沉默信息调节因子1对小鼠机械通气相关性肺损伤炎症反应的影响

Effect of silent information regulator 1 on the inflammatory response of ventilation-associated lung injury in mice

目的:评价沉默信息调节因子1 (silent information regulator 1, sirt1)对小鼠机械通气相关性肺损伤(ventilation-associated lung injury, VILI)炎症反应的影响。方法:SPF级健康雄性C57BL/6N小鼠40只,8周龄,体重20~25 g,采用随机数字表法分为4组(每组10只):假手术组(S组),小鼠仅插管不进行机械通气;VILI组(M组),小鼠插管后进行机械通气;sirt1敲低组(si-sirt1组),小鼠体内转染sirt1小干扰RNA;sirt1敲低+VILI组(si-sirt1+M组),小鼠体内转染sirt1小干扰RNA后再机械通气。机械通气参数为:通气频率60次/min,潮气量28 ml/kg,FiO 2 21%,吸呼比1∶2,时间4 h。机械通气结束后处死小鼠,收集肺组织和支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF):采用ELISA法测定BALF中IL-1β和IL-6浓度,肺组织H-E染色后观察病理学结果,计算肺组织湿重干重比(wet/dry, W/D),采用ELISA法检测肺组织中丙二醛(malondialdehyde, MDA)和超氧化物歧化酶(superoxide dismutase, SOD)水平,Western blot法检测肺组织sirt1、连环蛋白p120和紧密连接蛋白occludin的水平。 结果:与S组比较,M组、si-sirt1组和si-sirt1+M组肺组织出现明显损伤改变,W/D、IL-1β和IL-6水平升高( P<0.05),sirt1、p120和occludin蛋白水平下调( P<0.05),肺组织中MDA水平升高( P<0.05),SOD水平降低( P< 0.05)。与M组比较,si-sirt1+M组肺组织损伤改变加重,W/D、IL-1β、IL-6水平升高( P<0.05),sirt1、p120和occludin蛋白水平下调( P<0.05),肺组织中MDA水平升高( P<0.05),SOD水平降低( P<0.05)。 结论:小鼠低表达sirt1后,机械通气造成的肺组织炎症反应加重。

Objective To evaluate the effect of silent information regulator 1(sirt1)on inflammatory response to ventilation-associated lung injury(VILI)in mice.Methods A total of 40 SPF healthy male C57BL/6N mice,aged 8 weeks and weighing 20-25 g,were used.According to the random number table method,they were divided into four groups(n=10):a sham operation group(group S),where mice were intubated without mechanical ventilation;an VILI group(group M),where mice were mechanically ventilated after intubation;a sirt1 knockdown group(group si-sirt1),where mice were transfected with sirt1 small interference RNA;and a sirt1 knockdown and VILI group(group si-sirt1+M),where mice were transfected from sirt1 small interference RNA before mechanical ventilation.Mechanical ventilation parameters were listed as follows:ventilation frequency 60 bpm,tidal volume 28 ml/kg,the fraction of inspiratory oxygen 21%,inspiratory/expiratory ratio 1∶2,and ventilation time 4 h.After mechanical ventilation,the mice were sacrificed to collect lung tissue and bronchoalveolar lavage fluid(BALF).The concentrations of interleukin(IL)-1βand IL-6 in BALF were determined by enzyme-linked immunosorbent assay(ELISA).The pathological results were observed by hematoxylin-eosin(H-E)staining.The ratio of wet/dry(W/D)weight was calculated,while the contents of superoxide dismutase(SOD)and malondialdehyde(MDA)in lung tissues were determined by ELISA.The levels of sirt1,p120,and occludin in lung tissues were detected by Western blot.Results Compared with group S,group M,group si-sirt1,and group si-sirt1+M showed significant injury in lung tissues,and increases in W/D ratio,and IL-1β,and IL-6 concentrations(P<0.05),down-regulated expression of sirt1,p120 and occludin(P<0.05),increased MDA level in lung tissue(P<0.05),and decreased SOD level(P<0.05).Compared with group M,group si-sirt1+M showed more severe lung tissue injury,increases in W/D ratio,and IL-1β,and IL-6 concentrations(P<0.05),down-regulated expression of sirt1,p120 and occludin(P<0.05),increased MDA level in lung tissue(P<0.05),and decreased SOD level(P<0.05).Conclusions Low expression of sirt1 in mice causes aggravated inflammation in lung tissue caused by mechanical ventilation.