组织蛋白酶B在大鼠呼吸机相关性肺损伤中的作用:与NLRP3炎症小体的关系

Role of cathepsin B in mechanical ventilator-induced lung injury in rats:relationship with NLRP3 inflammasomes

目的评价组织蛋白酶B(CTSB)在大鼠呼吸机相关性肺损伤(VILI)中的作用及其与NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体的关系。方法SPF级健康雄性SD大鼠36只,6~8周龄,体重220~300 g,采用随机数字表法分为3组(n=12):对照组(C组)、VILI组(V组)和VILI+CA074-me组(Me组)。C组和V组大鼠气管插管前1 h腹腔注射等量生理盐水,Me组腹腔注射CA074-me 5 mg/kg。C组自主呼吸4 h,V组和Me组行机械通气4 h,通气参数:VT 20 ml/kg,通气频率80次/min,FiO221%,PEEP 0 cmH2O。气管插管前和自主呼吸或通气结束后采集股动脉血行动脉血气分析,记录PaO2,随后处死大鼠,收集支气管肺泡灌洗液(BALF)和取肺组织,确定肺组织湿重/干重(W/D)比值,HE染色法观察病理学结果并进行肺损伤评分,采用ELISA法检测BALF及血清IL-1β和IL-18浓度。采用qRT-PCR法检测肺组织CTSB、NLRP3、凋亡相关斑点样蛋白(ASC)和caspase-1的mRNA表达,Western blot法检测肺组织CTSB、NLRP3、ASC和caspase-1的表达。结果与C组比较,V组和Me组通气结束后PaO2降低,肺损伤评分和W/D比值、BALF及血清IL-1β和IL-18浓度升高,肺组织CTSB、NLRP3、ASC、caspase-1及其mRNA表达上调(P<0.01);与V组比较,Me组通气结束后PaO2升高,肺损伤评分和W/D比值、BALF及血清IL-18和IL-1β浓度降低,肺组织CTSB、NLRP3、ASC、caspase-1及其mRNA表达下调(P<0.01)。结论CTSB参与了大鼠VILI的过程,其机制与激活NLRP3炎症小体有关。

Objective To evaluate the role of cathepsin B(CTSB)in mechanical ventilator-induced lung injury(VILI)in rats and the relationship with NOD-like receptor pyrin domain containing 3(NLRP3)inflammasome.Methods Thirty-six SPF-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 220-300 g,were divided into 3 groups(n=12 each)by the random number table method:control group(group C),VILI group(group V)and VILI+CA074-me group(group Me).CA074-me 5 mg/kg was intraperitoneally injected in group Me,while the equal volume of normal saline was given instead in group C and group V.Group C kept spontaneous breathing for 4 h,and the animals were mechanically ventilated(tidal volume 20 ml/kg,respiratory rate 80 breaths/min,fraction of inspired oxygen 21%,PEEP 0 cmH2O).Blood samples from femoral artery were collected for arterial blood gas analysis before tracheal intubation and after spontaneous breathing or ventilation,and PaO2 was recorded.Rats were sacrificed,and bronchoalveolar lavage fluid(BALF)was collected and lung tissues were collected for determination of the wet/dry lung weight ratio(W/D ratio),serum interleukin-1beta(IL-1β)and IL-18 concentrations in BALF(by enzyme-linked immunosorbent assay),expression of CTSB,NLRP3,apoptosis-associated speck-like protein containing a caspase-1 recruitment domain(ASC)and caspase-1 mRNA in lung tissues(quantitative real-time polymerase chain reaction),and expression of CTSB,NLRP3,ASC and caspase-1 in lung tissues(by Western blot)and for microscopic examination of the pathological changes(using HE staining).Lung injury was assessed and scored.Results Compared with group C,PaO2 was significantly decreased after the end of ventilation,the lung injury score,W/D ratio and concentrations of IL-1βand IL-18 in serum and BALF were increased,and the expression of CTSB,NLRP3,ASC and caspase-1 protein and mRNA in lung tissues was up-regulated in group V and group Me(P<0.01).Compared with group V,PaO2 was significantly increased after the end of ventilation,the lung injury score,W/D ratio and concentrations of IL-1βand IL-18 in serum and BALF were decreased,and the expression of CTSB,NLRP3,ASC and caspase-1 protein and mRNA in lung tissues was down-regulated in group Me(P<0.01).Conclusions CTSB is involved in VILI in the rats,and the mechanism may be related to activation of NLRP3 inflammasomes.