c-Jun氨基端激酶介导细胞自噬参与肠缺血再灌注肺损伤

C-jun N-terminal kinase-mediated autophagy participates in intestinal ischemia/reperfusion lung injury in rats

目的探讨c-Jun氨基端激酶(JNK)信号通路活化在肠缺血再灌注(II/R)肺损伤的作用及其机制。方法夹闭6~8周龄雄性SD大鼠肠系膜上动脉[SCXK(京)2016-006]建立II/R肺损伤模型,首先用随机数据表法将36只大鼠随机分为假手术组(Sham)及肠缺血45 min后再灌注即刻(0)、2、6、8、16 h组(每组6只),蛋白质印迹法(Western blot)检测II/R肺组织磷酸化JNK(p-JNK)及活化蛋白-1(AP-1)蛋白表达;然后再取24只大鼠随机分为Sham组,II/R+溶剂对照组(II/R),II/R+JNK抑制剂组(JNK^(-)),II/R+JNK激动剂组(JNK^(+)),每组6只,JNK抑制剂SP600125或JNK激动剂Anisomycin预处理大鼠,II/R后6 h处死大鼠提取肺组织,Western blot方法检测肺磷酸化JNK(p-JNK)、AP-1蛋白水平,免疫荧光检测自噬相关蛋白Beclin-1、微管相关蛋白1轻链3B(LC3B)及p62表达,测量肺组织湿/干重比(W/D)及苏木精-伊红(HE)染色显微镜观察肺组织病理性学改变,观察II/R肺损伤情况。所有数据符合正态分布采用单因素方差分析。结果肠缺血后再灌注即刻、2、6、8、16 h肺组织p-JNK蛋白表达显著高于Sham组(1.51±0.01比1.000,q=42.2;2.32±0.01比1.000,q=109.1;4.70±0.03比1.000,q=305.6;3.71±0.04比1.000,q=223.5;2.43±0.01比1.000,q=118.0;P<0.01),II/R后肺组织W/D明显高于sham组(15.37±0.53比9.64±0.11,q=21.2,P<0.01),JNK^(-)组肺p-JNK明显低于II/R组(1.13±0.01比2.46±0.01,q=464.2,P<0.01)。免疫荧光检测显示SP600125预处理大鼠II/R后肺组织Beclin-1、LC3-BⅡ明显低于II/R组,p62蛋白表达高于II/R组,肺W/D显著低于II/R组(10.90±0.73比15.37±0.53,q=16.6,P<0.01),组织学检测肺损伤及炎细胞浸润减轻;而JNK^(+)组肺p-JNK及Beclin-1、LC3-BⅡ显著升高,p62明显降低,肺组织W/D升高,肺间质增厚、炎细胞浸润明显增多,部分肺泡内出血。结论II/R导致肺JNK/AP-1信号通路持续活化,通过自噬增强加重II/R肺损伤,抑制JNK信号通路活化通过减低自噬减轻II/R肺损伤。

Objective To investigate the mechanism of c-jun N-Terminal Kinase(JNK)signaling pathway activation on lung injury induced by intestinal ischemia/reperfusion(II/R).Methods The SD rat model of II/R lung injury was established by clipping the superior mesenteric artery.A total of 36 animals were randomly divided into Sham operation group(Sham)and reperfusion groups of 0,2,6,8 and 16 h after 45 min of intestinal ischemia,and then the dynamic changes of phosphorylation JNK(p-JNK)and AP-1 protein in II/R lung tissue were detected by Western blotting.A total of 24 rats were randomly divided into Sham group,II/R+DMSO control group(II/R),II/R+JNK inhibitor group(pretreated with JNK specific inhibitor SP600125)(JNK^(-)),II/R+JNK agonist group(pretreated with JNK agonist Anisomycin)(JNK^(+)).The animals were sacrificed at 6 h after II/R.The p-JNK and AP-1 protein expression was detected in lung tissue by Western blotting,and Beclin-1,microtubule-associated protein 1 light chain 3B(LC3B)and p62 proteins were detected by immunofluorescence assay.The wet/dry weight ratio(W/D)of the lung tissue was determined and the pulmonary pathological changes were observed under an optical microscope by hematoxylin and eosin(HE)staining.All data were analyzed by one-way ANOVA,and P<0.05 indicated that the difference was significant.Results The expression of p-JNK protein in lung tissue immediately,2,6,8 and 16 h after II/R was significantly higher than that in sham group(1.51±0.01 vs.1.00,q=42.2;2.32±0.01 vs.1.00,q=109.1;4.70±0.03 vs.1.00,q=305.6;3.71±0.04 vs.1.00,q=223.5;2.43±0.01 vs.1.00,q=118.0;P<0.01),and the W/D in lung tissue after II/R was significantly higher than that in sham group(15.37±0.53 vs.9.64±0.11,q=21.2,P<0.01),the lung p-JNK of JNK^(-)group was significantly lower than that of II/R(1.13±0.01 vs.2.46±0.01,q=464.2,P<0.01).The immunofluorescence test showed that Beclin-1 and LC3-BⅡin lung tissue of rats pretreated with SP600125 after II/R were significantly lower than those in II/R group,the expression of p62 protein was higher than that in II/R group,and the lung W/D was significantly lower than that in II/R group(10.90±0.73 vs.15.37±0.53,q=16.6,P<0.01).The histological examination showed that lung injury and inflammatory cell infiltration were alleviated;lung p-JNK,Beclin-1 and LC3-BⅡin the JNK^(+)group were significantly increased,p62 was significantly decreased,lung tissue W/D was increased,lung interstitial thickening,inflammatory cell infiltration was significantly increased,and partial alveolar hemorrhage was observed.Conclusion II/R leads to continuous activation of lung JNK/AP-1 signaling pathway,which aggravates II/R lung injury through autophagy enhancement,and inhibition of JNK signaling pathway activation reduces II/R lung injury by reducing autophagy.