咽立爽口含滴丸对慢性咽炎家兔免疫细胞亚群及NF-κB信号通路的影响

Effects of Pharyngeal Refreshing Pills on Immune Cell Subsets and NF-κB Signaling Pathway in Chronic Pharyngitis

目的探究咽立爽口含滴丸对慢性咽炎家兔模型的作用效果及机制。方法健康新西兰白兔42只,随机分为7组:空白组,模型组,慢严舒柠清喉利咽颗粒组(9 g/kg),咽立爽口含滴丸低(0.025 g/kg)、中(0.05 g/kg)、高(0.1 g/kg)剂量组和雾化组(0.025 g/kg)。除空白组外,其余家兔均采用化学刺激法建立慢性咽炎动物模型。造模成功后第2天起,5个治疗组每日分别以慢严舒柠清喉利咽颗粒、咽立爽口含滴丸相应剂量进行不同治疗,而空白组和模型组以蔗糖(0.025 g/kg)置于家兔口腔,连续给药7 d。从造模第3天开始,每天观察家兔咽喉壁局部情况,给药结束后进行采血,处死家兔后采样待检。HE染色观察咽喉壁黏膜组织病理学变化;流式细胞术检测血液CD3^(+)、CD4^(+)、CD8^(+),计算CD4^(+)/CD8^(+)水平;ELISA检测血清白细胞介素-6(IL-6)、白细胞介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)、C反应蛋白(CRP)和总抗氧化能力(T-AOC)水平;蛋白质印迹法检测咽喉壁黏膜组织IL-6、IL-1、TNF-α、核转录因子κB(NF-κB p65)、核转录因子-κB抑制因子(IκB-α)蛋白表达;实时荧光定量PCR检测咽喉壁黏膜组织NF-κB p65、IκB-αmRNA表达。结果与空白组比较,慢性咽炎家兔咽喉壁黏膜病理组织损伤;CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)和T-AOC水平显著降低,CD8^(+)、IL-6、IL-1、TNF-α、CRP、NF-κB p65和IκB-α水平明显升高(P<0.01)。与模型组比较,不同治疗组均可不同程度减轻咽喉壁黏膜组织病理损伤;咽立爽口含滴丸高剂量组及雾化组CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)和T-AOC水平显著升高,CD8^(+)、IL-6、IL-1、TNF-α、CRP、NF-κB p65和IκB-α水平均降低(P<0.05),效果优于低、中剂量及慢严舒柠清喉利咽颗粒组。结论咽立爽口含滴丸可显著改善慢性咽炎家兔模型的炎性指标,减轻组织病理损伤,调节NF-κB信号通路相关因子表达,且雾化用药的效果更明显。

Objective To explore the effect and mechanism of pharyngeal refreshing pills on an animal model of chronic pharyngitis.Methods A total of 42 healthy New Zealand white rabbits were randomly divided into seven groups:control,model,manyanshuning granules(9 g/kg),pharyngeal refreshing pills at low-(0.025 g/kg),medium-(0.05 g/kg),high-(0.1 g/kg)doses,and atomization(0.025 g/kg)groups.Animal models of chronic pharyngitis were established by chemical stimulation in all rabbits except the control group.From the second day after successful modeling,the five treatment groups were administered with manyanshuning granules and different doses of pharyngeal refreshing pills every day,while the control and model groups were fed with white sugar(0.025 g/kg)for 7 days.From the third day after successful modeling,the local condition of the pharyngeal wall of each rabbit was observed every day.Blood samples were collected at the end of drug administration and examined.Histopathological changes in the pharyngeal wall mucosa were examined by hematoxylin-eosin staining,and the levels of CD3^(+),CD4^(+),and CD8^(+)were detected by flow cytometry.The levels of interleukin-6(IL-6),IL-1,tumor necrosis factor-α(TNF-α),C-reactive protein(CRP),and total antioxidant capacity(T-AOC)in serum were detected by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the expression of IL-6,IL-1,TNF-α,nuclear factor-κappa B(NF-κB)p65 and NF-κB inhibitor(IκB-α)at the protein level in the pharyngeal wall mucosa.qRT-PCR was used to detect NF-κB p65 and IκB-αmRNA expression in the pharyngeal wall mucosa.Results Compared with the control group,the pathological tissues of the posterior pharyngeal mucosa of rabbits with chronic pharyngitis were significantly damaged;the levels of CD3^(+),CD4^(+),CD4^(+)/CD8^(+),and T-AOC were significantly decreased,while the levels of CD8^(+),IL-6,IL-1,TNF-α,CRP,NF-κB p65,and IκB-αwere significantly increased(all P<0.01).Compared with the model group,the pathological injury of pharyngitis could be alleviated to different degrees in the treatment group;the levels of CD3^(+),CD4^(+),CD4^(+)/CD8^(+),and T-AOC in the pharyngeal refreshing pills at high dose and atomization groups were significantly increased,while the levels of CD8^(+),IL-6,IL-1,TNF-α,CRP,NF-κB p65,and IκB-αwere significantly decreased(all P<0.05).The effect of high-dose pharyngeal refreshing pill administration was better than that of low-dose and middle-dose pharyngeal refreshing pills,and manyanshuning granules.Conclusion The pharyngeal refreshing pills can significantly improve the inflammatory index of chronic pharyngitis,reduce pathological injury,affect the expression of NF-κB signaling pathway-related factors,and the effect of atomization dose is more obvious.