右美托咪定对脓毒症小鼠急性肺损伤时DNA甲基转移酶表达的影响

Effect of dexmedetomidine on expression of DNA methyltransferases in septic mice with acute lung injury

目的评价右美托咪定对脓毒症小鼠急性肺损伤时DNA甲基转移酶表达的影响。方法清洁级健康雄性C57BL/6小鼠48只,6~8周龄,体质量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+右美托咪定组(Sham+DEX组)、脓毒症组(Sepsis组)和脓毒症+右美托咪定组(Sepsis+DEX组)。采用盲肠结扎穿孔法制备小鼠脓毒症模型。于制备模型前30 min,Sham+DEX组和Sepsis+DEX组腹腔注射右美托咪定0.05μg/g(生理盐水稀释至0.5 ml),Sham组和Sepsis组给予0.5 ml生理盐水。于盲肠结扎穿孔后24 h时处死小鼠取肺组织,确定肺湿质量/干质量(W/D)比值,HE染色法观察肺组织病理学结果,计算肺组织损伤评分,采用ELISA法测定IL-6、TNF-α、HMGB1含量、SOD、MPO活性和MDA含量,比色法测定全基因组DNA甲基化水平,采用Western blot法检测DNA甲基化转移酶(DNMTl、DNMT3a、DNMT3b)的表达。结果与Sham组比较,Sepsis组和Sepsis+DEX组小鼠肺组织损伤评分、肺W/D比值、IL-6、TNF-α、HMGB1、MDA含量、MPO活性和全基因组DNA甲基化水平升高,SOD活性降低,DNMT1和DNMT3a表达上调(P<0.05),Sham+DEX组上述各指标差异无统计学意义(P>0.05);与Sepsis组比较,Sepsis+DEX组小鼠肺组织损伤评分、肺W/D比值、IL-6、TNF-α、HMGB1、MDA含量、MPO活性和全基因组DNA甲基化水平降低,SOD活性升高,DNMT1和DNMT3a表达下调(P<0.05)。结论右美托咪定减轻脓毒症小鼠急性肺损伤的机制与抑制DNMT1和DNMT3a表达上调有关。

Objective To evaluate the effect of dexmedetomidine on the expression of DNA methyltransferases in septic mice with acute lung injury.Methods Forty-eight clean-grade 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+dexmedetomidine group(group Sham+DEX),sepsis group(group Sepsis)and sepsis+dexmedetomidine group(group Sepsis+DEX).Sepsis model was established by cecal ligation and puncture(CLP)in anesthetized mice.At 30 min before model preparation,dexmedetomidine 0.05μg/g(in 0.5 ml of normal saline)was administered in Sham+DEX and Sepsis+DEX groups,and normal saline 0.5 ml was given instead in Sham and Sepsis groups.The mice were sacrificed at 24 h after CLP,and the lung tissue was taken to determine the wet to dry lung weight ratio,contents of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α)and high mobility group box-1(HMGB-1),activities of superoxide dismutase(SOD)and myeloperoxidase(MPO),and content of malondialdehyde(MDA)(by enzyme-linked immunosorbent assay),global DNA methylation(by colorimetric assay),and expression of DNA methyltransferases(DNMTl,DNMT3a,DNMT3b)(by Western blot)and to examine the histopathological changes of lung tissues(by HE staining)which were scored.Results Compared with group Sham,the lung injury score,wet/dry lung weight ratio,contents of IL-6,TNF-αand HMGB1 and MDA,MPO activity and global DNA methylation were significantly increased,SOD activity was decreased,and the expression of DNMT1 and DNMT3a was up-regulated in group Sepsis and group Sepsis+DEX(P<0.05),and no significant change was found in the aforementioned indexes in group Sham+DEX(P>0.05).Compared with group Sepsis,the lung injury score,wet/dry lung weight ratio,contents of IL-6,TNF-αand HMGB1 and MDA,MPO activity and global DNA methylation were significantly decreased,SOD activity was increased,and the expression of DNMT1 and DNMT3a was down-regulated in group Sepsis+DEX(P<0.05).Conclusions The mechanism by which dexmedetomidine reduces acute lung injury is related to inhibition of up-regulation of DNMT1 and DNMT3a expression in septic mice.