鼻腔滴入γ干扰素对变应性鼻炎大鼠外周血Th17/Treg细胞及相关细胞因子的影响

Effects of intranasal infusion of IFN-γ on Th17/Treg cells in peripheral blood of rats with allergic rhinitis

目的探讨大鼠鼻腔滴入γ干扰素(IFN-γ)对变应性鼻炎(AR)大鼠外周血中辅助性T细胞17(Th17)、调节性T细胞(Treg)水平的影响。方法将40只Wistar雄性大鼠,按体质量编号、采用随机数字表法随机分为4组:对照组、致敏组、曲安奈德组、IFN-γ组,致敏阶段采用5级卵清蛋白(OVA)、氢氧化铝给予致敏组、曲安奈德组、IFN-γ组大鼠腹腔注射致敏以及OVA滴鼻激发造模,对照组腹腔注射以生理盐水替代,滴鼻以超纯水替代;给药阶段对照组不予处理,致敏组、曲安奈德组、IFN-γ组每只每侧鼻孔分别给予50μL PBS、3.5μg曲安奈德、1μg IFN-γ滴鼻,于末次给药结束24 h内行流式细胞术检测外周血Th17细胞、Treg细胞所占比例,1周内行定量酶联免疫吸附法(ELISA)检测外周血中白介素-17A、转化生长因子(TGF)-β1的浓度,并行苏木精-伊红(HE)染色,观察黏膜情况。结果与对照组[(0.2134±0.1495)%]相比,致敏组[(4.9757±1.5039)%]、曲安奈德组[(1.1693±0.3861)%]、IFN-γ组[(1.5320±0.5265)%]的Th17细胞比例都不同程度升高(F=51.532,P<0.05),除曲安奈德组、IFN-γ组间比较无统计学意义(P>0.05)外,其余各组间比较有统计学意义(P<0.05);与对照组[(22.9393±2.0995)%]相比,致敏组[(8.2232±2.1888)%]、曲安奈德组[(14.0875±1.3696)%]、IFN-γ组[(16.6143±1.62170)%]的Treg细胞比例不同程度降低(F=97.107,P<0.05),除曲安奈德组、IFN-γ组间比较无统计学意义(P>0.05)外,其余各组间比较有统计学意义(P<0.0083);与对照组[(5.0769±1.4356)pg/mL]相比,致敏组[(34.9005±11.6708)pg/mL]、曲安奈德组[(9.7338±1.8289)pg/mL]、IFN-γ组[(10.5246±2.1180)pg/mL]的IL-17A浓度升高(F=27.173,P<0.05),除曲安奈德组、IFN-γ组间比较无统计学意义(P>0.05)外,其余各组间比较有统计学意义(P<0.05);与对照组[(431.8816±67.3262)pg/mL]相比,致敏组[(173.8862±41.7082)pg/mL]、曲安奈德组[(246.0533±13.8412)pg/mL]、IFN-γ组[(290.0127±22.8856)pg/mL]的TGF-β1浓度降低(F=36.623,P<0.05),各组间比较有统计学意义(P<0.05)。与对照组大鼠黏膜相比,致敏组大鼠黏膜增厚、水肿,有大量炎性细胞浸润,细胞排列紊乱,杯状细胞增生,纤毛几乎不可见;曲安奈德组大鼠鼻黏膜层较致敏组变薄,水肿明显改善,炎性细胞明显降低,纤毛有所生长。IFN-γ组大鼠鼻黏膜层较致敏组变薄,水肿改善,仅见少量炎性细胞,纤毛有所生长。结论AR大鼠外周血中Th17、Treg细胞比例异常,伴有细胞因子IL-17A、TGF-β1表达水平的异常。IFN-γ可能通过下调AR大鼠外周血中Th17细胞比例及降低IL-17A水平,同时上调Treg细胞的比例及升高TGF-β1水平,对AR发挥治疗作用。

Objective To investigate the effects of interferon gamma(IFN-γ) on T helper cell 17(Th17) and regulatory T cell(Treg)(Th17/Treg) in peripheral blood of rats with allergic rhinitis(AR). Methods A total of 40 Wistar male rats were randomly divided into 4 groups: control group, sensitized group, triamcinolone acetonide group, and IFN-γ group. The control group was injected with saline intraperitoneally, and had ultra-pure water nasal drops. The other 3 groups were injected with ovalbumin(OVA) and aluminium hydroxide and had OVA nasal instillation to establish AR models. After that, the sensitized group was treated with 50 μL phosphate buffer(PBS), triamcinolone acetonide group was treated with 3.5 μg triamcinolone acetonide, and IFN-γ group was treated with 1 μg IFN-γ nasal drops on either nostril. The proportions of Th17 cells and Treg cells in peripheral blood were measured with flow cytometry within 24 hours after the last administration, and levels of interleukin-17 A(IL-17 A) and transforming growth factor(TGF-β1) were detected with quantitative enzyme-linked immunosorbent assay(ELISA) within 1 week. The nasal mucosa was observed with hematoxylin-eosin(HE) staining. Results Compared with control group [(0.213 4±0.149 5)%], the percentage of Th17 cells in sensitized group [(4.975 7±1.503 9) %], triamcinolone acetonide group [(1.169 3±0.386 1) %] and IFN-γ group [(1.532 0±0.526 5) %] increased in varying degrees(F=51.532, P<0.05), with no statistical difference between the triamcinolone acetonide group and IFN-γ group(P>0.05), but with differences among the other groups(P<0.05). Compared with control group [(22.939 3±2.099 5)%], the proportion of Treg cells in sensitized group [(8.223 2±2.1888)%], triamcinolone acetonide group [(14.087 5±1.369 6) %] and IFN-γ group [(16.614 3±1.621 7) %] decreased in varying degrees(F=97.107, P<0.05), with no difference between the triamcinolone acetonide group and IFN-γ group(P>0.05), but with differences among the other groups(P<0.008 3). Compared with control group [(5.076 9±1.435 6) pg/mL], the concentration of IL-17 A in sensitized group [(34.900 5±11.670 8)pg/mL], triamcinolone acetonide group [(9.733 8±1.828 9) pg/mL], and IFN-γ group [(10.524 6±2.118 0) pg/mL] increased(F=27.173, P<0.05), with no difference between the triamcinolone acetonide group and IFN-γ group(P>0.05), but with differences among the other groups(P<0.05). Compared with the control group [(431.881 6±67.326 2) pg/mL], the concentration of TGF-β1 in sensitization group [(173.886 2±41.708 2) pg/mL], triamcinolone acetonide group [(246.053 3±13.841 2) pg/mL] and IFN-γ group [(290.012 7±22.885 6) pg/mL] decreased(F=36.623, P<0.05), with no differences among the groups(P<0.05). Compared with control group, the nasal mucosa of sensitized group was thicker and more edematous, with infiltration of a large number of inflammatory cells, disordered cell arrangement, goblet cell proliferation and almost invisible cilia. The nasal mucosa of triamcinolone acetonide group was thinner, edema was improved, inflammatory cells were significantly reduced and cilia slightly grew. The nasal mucosa of IFN-γ group was thinner than that of sensitized group, edema was improved, only a few inflammatory cells were found and the cilia slightly grew. Conclusion The percentages of Th17 and Treg cells in peripheral blood of AR rats were abnormal, accompanied by abnormal expression of IL-17 A and TGF-β1. IFN-γ may play a therapeutic role in AR by decreasing the proportion of Th17 cells and level of IL-17 A, while increasing the proportion of Treg cells and level of TGF-β1.