盐酸戊乙奎醚对大鼠呼吸机相关性肺损伤时气道MUC5AC表达的影响及其与TLR4/MyD88信号通路的关系

Effect of penehyclidine hydrochloride on expression of airway MUC5AC during ventilator-induced lung injury and the relationship with TLR4/MyD88 signaling pathway in rats

目的评价盐酸戊乙奎醚对大鼠呼吸机相关性肺损伤(VILI)时气道黏蛋白5AC(MUC5AC)表达的影响及其与Toll样受体4/髓样分化因子88(TLR-4/MyD88)信号通路的关系。方法清洁级健康雄性SD大鼠36只,体重200~250 g,6~8周龄,采用随机数字表法分为3组(n=12):假手术组(S组)、VILI组和盐酸戊乙奎醚组(P组)。S组行气管切开术;VILI组和P组行气管切开术后连接小动物呼吸机行机械通气4 h,呼吸参数设置:VT 20 ml/kg,通气频率80次/min,I∶E 1∶1,FiO221%。机械通气前30 min时,P组尾静脉注射盐酸戊乙奎醚2 mg/kg,S组和VILI组给予等容量生理盐水。机械通气4 h时,取动脉血样,测定PaO2;然后处死大鼠,收集支气管肺泡灌洗液(BALF),采用ELISA法测定BALF中IL-1β、IL-6和TNF-α浓度。取肺组织,计算湿重/干重(W/D)比值,HE染色观察病理学结果并行肺损伤评分;采用免疫组化法测定MUC5AC表达,采用Western blot法检测TLR4、MyD88、p38丝裂原活化蛋白激酶(p38MAPK)和NF-κB表达水平,采用RT-PCR法测定MUC5AC mRNA表达水平。结果与S组比较,VILI组和P组PaO2降低,肺组织W/D比值和肺损伤评分升高,MUC5AC及其mRNA表达上调,BALF中IL-1β、IL-6和TNF-α浓度升高,肺组织TLR4、p38MAPK、MyD88和NF-κB表达上调(P<0.01)。与VILI组比较,P组PaO2升高,肺组织W/D比值和肺损伤评分降低,MUC5AC及其mRNA表达下调,BALF中IL-1β、IL-6和TNF-α浓度降低,肺组织TLR4、p38MAPK、MyD88和NF-κB表达下调(P<0.05)。结论盐酸戊乙奎醚可降低大鼠VILI时气道MUC5AC的表达,其机制可能与抑制TLR4/MyD88信号通路的激活有关。

Objective To evaluate the effect of penehyclidine hydrochloride on the expression of airway mucin 5AC(MUC5AC)during ventilator-induced lung injury(VILI)and the relationship with Toll-like receptor 4/myeloid differentiation factor 88(TLR4/MyD88)signaling pathway in rats.Methods Thirty-six clean-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 200-250 g,were divided into 3 groups(n=12 each)using a random number table method:sham operation group(group S),VILI group(group VILI),and penehyclidine hydrochloride group(group P).The rats were tracheotomized in group S.The rats were tracheotomized,connected to a small animal ventilator and mechanically ventilated for 4 h with the tidal volume of 20 ml/kg,respiratory rate 80 breaths/min,inspiratory/expiratory ratio 1∶1,and inspired oxygen fraction ratio 21%in VILI and P groups.At 30 min before mechanical ventilation,penehyclidine hydrochloride 2 mg/kg was injected via the tail vein in group P,and the equal volume of normal saline was given instead in S and VILI groups.At 4 h of mechanical ventilation,the arterial blood samples were taken for measurement of PaO2.The rats were then sacrificed,and broncho-alveolar lavage fluid(BALF)was collected for determination of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)concentrations by enzyme-linked immunosorbent assay.The lung specimens were collected for calculation of the wet/dry weight ratio(W/D ratio),for examination of pathological changes which were scored after haematoxylin and eosin staining(under a light microscope),and for determination of the expression of MUC5AC(by immunohistochemistry),expression of TLR4,MyD88,p38 mitogen-activated protein kinase(p38MAPK)and nuclear factor-kappa B(NF-κB)in lung tissues(by Western blot),and expression of MUC5AC mRNA in lung tissues(by real-time polymerase chain reaction).Results Compared with group S,PaO2 was significantly decreased,the W/D ratio and lung injury score were increased,the expression of MUC5AC protein and mRNA was up-regulated,the concentrations of IL-1β,IL-6 and TNF-αin BALF were increased,and the expression of TLR4,p38MAPK,MyD88 and NF-κB was up-regulated in VILI and P groups(P<0.01).Compared with group VILI,PaO2 was significantly increased,the W/D ratio and lung injury score were decreased,the expression of MUC5AC protein and mRNA was down-regulated,the concentrations of IL-1β,IL-6 and TNF-αin BALF were decreased,and the expression of TLR4,p38MAPK,MyD88 and NF-κB was down-regulated in group P(P<0.05).Conclusion Penehyclidine hydrochloride can decrease the expression of airway MUC5AC during VILI,and the mechanism may be related to inhibiting activation of TLR4/MyD88 signaling pathway in rats.