臭氧对变应性鼻炎鼻黏膜NF-κB p65核蛋白表达及炎性因子的影响

Effects of ozone on nuclear protein expression of NF-κB p65 in nasal mucosa and inflammatory factors in a rat model of allergic rhinitis

目的探讨臭氧吸入对变应性鼻炎(AR)鼻黏膜NF-κB p65核蛋白表达及炎性因子的影响。方法选取健康Sprague-Dawley雌性大鼠48只,每组8只,随机数字表法将其分为6组:正常对照组(NC组)、AR模型组(即AR组)、正常大鼠臭氧吸入暴露组(即NE组,浓度1 ppm)、AR低浓度臭氧吸入暴露组(即AREL组,浓度0.5 ppm)、AR中浓度臭氧吸入暴露组(即AREM组,浓度1 ppm)、AR高浓度臭氧吸入暴露组(即AREH组,浓度2 ppm)。采用卵清蛋白(ovalbumin,OVA)抗原联合氢氧化铝佐剂致敏法制备AR大鼠模型。在AR造模的全程同步进行臭氧吸入暴露。将臭氧吸入暴露实验组置入臭氧吸入暴露系统,进行不同浓度的臭氧暴露,2 h/d,共6周。观察各实验组AR症状,记录大鼠鼻部喷嚏、挠鼻次数,并测量鼻分泌物量。末次暴露的24 h后,收集鼻腔灌洗液和鼻黏膜组织。免疫蛋白印迹法(Western blot)检测鼻黏膜NF-κB p65核蛋白表达。实时定量PCR(qRT-PCR)检测鼻黏膜NF-κB靶基因IL-6、IL-8和肿瘤坏死因子-α(TNF-α)的mRNA表达。ELISA检测血清OVA特异性IgE(OVA-sIgE)和鼻腔灌洗液中的促炎因子IL-6、IL-8和TNF-α的蛋白含量。大鼠鼻黏膜经苏木素-伊红(HE)染色,观察鼻黏膜病理学改变。采用SPSS 20.0软件进行数据分析。结果AR大鼠不同浓度臭氧暴露组的喷嚏数量、挠鼻次数及鼻分泌物的量较NC和AR组均明显增加(P<0.05)。AR臭氧各浓度暴露组血清OVA-slgE含量高于AR组及NC组(P<0.05),且AREH组升高最明显。AREM和AREH组鼻黏膜的NF-κB p65胞核内蛋白表达高于AR(P<0.05)。AR臭氧暴露组鼻黏膜IL-6、IL-8和TNF-α的mRNA表达升高,鼻腔灌洗液中TNF-α、IL-6和IL-8蛋白含量升高,AREM和AREH组高于AR组及NC组(P<0.05),AREH组升高最明显。结论臭氧吸入暴露可促进AR的OVA-sIgE水平增高,促进炎性因子TNF-α、IL-6和IL-8的释放,导致鼻黏膜病理损害和AR症状加重。这一病理过程可能与臭氧激活AR大鼠鼻黏膜转录因子NF-κB核蛋白入核及其靶基因表达的变化有关。

Objective This study aimed to investigate the effect of ozone on the pathogenesis and inflammatory factors in a rat model of allergic rhinitis.Methods Forty-eight healthy female Sprague Dawley rats were randomly divided into six groups:normal control group(NC group),allergic rhinitis(AR)model group,normal rat ozone inhalation exposure group(NE group,1 ppm),AR model exposed to low concentration ozone group(AREL group and 0.5 ppm),AR model exposed to moderate concentration ozone group(AREM group,1 ppm),and AR model exposed to high ozone concentrations(AREH group,2 ppm).AR rat models were sensitized to ovalbumin(OVA).The rats were exposed to different concentrations of ozone using the prepared ozone inhalation exposure system for 2 h per day for 6 weeks consecutively.Within 15 min of the last OVA nasal challenge,the numbers of sneezes and scratches were recorded,and the amounts of nasal secretions were measured.Nasal lavage fluid and nasal mucosa were collected 24 h after the last exposure.Western blotting was performed to detect the protein expression of nuclear factor-kappa B(NF-κB)p65.Quantitative real-time PCR was used to detect the mRNA expression of the NF-κB target genes for tumor necrosis factor-alpha(TNF-α),interleukin(IL)-6,and IL-8 in the nasal mucosa.OVA-specific IgE levels and protein levels of the pro-inflammatory factors IL-6,IL-8,and TNF-αin nasal lavage fluid were determined by ELISA.Pathological changes in the nasal mucosa were assessed by hematoxylin and eosin staining.Statistical analyses and graphical representations were performed using SPSS 20.0.Results The frequency of sneezing,nasal scratching,and nasal secretion in rats with AR was higher in the ozone exposure group than in the AR and NC groups(P<0.05).The serum OVA slgE levels in the ARE group were higher than those in the AR and NC groups(P<0.05);the increase was most obvious in the AREH group.Nuclear protein expression of NF-κB p65 in the AREM and AREH groups was higher than that in the AR and NC groups(P<0.05).The mRNA expressions of IL-6,IL-8,and TNF-αin nasal mucosa and the protein content of IL-6,IL-8,and TNF-αin nasal lavage fluid were increased in the AR ozone exposure group.The nasal lavage fluid levels of IL-6,IL-8,and TNF-αwere higher in the AREL group than in the NC group,and those in the medium-and high-concentration exposure groups were higher than those in the AR and NC groups(P<0.05).Conclusion Ozone inhalation can increase the serum OVA slgE of AR;promote the release of inflammatory factors TNF-α,IL-6,and IL-8;and lead to the aggravation of AR symptoms.This pathological process may be related to the transfer of NF-κB into the nucleus and expression of its target genes in nasal mucosa activated by ozone in AR rats.