinese Medicine Fuzhou General Teaching Hospital of Bengbu Medical College,Fuzhou 350025,China;Department of Orthopaedics,Fuzhou General Clinical Medical College of Fujian Medical University 900th Hospital of Joint Logistics Support Force Dongfang Hospital Affiliated to Xiamen University Fuzhou General Teaching Hospital of Fujian University of Traditional Chinese Medicine Fuzhou General Teaching Hospital of Bengbu Medical College,Fuzhou 350025,China)
出处 《中华麻醉学杂志》 CAS CSCD 北大核心 2020年第3期355-361,共7页 Chinese Journal of Anesthesiology
基金 国家自然科学基金青年科学基金项目(31700740) 福建省科技创新联合资金项目(2017Y9126)。
关键词 乳糖合酶 甲烷 脊髓 神经元 细胞低氧 NF-E2相关因子2 Lactose synthase Methane Spinal cord Neurons Cell hypoxia NF-E2-Related Factor 2
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