Objective: To observe the effect of tumor necrosis factor-α (TNF-α) monoclonal antibody on autophagy in allergic rhinitis (AR) mice. Methods: Thirty six weeks old BALB/c mice were randomly divided by random number table method into five groups: control group, model group (AR group), TNF-α antibody intervention group (AR+TNF-α group), autophagy inhibitor (3-methylindole, 3-NA) intervention group (AR+3-MA group), TNF-α antibody combined with autophagy inducer rapamycin (RAP) intervention group (AR+TNF-α+RAP group), with 6 mice in each group. AR model was established by conventional method, the corresponding reagent was administered before nasal cavity stimulation sensitization and during the whole experiment. Behavioral scores of mice were obtained, blood was collected from the eye socket, and mice in each group were sacrificed to collect nasal mucosa tissue samples. Pathological changes of nasal mucosa were observed by hematoxylin-eosin staining. Expression levels of inflammatory factor and IgE in serum were detected by enzyme-linked immunosorbent assay (ELISA). Expressions of autophagy related indicators microtubule-associated protein-1 light chain-3B (LC3B), Beclin-1, sequestosome1 (p62), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7) were measured by Real-time PCR and Western blot. The aggregation of LC3B protein was observed by immunofluorescence. SPSS 19.0 software was used for statistical analysis. Results: Compared with the AR model group, symptoms of AR in AR+TNF-α group and AR+3-MA group were mild; the pathological changes of nasal mucosa were weak; the expression of IgE, TNF-α, interleukin 4 (IL-4), interferon-γ (IFN-γ) in serum significantly reduced (IgE: 666.19±78.35 (x±s) vs. 692.38±64.29 vs. 1 059.05±146.44, TNF-α: 112.06±12.95 vs. 113.17±15.43 vs. 161.22±17.96, IL-4: 54.05±7.14 vs. 58.26±5.67 vs. 79.95±6.33, IFN-γ: 28.58±4.51 vs. 30.67±2.60 vs. 39.83±3.31, all P<0.05), and the expression of LC3B Ⅱ/Ⅰ, Beclin-1, ATG5, ATG7 in nasal mucosa significantly decreased, the expression of p62 significantly elevated. After intervention with autophagy inducer RAP, the therapeutic effect of TNF-α monoclonal antibodies on AR was antagonized. Conclusion: TNF-α monoclonal antibody significantly improves nasal symptoms in AR mice by inhibiting autophagy levels.
目的： 观察肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）单克隆抗体对变应性鼻炎（allergic rhinitis，AR）模型小鼠自噬的影响。 方法： 30只6周龄雌性BALB/c小鼠，随机数字表法分为对照组、模型组（即AR组）、TNF-α抗体干预组（即AR+TNF-α组）、自噬抑制剂3-甲基嘌呤（3-methylindole, 3-MA)干预组（即AR+3-MA组）、TNF-α抗体联合自噬诱导剂雷帕霉素（rapamycin, RAP）干预组（即AR+TNF-α+RAP组）5个组，每组6只。常规方法制作AR模型，于鼻腔激发致敏前给予相应试剂，并持续整个实验过程。对小鼠进行行为学评分，眼窝静脉采血，处死小鼠收集鼻黏膜组织，苏木精-伊红染色观察鼻黏膜病理学变化，酶联免疫吸附测定法检测血清中炎性因子及IgE的表达，实时定量聚合酶链反应结合蛋白印迹法检测自噬相关指标微管相关蛋白1轻链3B（microtubule-associated protein-1 light chain-3B，LC3B）、Beclin-1、死骨片重组蛋白1（sequestosome1，p62）、自噬相关基因5（autophagy-related 5，ATG5）、自噬相关基因7（autophagy-related 7，ATG7）的表达，免疫荧光观察LC3B蛋白点状聚集情况。应用SPSS 19.0软件进行统计学分析。 结果： 同AR组比较，AR+TNF-α及AR+3-MA组小鼠AR症状较轻；鼻黏膜病理学变化较弱；血清IgE、TNF-α、白细胞介素4（interleukin 4，IL-4）、γ干扰素（interferon-γ，IFN-γ）的表达显著减少[IgE：666.19±78.35（x±s，下同）比692.38±64.29比1 059.05±146.44，TNF-α：112.06±12.95比113.17±15.43比161.22±17.96，IL-4∶54.05±7.14比58.26±5.67比79.95±6.33，IFN-γ：28.58±4.51比30.67±2.60比39.83±3.31，P值均<0.05]；鼻黏膜组织中LC3B Ⅱ/Ⅰ、Beclin-1、ATG5、ATG7的表达明显降低，p62的表达明显升高。采用自噬诱导剂RAP干预后，TNF-α单克隆抗体对AR的治疗作用被拮抗。 结论： TNF-α单克隆抗体可抑制自噬水平，显著改善AR小鼠鼻部症状。.
Keywords: Antibodies, monoclonal; Autophagy; Rhinitis, allergic; Tumor necrosis factor-alpha.