GSK484对小鼠呼吸机相关性肺损伤及中性粒细胞胞外诱捕网的影响

Effects of GSK484 on ventilator-induced lung injury and neutrophil extracelluar traps in mice

目的评价GSK484对小鼠呼吸机相关性肺损伤(VILI)及中性粒细胞胞外诱捕网(NETs)的影响。方法SPF级健康雄性C57BL/6小鼠48只,5~6周龄,体质量15~20 g,采用随机数字表法将小鼠分为4组(n=12):自主呼吸组(S组)、自主呼吸+GSK484干预组(SG组)、VILI组(V组)、VILI+GSK484干预组(VG组)。S组及SG组气管插管后自主呼吸4 h;V组及VG组机械通气4 h(潮气量30 ml/kg、呼吸频率75次/min、吸呼比1∶2、呼吸末正压0 mmHg、吸入氧气浓度21%)。于VILI建模前3 d,SG组及VG组腹腔注射GSK4844 mg/kg,1次/d,S组及V组每天以等容量生理盐水代替。小鼠自主呼吸或机械通气4 h时,采集腹主动脉血样,行血气分析,记录PaO_(2);随后处死小鼠,收集支气管肺泡灌洗液(BALF)及肺组织。确定肺组织湿重/干重比值(W/D);肺组织HE染色后,光镜下观察病理学结果并行损伤评分;采用ELISA法检测BALF IL-1β、IL-6、TNF-α及髓过氧化物酶(MPO)浓度;采用Western blot法检测肺组织肽酰基精氨酸脱亚氨酶4(PAD4)、中性粒细胞弹性蛋白酶(NE)、高迁移率族蛋白B1(HMGB1)及瓜氨酸化组蛋白3(Cit-H3)表达水平。结果与S组和SG组比较,V组和VG组肺损伤评分和W/D比值升高,PaO_(2)降低,BALF IL-1β、IL-6、TNF-α和MPO浓度升高,肺组织PAD4、NE、HMGB1和Cit-H3表达上调(P<0.05);与V组比较,VG组肺损伤评分和W/D比值降低,PaO_(2)升高,BALF IL-1β、IL-6、TNF-α和MPO浓度降低,肺组织PAD4、NE、HMGB1和Cit-H3表达下调(P<0.05)。结论GSK484可减轻小鼠VILI,机制与抑制PAD4表达,减少NETs产生,减轻肺组织炎症反应有关。

Objective To evaluate the effects of GSK484 on ventilator-induced lung injury(VILI)and neutrophil extracelluar traps(NETs)in mice.Methods Forty-eight SPF healthy male C57BL/6 mice,aged 5-6 weeks,weighing 15-20 g,were divided into 4 groups(n=12 each)by a random number table method:spontaneous breathing group(group S),spontaneous breathing+GSK484 intervention group(group SG),VILI group(group V),and VILI+GSK484 intervention group(group VG).The animals kept spontaneous breathing for 4 h after tracheal intubation in S and SG groups.The animals were mechanically ventilated for 4 h(tidal volume 30 ml/kg,respiratory rate 75 breaths/min,inspiratory/expiratory ratio 1∶2,positive end-expiratory pressure 0 mmHg,fraction of inspired oxygen 21%)in V and VG groups.At 3 days before developing the VILI model,GSK4844 mg/kg was intraperitoneally injected once a day in SG and VG groups,while the equal volume of normal saline was given instead in S and V groups.Blood samples were collected from the abdominal aorta for blood gas analysis at 4 h of spontaneous breathing or mechanical ventilation,and PaO_(2) was recorded.The mice were then sacrificed and bronchoalveolar lavage fluid(BALF)was collected and lung tissues were obtained for microscopic examination of the pathological changes(with a light microscope after HE staining)which were scored and for determination of wet to dry weight ratio(W/D ratio),concentrations of interleukin-1beta(IL-1β),IL-6,tumor necrosis factor-alpha(TNF-α)and myeloperoxidase(MPO)in BALF(by enzyme-linked immunosorbent assay),expression of peptidylarginine deiminase 4(PAD4),neutrophil elastase(NE),high mobility group box 1(HMGB1)and citrullinated-histone 3(Cit-H3)in lung tissues(by Western blot).Results Compared with S and SG groups,the lung injury score and W/D ratio were significantly increased,PaO_(2) was decreased,concentrations of IL-1β,IL-6,TNF-αand MPO in BALF were increased,and the expression of PAD4,NE,HMGB1 and Cit-H3 in lung tissues was up-regulated in V and VG groups(P<0.05).Compared with group V,the lung injury score and W/D ratio were significantly decreased,PaO_(2) was increased,the concentrations of IL-1β,IL-6,TNF-αand MPO in BALF were decreased,and the expression of PAD4,NE,HMGB1 and Cit-H3 was down-regulated in group VG(P<0.05).Conclusions GSK484 can alleviate VILI in mice,and the mechanism is associated with inhibition of PAD4,reduction of the production of NETs and attenuation of inflammatory responses in lung tissues.