NLRP3炎症小体在海水吸入性急性肺损伤中的表达和作用

Expression and effect of NLRP3 inflammasome in acute lung injury induced by seawater

目的探讨海水吸入型急性肺损伤大鼠肺组织中NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体表达的变化及介导的炎性因子在急性肺损伤(ALI)发生发展中的作用。方法将50只健康雄性SD大鼠随机分为5组,对照组,海水吸入1 h组,海水吸入3 h组,海水吸入6 h组,海水吸入9 h组,每组10只。采用经气管缓慢滴注(3 ml/kg)海水的方法制作大鼠损伤模型。制作大鼠肺脏石蜡切片并HE染色观察病理形态学变化。检测大鼠肺组织湿干比。ELISA检测测定各组肺组织中IL-1β和IL-18水平,RT-PCR检测肺组织中IL-1β、IL-18和NLRP3mRNA的表达。Western-blot检测肺组织中NLRP3蛋白表达。结果气管滴注海水后成功复制海水吸入性急性肺损伤模型。肺组织湿干比较对照组显著升高。病理形态学观察可见肺组织大量炎细胞浸润、水肿、间质增厚。各组大鼠血清中IL-1β和IL-18的水平随着时间增加逐渐升高,且在3～6 h达到顶峰,随后炎症因子的表达逐渐降低。与空白对照组比较,差异均有统计学意义(均P<0.05)。各组大鼠肺组织中IL-1β和IL-18的mRNA的表达水平与大鼠肺组织中IL-1β和IL-18的表达基本一致。与空白对照组比较,差异均有统计学意义(均P<0.05)。肺组织匀浆中NLRP3转录和翻译结果显示海水吸入刺激后,肺组织中NLRP3的mRNA和NLRP3蛋白含量变化含量随着时间明显逐渐增加,差异均有统计学意义(均P<0.05)。结论海水刺激下,NLRP3炎症小体介导的炎症反应参与了急性肺损伤发病过程并加重了肺损伤的程度,可能是海水急性肺损伤的发病机制之一,但其作用有待进一步证实。

Objective To investigate the expression and the role of NOD-like receptor thermal-protein domain related protein(NLRP3)in the lung tissues and its mediated inflammatory factors in the occurrence and development of acute lung injury(ALI).Methods 50 healthy male SD rats were randomly divided into 5 groups:the control group,the seawater inhalation 1h group,the seawater inhalation 3 h group,the seawater inhalation 6h group and the seawater inhalation 9 h group,with 10 rats in each group.The rat injury model was established by the transtracheal seawater slow infusion(3ml/kg).The rat lung paraffin sections were made and the pathomorphology was observed by HE staining.The wet/dry weight ratio of the lung tissue was detected in rats.The expressions of IL-1βand IL-18 in lung tissues were detected by ELISA.The expressions of IL-β,IL-18 and NLRP3 mRNA in lung tissue were detected by RT-PCR.The expression of NLRP3 protein in lung tissue was detected by Western-blot.Results The rat model of acute lung injury induced by seawater inhalation was successfully replicated.The wet/dry weight ratio of the lung tissue was significantly higher compared with the control group.There was a large number of inflammatory cell infiltration,edema and interstitial thickening in the lung tissue.The ELISA has revealed that the levels of IL-1βand IL-18 in the serum of rats gradually increase over time,reaching the peak in 3-6 hours,and then the expression of inflammatory factors gradually decreases.Compared with the results of the control group,the difference was statistically significant(P<0.05).The RT-PCR has found that the expression of mRNA of IL-1βand IL-18 in lung tissues of rats were similar to the expression of IL-1βand IL-18 in lung tissues of rats.Compared with results of the control group,the difference was statistically significant(P<0.05).Western Blot and RT-PCR have been used to detect the transcription and translation of NLRP3 in lung homogenate.The findings indicate that the mRNA and protein in NLRP3 in lung tissues increase gradually over time with the stimulation of seawater inhalation,and the difference was statistically significant(P<0.05).Conclusion NLRP3 inflammasome mediated inflammatory response may be involved in the pathogenesis of acute lung injury and aggravate the degree of lung injury,which may be one of the pathogenesis of acute lung injury induced by seawater.However,its role needs further confirmation.