DNA甲基化在小鼠脓毒症相关性脑病中的作用

Role of DNA methylation in sepsis-associated encephalopathy in mice

目的评价DNA甲基化在小鼠脓毒症相关性脑病中的作用。方法清洁级健康雄性C57BL/6小鼠144只,6~8周龄,体重20~25 g,采用随机数字表法分为4组(n=36):假手术组(Sham组)、脓毒症组(Sepsis组)、假手术+SAM组(Sham+SAM组)和脓毒症+SAM组(Sepsis+SAM组)。采用盲肠结扎穿孔(CLP)法制备小鼠脓毒症相关性脑病模型。Sham+SAM组和Sepsis+SAM组于术前1 h及术后12 h腹腔注射DNA甲基化的甲基供体S-腺苷甲硫氨酸(SAM)100 mg/kg,Sham组和Sepsis组腹腔注射等量生理盐水。于CLP后1、3和7 d时通过Y-迷宫和条件恐惧实验检测小鼠认知功能;于CLP后1、3和7 d时处死小鼠取海马组织,采用比色法测定全基因组DNA甲基化水平,采用实时荧光定量PCR法检测DNA甲基化转移酶(DNMTl、DNMT3a和DNMT3b)、TET(TET1、TET2和TET3)和TDG的mRNA表达水平。结果与Sham组比较,Sepsis组新异臂停留时间缩短,僵直时间百分比和各臂穿梭次数总和减少,CLP后1、3和7 d时小鼠海马组织全基因组甲基化水平降低,DNMT1、DNMT3a、TET1、TET2、TET3和TDG mRNA表达上调,DNMT3b mRNA表达下调(P<0.05);与Sepsis组比较,Sepsis+SAM组新异臂停留时间延长,僵直时间百分比和各臂穿梭次数总和明显增加,各时点DNMT1、DNMT3a、TET1、TET2、TET3和TDG的mRNA表达下调,DNMT3b mRNA表达上调(P<0.05)。结论海马DNA甲基化参与了小鼠脓毒症相关性脑病发生的过程。

Objective To evaluate the role of DNA methylation in sepsis-associated encephalopathy in mice.Methods A total of 144 clean-grade healthy male C57BL/6 mice,aged 6-8 weeks,weighing 20-25 g,were divided into 4 groups(n=36 each)using a random number table method:sham operation group(group Sham),sepsis group(group Sepsis),sham operation plus S-adenosyl methionine(SAM)group(group Sham+SAM)and sepsis plus SAM group(group Sepsis+SAM).Sepsis was produced by cecum ligation and puncture(CLP).In Sham+SAM and Sepsis+SAM groups,DNA methylated methyl donor SAM 100 mg/kg was intraperitoneally injected at 1 h before operation and 12 h after operation,while the equal volume of normal saline was given instead in Sham and Sepsis groups.The cognitive function was assessed using Y-maze and contextual fear conditioning test at 1,3 and 7 days after CLP.Mice were sacrificed at 1,3 and 7 days after CLP,and the hippocampal tissues were taken for determination of genome-wide DNA methylation(by colorimetric assay)and expression of DNA methyltransferase enzymes(DNMT1,DNMT3a,and DNMT3b),ten-eleven translocation(TET)enzymes(TET1,TET2 and TET3)and thymine-DNA glycosylase(TDG)mRNA(by fluorescent quantitative real-time).Results Compared with group Sham,the time of staying at the novel arm was significantly shortened,the percentage of time spent freezing and the total number of entries into each arm were reduced,genome-wide DNA methylation in hippocampal tissues was decreased at 1,3 and 7 days after CLP,the expression of DNMT1,DNMT3a,TET1,TET2,TET3 and TDG was up-regulated,and the expression of DNMT3b was down-regulated in group Sepsis(P<0.05).Compared with group Sepsis,the time of staying at the novel arm was significantly prolonged,the percentage of time spent freezing and the total number of entries into each arm were increased,the expression of DNMT1,DNMT3a,TET1,TET2,TET3 and TDG mRNA was down-regulated,and the expression of DNMT3b was up-regulated in group Sepsis+SAM(P<0.05).Conclusion DNA methylation is involved in the development of sepsis-associated encephalopathy in mice.