TNF-α单抗对大鼠矽肺纤维化中NF-κB活化和iNOS表

Effects of tumor necrosis factor-α monoclonal antibody on nuclear factor-κB activation and inducible nitric oxide synthase expression in rats with silicotic fibrosis

目的探讨肿瘤坏死因子-α（TNF-α）单克隆抗体对矽尘诱导的大鼠肺纤维化中NF-κB活化和诱导型一氧化氮合酶（iNOS）表达的影响。方法将48只雄性wistar大鼠随机分为干预组、染尘组、对照组，各16只。干预组：气管内一次性注入50 mg二氧化硅粉尘，建模2~6 d，每天给予3 mg/kg，连续5 d TNF-α单克隆抗体皮下注射；染尘组：同样方法建模，等量生理盐水皮下注射；对照组：等量生理盐水气管内注入和皮下注射。各组分别于建模后7、14 d处死大鼠各8只。HE、Masson染色观察肺组织的形态学变化；ELISA法分别检测血中TNF-α含量；IHC检测肺组织NF-κBp65的表达；Western-blot法检测肺组织IκB蛋白表达量；RT-qPCR检测肺组织iNOS mRNA的转录水平。结果与对照组比较，染尘后7、14 d，肺组织炎症评分（3.375±1.061，2.500±0.535），血清TNF-α含量（86.405±20.494，77.064±11.829）ng/L，肺组织NF-κBp65阳性细胞吸光度值（0.297±0.05，0.287±0.039），iNOS mRNA表达（12.906±0.590，12.600±0.517），14 d纤维化程度评分（3.250±0.707）均明显升高，I-κB蛋白表达量（0.579±0.141，0.748±0.081）下调，差异有统计学意义（P〈0.05）。与染尘组比较，干预组肺组织7 d炎症程度评分（2.375±0.916），14 d纤维化评分（2.375±1.061），7、14 d血清TNF-α含量（66.565±19.850，58.734±16.335）ng/L，肺组织NF-κBp65阳性细胞吸光度值（0.248±0.028，0.238±0.027），7 d、14 d iNOS mRNA表达（11.656±0.405，12.025±0.618）下调，7、14 d I-κB蛋白表达量（0.802±0.165，0.888±0.144）上调，差异有统计学意义（P〈0.05）。结论TNF-α单克隆抗体通过抑制NF-κB信号通路的活化，下调iNOS表达，对矽尘诱导的大鼠肺组织起一定保护作用。

ObjectiveTo investigate the effects of tumor necrosis factor-α （TNF-α） monoclonal anti-body on nuclear factor-κB （NF-κB） activation and inducible nitric oxide synthase （iNOS） expression in rats with pulmonary fibrosis induced by silica dust.MethodsA total of 48 male Wistar rats were randomly divided into intervention group, silica dust exposure group, and control group, with 16 rats in each group. The rats in the intervention group were given intratracheal injection of 50 mg silicon dioxide dust once to establish a rat model and then treated with subcutaneously injected TNF-α monoclonal antibody 15 mg/kg for 5 consecutive days at 2-6 days after the establishment of the model. The rats in the silica dust exposure group were treated with the same method to establish the model and then given subcutaneous injection of the same volume of normal saline. The rats in the control group were given intratracheal and subcutaneous injection of normal saline. In both groups, 8 rats each were sacrificed at 7 and 14 days after the establishment of the model. Hematoxylin-eosin staining or Masson staining was used to observe morphological changes in lung tissue, ELISA was used to measure the serum level of TNF-α, IHC was used to measure the expression of NF-κBp65 in lung tissue, Western blot was used to measure the protein expression of I-κB in lung tissue, and RT-qPCR was used to measure the transcriptional level of iNOS mRNA in lung tissue.ResultsCompared with the control group, the silica dust exposure group had significant increases in the lung inflammation score （3.375±1.061 and 2.500±0.535） , serum TNF-α level （86.405±20.494 and 77.064±11.829） , absorbance of cells with positive NF-κBp65 in lung tissue （0.297±0.05 and 0.287±0.039） , and mRNA expression of iNOS （12.906±0.590 and 12.600±0.517） at 7 and 14 days after dust exposure, a significant increase in pulmonary fibrosis score at 14 days （3.250±0.707） , and significant reductions in the protein expression of I-κB at 7 and 14 days （0.579±0.141 and 0.748±0.081） （P〈0.05） . Compared with the silica dust exposure group, the intervention group had significant reductions in the lung inflammation score at 7 days （2.375±1.061） , pulmonary fibrosis score at 14 days （2.375±1.061） , serum level of TNF-α at 7 and 14 days （66.565±19.850 and 58.734±16.335） , absorbance of cells with positive NF-κBp65 in lung tissue at 7 and 14 days （0.248±0.028 and 0.238±0.027） , and mRNA expression of iNOS at 7 and 14 days （11.656±0.405 and 12.025±0.618） , as well as significant increases in the protein expression of I-κB at 7 and 14 days （0.802±0.165 and 0.888±0.144） （P〈0.05） .ConclusionTNF-α monoclonal antibody can inhibit the activation of the NF-κB signaling pathway and down-regulate the expression of iNOS, and thus exerts a certain protective effect on lung tissue in rats with pulmonary fibrosis induced by silica dust.