调控Rac1/AKT/NF-κB信号通路对呼吸机相关性肺损伤大鼠的影响

Effect of the regulation of Rac1/AKT/NF-κB signaling pathway on ventilator-induced lung injury in rats

目的:探讨调控Ras相关C3肉毒素底物1/丝氨酸—苏氨酸蛋白酶/核转录因子-κB(Rac1/AKT/NF-κB)信号通路对呼吸机相关性肺损伤(VILI)大鼠的影响。方法:将30只SD大鼠随机分为3组:自主呼吸组、高潮气量(VT)组和Rac1抑制剂(NSC23766)组,每组10只。NSC23766组大鼠经气管导管缓慢滴入200μL NSC23766预处理1h后,与高VT组大鼠同时以40mL/kg机械通气4h,建立VILI模型。计算各组肺湿/干重比(W/D)值,观察肺组织病理学结构改变。采用BCA法测定BALF中总蛋白水平,酶联免疫吸附试验(ELISA)法检测血清和支气管肺泡灌洗液(BALF)中白细胞介素(IL)-1β、IL-6及Rac1含量,qPCR法检测肺组织NF-κB、Rac1 mRNA相对表达量,western blotting法检测肺组织AKT、磷酸化(p)-AKT和NF-κB蛋白表达水平。结果:自主呼吸组大鼠肺部无明显病理变化;高VT组可见明显肺泡腔融合,肺泡间隔增宽,大量炎性细胞聚集;NSC23766组仅见轻微水肿及少量炎性细胞浸润。与自主呼吸组比较,高VT组和NSC23766组肺组织W/D值、BALF中总蛋白和Rac1含量及血清和BALF中IL-1β、IL-6水平均明显升高(P<0.05),Rac1、NF-κB mRNA相对表达及AKT、p-AKT、NF-κB蛋白表达均明显上调(P<0.05),且高VT组上述指标均明显高于NSC23766组(P<0.05)。结论:Rac1/AKT/NF-κB通路参与了VILI的发生发展,应用该通路抑制剂NSC23766可减少炎性因子的释放,在一定程度上影响VILI的进展。

Objective:To explore whether Ras related C3 botulinum toxin substrate 1/protein kinase B/nuclear factor-κB(Rac1/AKT/NF-κB)pathway is involved in the process of ventilator induced lung injury(VILI)in rats.Methods:Thirty Sprague-Dawley(SD)rats were randomly divided into 3 groups(n=10 per group):spontaneous respiratory group,high tidal volume(VT)group and NSC23766 group.Rats in the NSC23766 group were given 200μL NSC23766 through tracheal catheter for 1 h,then received mechanical ventilation with 40 mL/kg VT to establish a VILI model.The lung wet-to-dry weight ratio(W/D)was calculated.The pathological changes of lung tissue was observed.The total protein content in bronchoalveolar lavage fluid(BALF)was measured using BCA method.The concentrations of interleukin(IL)-1βand IL-6 in serum and Rac1 in BALF were determined by enzyme linked immunosorbent assay(ELISA).The mRNA expressions of NF-κB and Rac1 were detected by fluorescent quantitative PCR(qPCR).The protein expressions of AKT,phosphorylated(p)-AKT,and NF-κB were determined by western blotting.Results:No obvious pathological changes of lung tissues were found in spontaneous respiratory group,while pulmonaryalveoli fusion,alveoli septum thickening,and inflammatory cells infiltration were observed in high VT group.In NSC23766 group,the lung tissue exhibited slight edema and infiltration of inflammatory cells.Compared with the spontaneous respiratory group,the W/D ratio,the total protein and Rac1 in BALF,and inflammatory cytokines(IL-1βand IL-6)levels in serum and BALF were significantly increased in high VT group and NSC23766 group(P<0.05),meanwhile,the expressions of NF-κB and Rac1 mRNA as well as the protein expressions of AKT,p-AKT and NF-κB were up-regulated(P<0.05),but the changes were more significant in high VT group(P<0.05).Conclusion:Rac1/AKT/NF-κB pathway was involved in the development of VILI.Rac1 inhibitor NSC23766 attenuated the release of inflammatory factors,thereby affecting the progress of VILI to some extent.