抗补体5a受体抗体对变应性鼻炎大鼠Treg/Th17免疫失衡的调控作用

Regulation of anti-complement 5a receptor antibody on Treg/Th17 immune imbalance in rats with allergic rhinitis

目的探究抗补体5a受体(C5aR)抗体对变应性鼻炎(AR)大鼠调节性T细胞(Treg)/辅助T淋巴细胞17(Th17)免疫失衡的调控作用及机制初探。方法60只Wistar大鼠随机分为对照组、模型组、抗C5aR抗体组、抗C5aR抗体+Jagged1组,每组15只,除对照组外,其余各组大鼠均用卵清蛋白(OVA)和氢氧化铝[Al(OH)_(3)]致敏法建立AR模型。造模成功后,抗C5aR抗体组腹腔注射10 mg/kg抗C5aR抗体,抗C5aR抗体+Jagged1组腹腔注射10 mg/kg抗C5aR抗体和5μL的10μg/mL Jagged1钛,对照组和模型组注射等体积的生理盐水,每周注射1次,持续4周。采用酶联免疫吸附试验(ELISA)检测血清免疫球蛋白E(IgE)、干扰素-γ(IFN-γ)、白细胞介素(IL)-6、IL-10和IL-17水平,HE染色和PAS染色观察鼻腔黏膜组织嗜酸性粒细胞与杯状细胞数目,免疫组织化学染色检测Notch1表达,Western blot检测RORγt、Foxp3、Notch1及Jagged1蛋白表达,流式细胞术检测外周血Treg和Th17亚群比例。结果各处理组大鼠建模后表观行为学评分明显高于对照组(P<0.05);与对照组比较,模型组血清中IgE水平升高,IFN-γ、IL-10水平下降,IL-6、IL-17水平升高,鼻腔黏膜组织中嗜酸性细胞粒数目和杯状细胞数目增加,RORγt蛋白表达上调,Foxp3蛋白表达则下调,Th17细胞比例增加,Treg细胞比例减少,Th17/Treg比值上升,Notch1与Jagged1蛋白表达升高,差异均有统计学意义(P<0.05);与模型组比较,抗C5aR抗体组血清IgE水平下降,IFN-γ、IL-10水平升高而IL-6、IL-17水平下降,鼻腔黏膜组织中嗜酸性细胞粒数目和杯状细胞数目减少,RORγt蛋白表达下调、Foxp3蛋白表达上调,Th17细胞比值减少,Treg细胞比例增加,Th17/Treg比例降低,Notch1与Jagged1蛋白表达降低,差异均有统计学意义(P<0.05);而相较于抗C5aR抗体组,经过抗C5aR抗体与Jagged1肽作用的大鼠,各检测指标改善情况又受到明显抑制,差异均有统计学意义(P<0.05)。结论抗C5aR抗体可改善AR大鼠的症状,并且可调节机体内Treg/Th17免疫平衡,抑制Notch1-Jagged1信号途径的激活。

Objective To explore the regulation and mechanism of anti-complement 5a receptor(C5aR)antibody on regulatory T cells/helper T lymphocytes 1(Treg/Th17)immune imbalance in rats with allergic rhinitis(AR).Methods A total of 60 Wistar rats were randomly divided into control group,model group,anti-C5aR antibody group,anti-C5aR antibody+Jagged1 group,15 rats in each group.Except for the control group,all rats in the other groups were treated with ovalbumin(OVA)and aluminum hydroxide[Al(OH)_(3)]sensitization method to establish AR model.After successful modeling,the anti-C5aR antibody group was intraperitoneally injected with 10 mg/kg anti-C5aR antibody,and the anti-C5aR antibody+Jagged1 group was intraperitoneally injected with 10 mg/kg anti-C5aR antibody and 5μL of 10μg/mL Jagged1 titanium,and the control and model groups were injected an equal volume of normal saline was injected once a week for 4 weeks.Enzyme linked immunosorbent assay(ELISA)method was used to detect serum immunoglobulin E(IgE),interferon-γ(IFN-γ),interleukin(IL)-6,IL-10 and IL-17 levels,HE staining and PAS staining were used to observe the number of eosinophils and goblet cells in nasal mucosa tissue,immunohistochemical staining was used to detect Notch1 expression,Western blot was used to detect RORγt,Foxp3,Notch1 and Jagged1 protein expression,and flow cytometry was used to detect the proportion of peripheral blood Treg and Th17 subsets.Results The behavioral scores of rats in each treatment group after modeling were significantly higher than those in the control group(P<0.05);compared with the control group,the serum IgE level in the model group increased,the IFN-γand IL-10 levels decreased,and the IL-6 and IL-17 levels increased,the number of eosinophil granules and goblet cells in the nasal mucosa tissue increased,the expression of RORγt protein was up-regulated,and the expression of Foxp3 protein was down-regulated,the proportion of Th17 cells increased,the proportion of Treg cells decreased,and the ratio of Th17/Treg increased,the expression of Notch1 and Jagged1 protein increased,and the differences were statistically significant(P<0.05).Compared with the model group,serum IgE levels in the anti-C5aR antibody group decreased,IFN-γ,IL-10 levels increased while IL-6,IL-17 levels decreased,and the number of eosinophil granules and goblet cells in the nasal mucosa tissue decreased,RORγt protein expression was down-regulated,Foxp3 protein expression was up-regulated,the proportion of Th17 cells decreased,the proportion of Treg cells increased,Th17/Treg ratio decreased,Notch1 and Jagged1 protein expression decreased,the differences were statistically significant(P<0.05).Compared with the anti-C5aR antibody group,the rats treated with anti-C5aR antibody and Jagged1 peptide showed significant inhibition of the improvement of each detection index,and the differences were statistically significant(P<0.05).Conclusion Anti-C5aR antibody could improve the symptoms of AR rats,regulate the immune balance of Treg/Th17 in the body,and inhibit the activation of Notch1-Jagged1 signal pathway.