CaN/NFATc4信号通路在VILI大鼠肺组织炎症反应中的作用

Role of CaN/NFATc4 signaling pathway in ventilator-induced lung injury

目的评价钙调磷酸酶/活化T细胞核因子4(CaN/NFATc4)信号通路在机械通气相关性肺损伤(VILI)大鼠肺组织炎症反应中的作用.方法清洁级健康雄性Wistar大鼠24只,5~8周龄,体重220~250 g,采用随机数字表法分为3组(n=8):对照组(C组)、VILI组及环孢霉素A+VILI组(CsA+VILI组).采用VT 40 ml/kg、通气频率40次/min、机械通气6 h的方法制备VILI模型;CsA+VILI组机械通气前1 h腹腔注射CaN特异性抑制剂环孢霉素A 10 mg/kg.机械通气结束即刻,取肺组织行HE染色,行肺损伤评分;收集支气管肺泡灌洗液(BALF),采用ELISA法检测TNF-α、IL-1β及IL-6浓度;取肺组织,确定湿重/干重比值(W/D比值),采用RT-PCR法检测肺组织细胞间黏附分子-1(ICAM-1)mRNA及血管细胞粘附分子-1(VCAM-1)mRNA表达,采用Western blot法检测CaN和NFATc4的表达.结果与C组比较,VILI组肺组织W/D比值、肺损伤评分、BALF IL-1β、IL-6及TNF-α浓度升高,肺组织CaN、NFATc4、ICAM-1 mRNA及VCAM-1 mRNA表达上调(P<0.05).与VI-LI组比较,CsA+VILI肺组织W/D比值、肺损伤评分、BALF IL-1β、IL-6及TNF-α浓度降低,肺组织CaN、NFATc4、ICAM-1 mRNA及VCAM-1 mRNA表达下调(P<0.05).结论 CaN/NFATc4信号通路介导了VILI大鼠肺组织炎症反应.

Objective To evaluate the role of calcineurin ( CaN)/nuclear factor of activated T cell cytoplasmic 4 protein ( NFATc4) signaling pathway in inflammatory responses in lung tissues of rats with ventilator-induced lung injury ( VILI). Methods Twenty-four clean-grade healthy male Wistar rats, aged 5-8 weeks, weighing 220-250 g, were divided into 3 groups ( n=8 each) using a random number table method: control C (group C), VILI group and cyclosporine A plus VILI group (group CsA+VILI). The animals were anesthetized with pentobarbital and tracheostomized. The rats were mechanically ventilated for 6 h with the tidal volume set at 40 ml/kg and respiratory rate at 40 breaths/min to establish the model of VI-LI. The rats kept spontaneous breathing in group C. CaN specific inhibitor cyclosporine A 10 mg/kg was in-traperitoneally injected at 1 h before ventilation in group CsA+VILI. Rats were sacrificed immediately after ventilation, lung tissues were obtained and stained with hematoxylin and eosin to evaluate lung injury, broncho-alveolar lavage fluid was collected for determination of tumor necrosis factor-alpha ( TNF-α), inter-leukin-1beta ( IL-1β) and IL-6 concentrations by enzyme-linked immunosorbent assay, and the lungs were removed for determination of the wet to dry weight ratio ( W/D ratio), expression of intercellular adhesion molecule-1 ( ICAM-1) and vascular cell adhesion molecule-1 ( VCAM-1)( by real-time polymerase chain reaction), and expression of calcineurin and NFATc4 in lung tissues ( using Western blot ). Results Compared with group C, the W/D ratio, lung injury scores and concentrations of IL-1β, IL-6 and TNF-αin BALF were significantly increased, and the expression of CaN, NFATc4, ICAM-1 mRNA and VCAM-1 mRNA was up-regulated in group VILI ( P<0. 05). Compared with group VILI, the W/D ratio, lung injury scores and concentrations of IL-1β, IL-6 and TNF-αin BALF were significantly decreased, and the expres-sion of CaN, NFATc4, ICAM-1 mRNA and VCAM-1 mRNA was down-regulated in group CsA+VILI ( P<0. 05). Conclusion CaN/NFATc4 signaling pathway mediates inflammatory responses in lung tissues of rats with VILI.