NEMO结合域肽对急性呼吸窘迫综合征小鼠肺组织炎症与细胞凋亡的影响及机制研究

Impact and mechanism of NEMO binding domain peptide on pulmonary inflammation and apoptosis of lung tissues in mice with acute respiratory distress syndrome

目的探讨NEMO结合域肽(NBDP)对急性呼吸窘迫综合征(ARDS)小鼠肺组织炎症和细胞凋亡的影响及其机制。方法采用随机数字表法将36只雄性BALB/c小鼠分为生理盐水(NS)对照组、ARDS模型组、NBDP阴性对照组及6、12、18μg NBDP预处理组,每组6只。雾化吸入脂多糖(LPS)50μL制备ARDS小鼠模型;NS对照组吸入等量NS。在雾化吸入LPS前30 min,NBDP阴性对照组吸入非功能性NBDP类似物;NBDP预处理组分别吸入6、12、18μg NBDP 50μL。吸入LPS 6 h后处死小鼠取肺组织,观察肺组织病理损伤及水肿程度;采用蛋白质免疫印迹试验(Western blotting)检测肺组织中核转录因子-κB(NF-κB)信号通路蛋白〔NF-κB抑制蛋白(IκB)激酶α/β(IKKα/β)、IκBα和NF-κB p65〕的磷酸化(p-IKKα/β、p-IκBα、p-p65)水平及凋亡蛋白天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)的表达。收集支气管肺泡灌洗液(BALF),采用酶联免疫吸附试验(ELISA)检测髓过氧化物酶(MPO)、白细胞介素(IL-1β、IL-8)和肿瘤坏死因子-α(TNF-α)等炎症指标的水平。结果光镜下显示,ARDS模型组存在明显水肿、出血,肺泡结构破坏,透明膜形成等,符合ARDS肺组织病理学特征,说明ARDS模型制备成功。ELISA显示,ARDS模型组BALF中MPO、IL-1β、IL-8、TNF-α水平均明显高于NS对照组;NBDP阴性对照组上述炎症指标与ARDS模型组差异无统计学意义;NBDP预处理组MPO、IL-1β、IL-8、TNF-α水平均明显低于ARDS模型组,并呈一定剂量依赖性,以18μg NBDP作用更加显著,与ARDS模型组比较差异有统计学意义〔MPO(ng/L):393.32±19.35比985.87±101.50,IL-1β(ng/L):43.05±5.11比97.68±10.88,IL-8(ng/L):84.64±2.32比204.00±17.37,TNF-α(ng/L):229.13±17.03比546.73±62.72,均P<0.05〕。Western blotting显示,ARDS模型组p-IKKα/β、p-IκBα、p-p65及caspase-3蛋白表达均较NS对照组明显升高;NBDP阴性对照组NF-κB信号通路蛋白及凋亡蛋白表达与ARDS模型组差异无统计学意义;NBDP预处理组p-IKKα/β、p-IκBα、p-p65和caspase-3蛋白表达均较ARDS模型组明显降低,且呈一定剂量依赖性,以18μg NBDP作用更加显著,与ARDS模型组比较差异有统计学意义〔p-IKKα/β蛋白(p-IKKα/β/β-actin):0.15±0.02比0.42±0.04,p-IκBα蛋白(p-IκBα/β-actin):0.10±0.01比0.93±0.30,p-p65蛋白(p-p65/β-actin):0.22±0.05比1.37±0.21,均P<0.05〕。结论NBDP可剂量依赖性抑制ARDS肺组织炎症反应和细胞凋亡,其机制与干扰NF-κB信号通路转导有关。

Objective To investigate the effect of NEMO binding domain peptide(NBDP)on lung inflammation and apoptosis in mice with acute respiratory distress syndrome(ARDS)and its mechanism.Methods Thirty-six male BALB/c mice were divided into normal saline(NS)control group,ARDS model group,NBDP negative control group and 6,12 and 18μg NBDP pretreatment group by random number table method,with 6 mice in each group.ARDS mouse model was reproduced by aerosol inhalation lipopolysaccharide(LPS)50μL.An equivalent among of NS was inhaled in NS control group.The mice in NBDP negative control group were inhaled the materials similar to the non-functional NBDP 30 minutes before the aerosol inhalation LPS;6,12 and 18μg of NBDP 50μL were respectively inhaled in NBDP pretreatment groups.After inhalation of LPS for 6 hours,mice were sacrificed to get lung tissue and observe the degree of pathological injury and edema.Western blotting was used to detect the phosphorylation of nuclear factor-κB(NF-κB)pathway related proteins[NF-κB inhibitor(IκB)kinaseα/β(IKKα/β),IκBαand NF-κB p65;p-IKKα/β,p-IκBα,p-p65]and the expression of caspase-3 in lung tissue.The bronchoalveolar lavage fluid(BALF)was collected and the levels of inflammatory markers such as myeloperoxidase(MPO),interleukins(IL-1β,IL-8),and tumor necrosis factor-α(TNF-α)were detected by enzyme linked immunosorbent assay(ELISA).Results ARDS model group had severe edema and hemorrhage,alveolar structure destruction,pulmonary hemorrhage and hyaline membrane formation etc.under light microscope,consistent with the pathological characteristics of ARDS lung tissue,suggesting that the ARDS model was successfully reproduced.ELISA showed that MPO,IL-1β,IL-8 and TNF-αlevels of BALF in ARDS model group were obviously higher than those in NS control group.There were no significant differences in the above inflammatory indicators between NBDP negative control group and ARDS model group.The levels of MPO,IL-1β,IL-8 and TNF-αin NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner,especially in 18μg NBDP,the differences were statistically significant as compared with ARDS model group[MPO(ng/L):393.32±19.35 vs.985.87±101.50,IL-1β(ng/L):43.05±5.11 vs.97.68±10.88,IL-8(ng/L):84.64±2.32 vs.204.00±17.37,TNF-α(ng/L):229.13±17.03 vs.546.73±62.72,all P<0.05].Western blotting showed that p-IKKα/β,p-IκBα,p-p65 and caspase-3 protein expressions in ARDS model group were significantly higher than those in NS control group.There was no significant difference in above NF-κB pathway and apoptosis-related protein expression between the NBDP negative control group and ARDS model group.The p-IKKα/β,p-IκBα,p-p65 and caspase-3 protein expression in NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner,especially in 18μg NBDP,the differences were statistically significant as compared with ARDS model group[p-IKKα/βprotein(p-IKKα/β/β-actin):0.15±0.02 vs.0.42±0.04,p-IκBαprotein(p-IκBα/β-actin):0.10±0.01 vs.0.93±0.30,p-p65 protein(p-p65/β-actin):0.22±0.05 vs.1.37±0.21,all P<0.05].Conclusion NBDP can inhibit inflammatory response and apoptosis in ARDS lung tissue in a dose-dependent manner,and its mechanism is associated with interference NF-κB signaling pathway transduction.