PKC-δ在大鼠机械通气相关性肺损伤中的作用:与NLRC4的关系

Role of PKC-δin ventilator-induced lung injury in rats:relationship with NLRC4

目的评价蛋白激酶C(PKC)-δ在大鼠机械通气相关性肺损伤中的作用及其与NOD样受体包含CARD结构域蛋白4(NLRC4)的关系。方法清洁级健康雄性SD大鼠36只,6~8周龄,体重200~250 g,采用随机数字表法分为3组(n=12):对照组(C组)、机械通气相关性肺损伤组(V组)和机械通气相关性肺损伤+KAI 9803组(VK组)。气管切开后置入气管导管行机械通气,设置VT 40 ml/kg,通气频率60次/min,I∶E为1∶2,吸入空气。C组气管插管后不行机械通气。气管插管完成后即刻VK组于气管内滴注PKC-δ特异性抑制剂KAI 9803200μg/kg,余2组滴入等量PBS。机械通气4 h时,股动脉采血行血气分析,记录PaO2。开胸取肺组织,行左侧肺灌洗,收集肺泡灌洗液。光镜下观察肺组织病理学结果并行肺损伤评分;采用ELISA法检测肺泡灌洗液IL-1β和IL-18浓度,Western blot法检测右肺下叶NLRC4、caspase-1和PKC-δ表达,RT-PCR法检测右肺下叶NLRC4 mRNA表达,计算右肺中叶湿重/干重(W/D)比值。结果与C组比较,余2组肺损伤评分和W/D比值升高,PaO2降低,肺泡灌洗液IL-18和IL-1β浓度升高,肺组织NLRC4、caspase-1和NLRC4 mRNA表达上调,V组肺组织PKC-δ表达上调(P<0.01),肺泡腔内可见大量水肿液渗出并伴炎性细胞浸润;与V组比较,VK组肺损伤评分和W/D比值降低,PaO2升高,肺泡灌洗液IL-18和IL-1β浓度降低,肺组织NLRC4、caspase-1、PKC-δ和NLRC4 mRNA表达下调(P<0.05),肺泡腔液体渗出和炎性细胞浸润程度减轻。结论PKC-δ参与了大鼠机械通气相关性肺损伤的过程,与抑制NLRC4表达有关。

Objective To evaluate the role of protein kinase C-delta(PKC-δ)in ventilator-induced lung injury(VILI)and the relationship with NLR family CARD domain-containing protein 4(NLRC4)in rats.Methods Thirty-six clean-grade healthy adult male Sprague-Dawley rats,weighing 200-250 g,were divided into 3 groups(n=12 each)using a random number table method:control group(group C),VILI group(group V)and VILI plus KAI 9803 group(group VK).In V and VK groups,tracheal tubes were placed for mechanical ventilation after tracheotomy,ventilator settings were adjusted with a tidal volume of 40 ml/kg,respiratory rate of 60 breaths/min,and inspiratory/expiratory ratio of 1∶2,and air was inhaled.Group C received no mechanical ventilation after tracheal intubation.Immediately after completion of intubation,PKC-δspecific inhibitor KAI 9803200μg/kg was intratracheally injected in group VK,and the equal volume of phosphate buffer saline was given instead in the other two groups.Blood samples were taken from the femoral artery at 4 h of mechanical ventilation to record PaO2.The chest was opened at the end of mechanical ventilation,lung tissues were removed,and the left lung tissues were lavaged to collect bronchoalveolar lavage fluid(BALF).The pathological changes of lung tissues were examined with a light microscope and scored.Enzyme-linked immunosorbent assay was used to detect the concentrations of interleukin-1beta(IL-1β)and IL-18 in BALF,Western blot was used to detect the expression of NLRC4,caspase-1 and PKC-δin the right lower lobe of the lung,and the expression of NLRC4 mRNA in the right lower lobe of the lung was determined by real-time polymerase chain reaction,and the wet/dry weight ratio(W/D ratio)of the right middle lobe of the lung was calculated.Results Compared with group C,the pathological score and W/D ratio were significantly increased,PaO2 was decreased,the concentrations of IL-1βand IL-18 in BALF were increased,and the expression of NLRC4,caspase-1 and NLRC4 mRNA was up-regulated in V and VK groups,and the expression of PKC-δwas significantly up-regulated(P<0.01),and a large amount of edema fluid was seen in the alveolar space,with inflammatory cell infiltration in group V(P<0.01).Compared with group V,the pathological score and W/D ratio were significantly decreased,PaO2 was increased,the concentrations of IL-1βand IL-18 in BALF were decreased,the expression of NLRC4,caspase-1,PKC-δand NLRC4 mRNA was down-regulated(P<0.05),and fluid exudation in the alveolar space and the degree of inflammatory cell infiltration were significantly attenuated in group VK.Conclusion PKC-δis involved in VILI,which is related to inhibiting NLRC4 expression in rats.