氯胺酮通过增强星形胶质细胞KAT6B基因表达提高抗氧化应激水平  

Ketamine enhances the ability of antioxidative stress through increasing the expression of KAT6B gene in astrocytes

作　　者：刘行 陈嘉鑫 冯建国 贾静 王晓斌 LIU Xing;CHEN Jiaxin;FENG Jianguo;JIA Jing;WANG Xiaobin(Department of Anesthesiology,Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学附属医院麻醉科,泸州646000

出　　处：《山西医科大学学报》2020年第8期801-806,共6页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(81271478);四川省科技计划项目-面上项目(应用基础研究项目)(20YYJC2046)。

摘　　要：目的探讨氯胺酮提高星形胶质细胞抗氧化应激水平的作用及分子机制。方法氯胺酮(50μmol/L)处理人星形胶质细胞(HA1800)后,分别使用比色法、WST-1法及钼酸胺法检测谷胱甘肽过氧化物酶(GSH-Px)、总超氧化物歧化酶(T-SOD)和过氧化氢酶(CAT)活力等氧化应激指标的改变。Western blot检测转录因子E2相关因子2(Nrf2)、蛋白组乙酰化和KAT6B蛋白水平。免疫荧光染色检测乙酰化水平和Nrf2在星形胶质细胞中的表达及定位。RT-PCR检测氯胺酮对KAT6B基因的mRNA和circRNA表达的影响。KAT6B基因siRNA转染沉默基因表达,复合氯胺酮处理,检测Nrf2、蛋白质乙酰化、GSH-Px、T-SOD和CAT水平。结果50μmol/L氯胺酮处理12 h可显著提高HA1800细胞GSH-Px、T-SOD和CAT活力(P<0.05)。50μmol/L氯胺酮处理3,6,12,24 h,HA800细胞内Nrf2、乙酰化和KAT6B的表达均明显增加(P<0.05),且以12 h最明显。免疫荧光结果显示,与0μmol/L比较,50μmol/L氯胺酮处理12 h,HA800细胞内乙酰化表达明显增加(P<0.05),且Nrf2蛋白向细胞核内转移。RT-PCR结果显示,50μmol/L氯胺酮处理HA1800细胞12 h,KAT6B mRNA的表达明显增加(P<0.05),KAT6B circRNA表达明显降低(P<0.05)。siRNA沉默KAT6B实验结果发现,沉默KAT6B基因表达可显著抑制氯胺酮提高星形胶质细胞氧化应激水平的作用,显著降低KAT6B、Nrf2和乙酰化蛋白表达(P<0.05),以及T-SOD、CAT活力(P<0.05)。结论氯胺酮可通过降低circKAT6B水平、增强KAT6B基因表达、促进Nrf2激活进而提升星形胶质细胞抗氧化应激水平提高。Objective To elucidate the role and mechanism of ketamine increasing antioxidative stress in astrocytes.Methods After human astrocyte cell line HA1800 cells were cultured with 50μmol/L,the activities of antioxidant enzymes in astrocytes were evaluat-ed,including glutathione peroxidase(GSH-Px),total superoxide dismutase(T-SOD)and catalase(CAT).The expression levels of nuclear factor erythroid 2-related factor 2(Nrf2),acetylation and KAT6B protein were measured by Western blot analysis.The expression level of acetylation and the location of Nrf2 were detected by immunofluorescence technique.The relative levels of KAT6B mRNA and circRNA in HA1800 cells were measured by RT-PCR.After siRNA of KAT6B was transfected and HA1800 cells were treated with ketamine,the expression of Nrf2 and acetylation was detected by Western blot analysis,and the activities of T-SOD and CAT were evaluated.Results After treatment with 50μmol/L keatmine for 12 h,the antioxidant enzyme activities were increased,including GSH-Px,T-SOD,and CAT(P<0.05).The expression levels of Nrf2,acetylation and KAT6B protein in astrocytes were significantly increased after 50μmol/L ketamine treatment for 3,6,12,24 h(P<0.05),especially 12 h.The 50μmol/L ketamine for 12 h significantly increased the expression of acetylation in HA1800 cells(P<0.05),and induced the translocation of Nrf2 into nucleus from cytoplasm.The 50μmol/L ketamine for 12 h significantly increased KAT6B mRNA in HA1800 cells(P<0.05),but decreased KAT6B circRNA(P<0.05).After silencing the expression of KAT6B gene,the ability of ketamine increasing antioxidaive stress was suppressed,the expression levels of KAT6B,Nrf2 and acetylation protein were significantly decreased(P<0.05),and the activities of T-SOD and CAT were significantly decreased(P<0.05).Conclusion Ketamine can increase the ability of antioxidative stress of astrocytes through activating Nrf2,decreasing the circKAT6B levels and enhancing the expression of KAT6B gene.

关 键 词：氯胺酮 星形胶质细胞 KAT6B 抗氧化应激 NRF2 

分 类 号：R966[医药卫生—药理学]