程序性坏死在幼鼠高氧急性肺损伤中的作用

Role of necroptosis in hyperoxia-induced acute lung injury in preadolescent rats

目的评价程序性坏死在幼鼠高氧急性肺损伤中的作用。方法清洁级健康雄性SD大鼠72只,14日龄,体重40~50 g,采用随机数字表法分为3组(n=24):对照组(C组)、高氧急性肺损伤组(ALI组)和高氧急性肺损伤+necrostatin-1组(ALI+N组)。ALI+N组腹腔注射程序性坏死抑制剂necrostatin-11.0 mg/kg,C组和ALI组分别腹腔注射二甲基亚砜0.2 ml/kg,1次/d,连续给药3 d。吸氧72 h时处死大鼠,收集支气管肺泡灌洗液(BALF),采用ELISA法检测BALF中IL-6和IL-8浓度,采用黄嘌呤氧化酶法测定SOD活性,采用硫代巴比妥酸法测定MDA浓度。取肺组织,测定湿重/干重比值(W/D比值),光镜下观察病理学结果,并计算肺泡损伤率(IAR);电镜下观察超微结构。采用Western blot法测定肺组织受体相互作用蛋白激酶1(RIPK1)、RIPK3和混合系列蛋白酶样结构域(MLKL)表达水平。结果与C组比较,ALI组BALF中IL-6、IL-8和MDA浓度升高,SOD活性降低,肺组织W/D比值和IAR升高,RIPK1、RIPK3和MLKL表达上调(P<0.05),肺组织病理学损伤加重。与ALI组比较,ALI+N组BALF中IL-6、IL-8和MDA浓度降低,SOD活性升高,肺组织W/D比值和IAR降低,RIPK1、RIPK3和MLKL表达下调(P<0.05),肺组织病理学损伤减轻。结论细胞程序性坏死参与了幼鼠高氧急性肺损伤的病理生理过程。

Objective To evaluate the role of necroptosis in hyperoxia-induced acute lung injury(ALI)in preadolescent rats.Methods A total of 72 clean-grade healthy male Sprague-Dawley rats,aged 14 days,weighing 40-50 g,were divided into 3 groups(n=24 each)by using a random number table method:control group(group C),hyperoxia-induced ALI group(group ALI)and hyperoxia-induced ALI and necrostatin-1 group(group ALI+N).The rats of group ALI+N was intraperitoneally injected with necrostatin-11.0 mg/kg once a day for 3 consecutive days.The rats were intraperitoneally injected with dimethyl sulfoxide 0.2 ml/kg once a day for 3 consecutive days in C and ALI groups.The animals were sacrificed at 72 h after inhaling oxygen,and bronchoalveolar lavage fluid(BALF)was collected for determination of interleukin-6(IL-6)and IL-8 concentrations(by enzyme-linked immunosorbent assay),superoxide dismutase(SOD)activity(by xanthine oxidase method),and malondialdehyde(MDA)concentration(by thiobarbituric acid method).Lung tissues were taken for measurement of wet/dry weight ratio(W/D ratio)and for examination of the pathological changes(with a light microscope)and ultrastructure of lung tissues(with an electron microscope).The injured alveolus rate(IAR)was calculated.The expression of receptor-interacting protein kinase 1(RIPK1),RIPK3 and mixed-lineage kinase domain-like protein(MLKL)in lung tissues was detected by Western blot.Results Compared with group C,the concentrations of IL-6,IL-8 and MDA in BALF were significantly increased,the activity of SOD in BALF was decreased,the W/D ratio and IAR of lung tissues were increased,the expression of RIPK1,RIPK3 and MLKL in lung tissues was up-regulated(P<0.05),and the pathological damage was accentuated in group ALI.Compared with group ALI,the concentrations of IL-6,IL-8 and MDA in BALF were significantly deceased,the activity of SOD in BALF was increased,the W/D ratio and IAR of lung tissues were decreased,the expression of RIPK1,RIPK3 and MLKL in lung tissues was down-regulated(P<0.05),and the pathological damage was significantly attenuated in group ALI+N.Conclusion Necroptosis is involved in the pathophysiological process of hyperoxia-induced ALI in preadolescent rats.