miR-155干预烧伤急性肺损伤模型大鼠:核转录因子κB通路的变化

Effect of miR-155 on acute lung injury in burned rats: changes of nuclear factor-kappa B pathway

背景:目前有研究关注核转录因子κB通路在烧伤大鼠急性肺损伤病理过程中的作用及机制,如miR-155靶向抑制KB激酶,进而减弱核转录因子κB活性,在烧伤大鼠急性肺损伤发挥作用,然而仍存在病理机制有待研究和确认。目的:观察miR-155通过核转录因子κB通路对烧伤大鼠急性肺损伤的影响。方法:采用温水水浴模拟烫伤法建立烧伤急性肺损伤大鼠模型,将烧伤大鼠随机分为烧伤急性肺损伤组、miR-155增强试剂组和miR-155抑制试剂组,液体复苏后,miR-155增强试剂组、miR-155抑制试剂组大鼠分别尾静脉注入5μL的miR-155-mimics和miR-155-inhibitions。酶联免疫吸附剂测定肺泡灌洗液中肿瘤坏死因子α、白细胞介素1β的变化情况;苏木精-伊红染色法观察3组肺组织形态变化;Western blot法检测核转录因子κB及环氧化酶2蛋白表达;免疫组织化学染色检测肺组织核转录因子κB蛋白表达。结果与结论:①苏木精-伊红染色结果表明,miR-155抑制试剂组、烧伤急性肺损伤组及miR-155增强试剂组肺组织损伤程度,逐渐加重(P<0.05);②酶联免疫吸附实验结果表明,与烧伤急性肺损伤组相比,miR-155增强试剂组肿瘤坏死因子α、白细胞介素1β表达增加(P<0.05),而miR-155抑制试剂组肿瘤坏死因子α、白细胞介素1β表达降低(P<0.05);③Western结果表明,与烧伤急性肺损伤组相比,miR-155增强试剂组核转录因子κB、环氧化酶2蛋白表达增加(P<0.05),而miR-155抑制试剂组核转录因子κB、环氧化酶2蛋白表达降低(P<0.05);④免疫组织化学染色结果显示,miR-155抑制试剂组核转录因子κB蛋白表达增强,呈深棕色,中性粒细胞、单核巨噬细胞、肺泡上皮细胞的胞质及胞核内核转录因子κB表达最明显;⑤上述数据证实,肺组织细胞中核转录因子κB活性降低,可以通过下调miR-155来实现,从而降低肺损伤组织间的炎症反应。实验于2018年6月经内江市第一人民医院动物伦理委员会批准(批准号:1801270)。

BACKGROUND:Currently,studies have focused on the role and mechanism of nuclear factor-kappa B pathway in the pathological process of acute lung injury in burned rats,such as the targeting inhibition ofκB kinase by miR-155,which further weakens the activity of nuclear factor-κB and plays a role in acute lung injury in burned rats.However,there are still some pathological mechanisms to be studied and confirmed.OBJECTIVE:To investigate the effect of miR-155 on acute lung injury in burned rats through nuclear factor-κB pathway.METHODS:The rat models of acute lung injury were established by warm water bath simulating burn injury.The burned rats were divided into acute lung injury,miR-155-mimics and miR-155-inhibitor groups.After fluid resuscitation,the rats in the miR-155-mimics and miR-155-inhibitor groups were injected into the tail vein of 5μL of miR-155-mimics and miR-155-inhibitions,respectively.The expression levels of tumor necrosis factor-αand interleukin-1βin bronchoalveolar lavage fluid were detected by ELISA.The lung morphology in the three groups was observed by hematoxylin-eosin staining.The protein expression levels of nuclear factor-κB and cyclooxygenase 2 were evaluated by western blot assay.The nuclear factor-κB protein in lung tissues was detected by immunohistochemistry.RESULTS AND CONCLUSION:(1)The results of hematoxylin-eosin staining showed that the severity of lung injury in the miR-155-inhibitor group,acute lung injury group and the miR-155-mimics group was increased gradually(P<0.05).(2)ELISA results showed that compared with the acute lung injury group,the expression levels of tumor necrosis factor-αand interleukin-1βwere increased in the miR-155-mimics group(P<0.05),and decreased in the miR-155-inhibitor group(P<0.05).(3)Western blot assay results showed that compared with the acute lung injury group,the expression levels of nuclear factor-κB and cyclooxygenase 2 proteins were increased in the miR-155-mimics group(P<0.05),and decreased in the miR-155-inhibitor group(P<0.05).(4)Immunohistochemical results showed that the expression level of nuclear factor-κB was increased in the miR-155-inhibitor group,which was dark brown.The expression of nuclear factor-κB in cytoplasm and nucleus of neutrophils,mononuclear macrophages,alveolar epithelial cells was the most obvious.(5)These results indicate that in lung tissue cells,decreased miR-155 can down-regulate nuclear factor-κB activity,which reduces the inflammatory response of the lung between the damaged tissue.The study was approved by the Laboratory Animal Ethics Committee of the First People’s Hospital of Neijiang,approval No.1801270.