腹腔巨噬细胞Sirt3表达在右美托咪定抑制脓毒症小鼠炎症反应中的作用

Role of Sirt3 expression in peritoneal macrophages in dexmedetomidine-induced inhibition of inflammatory responses in septic mice

目的评价腹腔巨噬细胞NAD依赖性蛋白脱乙酰酶3(Sirt3)表达在右美托咪定抑制脓毒症小鼠炎症反应中的作用。方法SPF级健康雄性C57BL6小鼠64只,7周龄,体重约20 g。采用随机数字表法分为4组(n=16):假手术组(S组)、脓毒症组(Sep组)、右美托咪定组(DEX组)和右美托咪定+Sirt3抑制剂3-TYP组(TYP组)。采用盲肠结扎穿孔的方法制备小鼠脓毒症模型,DEX组于制备模型前1 h时腹腔注射生理盐水0.25 ml,30 min后腹腔注射右美托咪定50μg/kg(生理盐水稀释至0.25 ml);TYP组于制备模型前1 h时腹腔注射3-TYP 5 mg/kg(生理盐水稀释至0.25 ml),30 min后腹腔注射右美托咪定50μg/kg。S组和Sep组于制备模型前1 h和30 min分别腹腔注射等量生理盐水。S组仅进行开腹、取出盲肠操作,不结扎穿孔。于术后24 h时腹腔灌洗,提取腹腔巨噬细胞贴壁培养,采用qRT-PCR法检测巨噬细胞Sirt3、IL-1β和TNF-αmRNA表达,Western blot法检测Sirt3的表达,ELISA法测定腹腔灌洗液IL-1β和TNF-α浓度,观察各组小鼠10 d生存情况。结果与S组比较,Sep组、DEX组和TYP组腹腔巨噬细胞Sirt3及其mRNA表达下调,IL-1β和TNF-αmRNA表达上调,腹腔灌洗液IL-1β和TNF-α浓度升高,小鼠10 d生存率下降(P<0.05);与Sep组比较,DEX组腹腔巨噬细胞Sirt3及其mRNA表达上调,IL-1β和TNF-αmRNA表达下调,腹腔灌洗液IL-1β和TNF-α浓度降低,小鼠10 d生存率升高(P<0.05);与DEX组比较,TYP组腹腔巨噬细胞Sirt3及其mRNA、IL-1β和TNF-αmRNA表达上调,腹腔灌洗液IL-1β和TNF-α浓度升高,小鼠10 d生存率降低(P<0.05)。结论腹腔巨噬细胞Sirt3表达上调参与了右美托咪定抑制脓毒症小鼠炎症反应的过程。

Objective To evaluate the role of nicotinamide adenine dinucleotide-dependent deacetylase Sirtuin3(Sirt3)expression in peritoneal macrophages in dexmedetomidine-induced inhibition of inflammatory responses in septic mice.Methods Sixty-four healthy male C57BL/6 mice,aged 7 weeks,weighing about 20 g,were divided into 4 groups(n=16 each)using a random number table method:sham operation group(S group),sepsis group(Sep group),dexmedetomidine group(DEX group),and dexmedetomidine plus Sirt3 inhibitor 3-TYP group(TYP group).Sepsis was induced by cecal ligation and puncture.In group DEX,normal saline 0.25 ml was intraperitoneally injected at 1 h before development of the model,and 30 min later dexmedetomidine 50μg/kg(in 0.25 ml of normal saline)was intraperitoneally injected.In group S and group Sep,the equal volume of normal saline was intraperitoneally injected at 1 h and 30 min before development of the model,respectively.In group S,laparotomy and cecal extraction were performed without ligation and perforation.Peritoneal lavage was obtained at 24 h after operation,and peritoneal macrophages were cultured for 1 h.The expression of Sirt3,interleukin-1beta(IL-1β)and tumor necrosis factor-alpha(TNF-α)mRNA in peritoneal macrophages was detected using quantitative real-time polymerase chain reaction.Western blot was used to detect the expression of Sirt3 in peritoneal macrophages.The concentrations of IL-1βand TNF-αin peritoneal fluid were measured by enzyme-linked immunosorbent assay.The 10-day survival of mice in each group was observed,and the survival rates were calculated.Results Compared with group S,the expression of Sirt 3 protein and mRNA in peritoneal macrophages was significantly down-regulated,the expression of IL-1βand TNF-αmRNA was up-regulated,the concentrations of IL-1βand TNF-αin peritoneal lavage were increased,and the 10-day survival rates were decreased in Sep,DEX and TYP groups(P<0.05).Compared with group Sep,the expression of Sirt3 protein and mRNA in peritoneal macrophages was significantly up-regulated,the expression of IL-1βand TNF-αmRNA was down-regulated,the concentrations of IL-1βand TNF-αin the peritoneal lavage were decreased,and the 10-day survival rate was increased in group DEX(P<0.05).Compared with group DEX,the expression of Sirt3 protein and mRNA and IL-1βand TNF-αmRNA in peritoneal macrophages was significantly up-regulated,the concentrations of IL-1βand TNF-αin peritoneal lavage were increased,and the 10-day survival rate was decreased in group TYP(P<0.05).Conclusions Up-regulation of Sirt3 expression in peritoneal macrophages is involved in the process of dexmedetomidine-induced inhibition of inflammatory responses in septic mice.