DNA甲基转移酶在脓毒症小鼠急性肺损伤中的作用  

Role of DNA methyltransferase in acute lung injury in septic mice

作　　者：李佩[1,2] 于明懂 张英立[3] 任万陆[2] 余剑波 Li Pei;Yu Mingdong;Zhang Yingli;Ren Wanlu;Yu Jianbo(Tianjin Medical University,Tianjin 300070,China;Department of Anesthesiology,Tianjin Hospital,Tianjin 300211,China;Department of Anesthesiology,The Second Hospital of Tianjin Medical University,Tianjin 300211,China;Department of Anesthesiology,Tianjin Nankai Hospital,Tianjin 300100,China)

机构地区：[1]天津医科大学,天津300070 [2]天津市天津医院麻醉科,天津300211 [3]天津医科大学第二医院麻醉科,天津300211 [4]天津市南开医院麻醉科,天津300100

出　　处：《中华麻醉学杂志》2022年第4期447-450,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(82074153);天津医科大学第二医院青年科研基金项目(2020ydey11)。

摘　　要：目的评价DNA甲基转移酶在脓毒症小鼠急性肺损伤中的作用。方法健康雄性C57BL/6小鼠48只,6~8周龄,体重20~25 g,采用随机数字表法分为4组(n=12):假手术组(Sham组)、假手术+DNA甲基转移酶抑制剂组(Sham+5-Aza组)、脓毒症组(Sepsis组)和脓毒症+DNA甲基转移酶抑制剂组(Sepsis+5-Aza组)。采用盲肠结扎穿孔(CLP)法制备脓毒症小鼠急性肺损伤模型。于CLP后24 h时处死小鼠取肺组织,提取DNA利用比色法测定全基因组DNA甲基化水平,提取RNA采用实时荧光定量PCR法检测DNA甲基转移酶(DNMTl、DNMT3a、DNMT3b)的mRNA表达,确定肺湿重/干重比值,HE染色观察肺组织病理学结果,采用ELISA法测定IL-6、TNF-α、高迁移率族蛋白B1(HMGB1)、MDA含量、SOD和过氧化氢酶(CAT)活性。结果与Sham组比较,Sepsis组和Sepsis+5-Aza组小鼠CLP后24 h时肺组织全基因组甲基化水平升高,DNMT1和DNMT3a的mRNA表达上调,肺组织病理学损伤评分和肺湿重/干重比值升高,IL-6、TNF-α、HMGB1和MDA含量升高,SOD和CAT活性降低(P<0.05),Sham+5-Aza组上述各指标差异无统计学意义(P>0.05);与Sepsis组比较,Sepsis+5-Aza组小鼠肺组织全基因组甲基化水平降低,DNMT1和DNMT3a的mRNA表达下调,肺组织病理学损伤评分、肺湿重/干重比值、IL-6、TNF-α、HMGB1和MDA含量降低,SOD和CAT活性增加(P<0.05)。结论DNMT1和DNMT3a介导的DNA高甲基化参与了脓毒症小鼠急性肺损伤的过程。Objective To evaluate the role of DNA methyltransferase in acute lung injury in septic mice.Methods Forty-eight healthy male C57BL/6 mice,aged 6-8 weeks,weighing 20-25 g,were divided into 4 groups(n=12 each)using a random number table method:sham operation group(group Sham),sham operation+DNA methyltransferase inhibitor group(group Sham+5-Aza),sepsis group(group Sepsis)and sepsis+DNA methyltransferase inhibitor group(group Sepsis+5-Aza).Sepsis model was developed by cecal ligation and puncture(CLP)in anesthetized mice.Mice were sacrificed at 24 h after CLP,and lung tissues were obtained,DNA was extracted to determine the global DNA methylation by colorimetry,and RNA was extracted to detect the expression of DNA methyltransferase(DNMTl,DNMT3a,DNMT3b)mRNA by real-time fluorescent quantitative polymerase chain reaction,the wet/dry lung weight ratio(W/D ratio)was measured,the histopathological changes of lung tissues were determined by HE staining,the contents of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),high-mobility group box 1 protein(HMGB1)and malondialdehyde(MDA)and activities of superoxide dismutase(SOD)and catalase were measured by enzyme-linked immunosorbent assay.Results Compared with group Sham,the global DNA methylation was significantly increased,the expression of DNMT1 and DNMT3a mRNA was up-regulated,the lung injury score,W/D ratio,and contents of IL-6,TNF-α,HMGB1 and MDA were increased,and activities of SOD and CAT were decreased at 24 h after CLP in group Sepsis and group Sepsis+5-Aza(P<0.05),and no significant change was found in the indexes mentioned above in group Sham+5-Aza(P>0.05).Compared with group Sepsis,the global DNA methylation was significantly decreased,the expression of DNMT1 and DNMT3a mRNA was down-regulated,the lung injury score,W/D ratio,contents of IL-6,TNF-α,HMGB1 and MDA were decreased,and the activities of SOD and CAT were increased in group Sepsis+5-Aza(P<0.05).Conclusions DNA hypermethylation mediated by DNMT1 and DNMT3a is involved in the process of acute lung

关 键 词：DNA甲基化 脓毒症 急性肺损伤 

分 类 号：R459.7[医药卫生—急诊医学]