目的:观察肿瘤坏死因子α(TNF-α)拮抗剂依那西普对博来霉素诱导的肺纤维化小鼠的抑制纤维化作用,并探讨依那西普治疗肺纤维化的可能机制.方法:将45只SPF级雌性昆明小鼠随机分为3组:对照组(气管内雾化生理盐水)、纤维化组(气管内博来霉素3 mg/kg溶于100 μL生理盐水内雾化)和依那西普干预组(气管内雾化博来霉素后,4 mg/kg依那西普溶于100 μL生理盐水内腹腔注射,每3d注射1次).处理后第28 d收集样本,小鼠左肺置于10％中性甲醛固定,石蜡包埋切片后行HE与Masson染色；右肺碱水解法检测组织羟脯氨酸(HYP)的含量；酶联免疫法检测血清TNF-α和转化生长因子β(TGF-β)的含量；提取肺组织总蛋白,Western blotting检测磷酸化ERK1/2、JNK和p38的表达.结果:依那西普干预组肺组织病理损伤及气道上皮下胶原沉积较纤维化组减轻,肺叶炎症损伤评分和纤维化评分明显下降(均P＜0.01),肺组织HYP含量显著降低(P＜0.05),血清TNF-α和TGF-β的浓度明显减少(均P＜0.01),肺组织ERK1/2、JNK和p38蛋白的磷酸化水平也显著下降(P＜0.01,P＜0.05,P＜0.01).结论:依那西普能显著下调TNF-α和TGF-β的水平,从而抑制ERK1/2、JNK和p38的活化,缓解博来霉素诱导的小鼠肺纤维化病变.%AIM:To evaluate the effect of tumor necrosis factor α (TNF-α) antagonist etanercept on bleomycin-induced lung fibrosis in mice..METHODS:Forty-five Kunming female mice were randomly divided into 3 groups:the mice in control group were intraperitoneally injected with vehicle and intratracheally administered with saline aerosol,the mice in bleomycin group were intraperitoneally injected with vehicle and intratracheally administered with bleomycin (3 mg/kg) aerosol,and the mice in bleomycin + etanercept group were intraperitoneally injected with etanercept (4 mg/kg) every 3 d and intratracheally administered with bleomycin aerosol.All animals were sacrificed 28 d after treatments.The left lung was fixed in 10％ neutral formalin and then stained with hematoxylin-eosin or Masson' s trichrome for the pathological examination.The tissues of right lung were sampled for measuring the content of hydroxyproline (HYP) by the method of alkaline hydrolysis.The serum concentrations of TNF-α and TGF-β were detected by ELISA.Total tissue protein was extracted for examination of ERK1/2,JNK and p38 by Western blotting.RESULTS:Etanercept prevented the collagen accumulation under the airway epithelium and decreased the scores of lung inflammation and fibrosis induced by bleomycin with significantly reduced the levels of tissue HYP,serum TNF-α and serum TGF-β.The protein phosphorylations of ERK/JNK/p38 in the lung tissues were remarkably decreased compared with BLM group.CONCLUSION:Etanercept decreases the phosphorylations of ERK1/2/JNK/p38 via inhibiting the expression of TNF-α and TGF-β.Etanercept might be useful in the treatment of pulmonary fibrosis.
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