Effects of Immuno-modulation by Qingqiao Capsule on Secretory Otitis Media in Rats
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维普
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目的 探讨清窍胶囊通过AKT/mTOR/HIF-1α途径对大鼠分泌性中耳炎(secretory otitis media,SOM)的中耳黏膜免疫的调节作用.方法 将24只大鼠平均分为对照组、模型组、清窍胶囊低剂量治疗组、清窍胶囊高剂量治疗组,除对照组外,其余大鼠均建立中耳炎大鼠模型,采用苏木精-伊红染色观察大鼠中耳及咽鼓管的病理变化,采用免疫组化观察中耳黏膜组织中HIF-1α的含量,采用HypoxyprobeTM-1 Plus Kit检测中耳黏膜组织缺氧情况,采用流式细胞仪检测外周血辅助性T细胞的含量,采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELI-SA)检测中耳灌洗液中血管内皮生长因子(vascular endothelial growth factor,VEGF)、转化生长因子-β(transforming growth factor-β,TGF-β)、白细胞介素-23(interleukin-23,IL-23)、白细胞介素-10(interleukin-10,IL-10)、干扰素-γ(interferon-γ,IFN-γ)、白细胞介素-17(interleukin-17,IL-17)、白细胞介素-4(interleukin-4,IL-4)含量,采用免疫印迹检测中耳黏膜组织中蛋白激酶B(protein binase B,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)/缺氧诱导因子-1α(hypoxia-inducible factor 1α,HIF-1α)相关蛋白的表达.结果 清窍胶囊低剂量组、清窍胶囊高剂量组黏膜上皮坏死细胞、炎性细胞及纤维组织增生减少,中耳黏膜组织中HIF-1α含量,外周血中辅助性T细胞 17(T helper 17,Th17)/调节性T细胞(regulatory T Cell,Treg)和辅助性T细胞 1 型细胞(type 1 helper T Cell,Th1)/辅助性T细胞2 型细胞(type 2 helper T cells,Th2),中耳灌洗液中 IL-4、IL-10、IL-17、IL-23、IFN-γ、VEGF、TGF-β含量,中耳黏膜组织中p-AKT/AKT、p-mTOR/mTOR、HIF-1α蛋白表达均低于模型组,差异有统计学意义(P＜0.05).结论 清窍胶囊能够通过抑制AKT/mTOR/HIF-1α途径,调节免疫细胞因子分泌,从而缓解分泌性中耳炎大鼠的炎症反应及组织缺氧情况.
Objective To investigate the regulatory effects of Qingqiao capsule on ear mucosal immunity in rats with secretory otitis media(SOM)through the AKT/mTOR/HIF-1α pathway.Methods Twenty-four rats were equally di-vided into a control group,a modeling group,a low-dose(0.36 g/100 mL)Qingqiao capsule treatment group,and a high-dose(0.72 g/100 mL)Qingqiao capsule treatment group(n=6 each).Otitis media was modeled in all groups except the control group and HE staining was used to observe pathological changes in the middle ear and eustachian tube.Immuno-histochemical staining was used to observe expression of HIF-1α and HypoxyprobeTM-1 Plus Kit was used to detect tis-sue hypoxia in middle ear mucosa.Peripheral blood helper T cells counts were obtained using flow cytometry.ELISA was used to label presence of VEGF,TGF-β,IL-23,IL-10,IFN-γ,IL-17 and IL-4.Immunoblotting was used to detect expression of AKT/mTOR/HIF-1α related proteins in middle ear mucosa.Results Compared to no treatment,middle ear tissue specimens from Qingqiao treated animals(both low and high dose groups)demonstrated reduced epithelial cells necrosis,inflammatory cells and fibrous hyperplasia;decreased HIF-1α expression;less presence of Th1,Th2,Th17,Treg and Th17/Treg and Th1/Th2;less IL-4,IL-10,IL-17,IL-23,IFN-γ,VEGF and TGF-β in middle ear lavage;and de-creased expression of p-AKT,p-mTOR and HIF-1α proteins,with no significant difference in of AKT and mTOR pro-teins expressions.Conclusion Qingqiao Capsule can regulate the immune responses in secretory otitis media in rats by inhibiting the AKT/mTOR/HIF-1α pathway,mitigate inflammatory response and tissue hypoxia.
维普
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