党参多糖调控NF-κB信号通路对慢性阻塞性肺疾病大鼠T细胞免疫紊乱和气道炎症的影响

Codonopsis pilosula polysaccharides regulate NF-κB signaling pathway to alleviate T cell immune disorders and airway inflammation in rats with chronic obstructive pulmonary disease

关键词:  党参多糖  慢性阻塞性肺疾病  肺脾气虚证  T细胞免疫  气道炎症  NF-κB信号通路

Key words:  codonopsis pilosula polysaccharides  chronic obstructive pulmonary disease  syndrome of qi deficiency of spleen and lung  T cell immunity  airway inflammation  NF-κB signal pathway

摘要:

[目的] 研究党参多糖通过调控核转录因子κB（NF-κB）信号通路对慢性阻塞性肺疾病（简称慢阻肺）大鼠T细胞免疫紊乱和气道炎症的影响。[方法] 从60只SD大鼠中随机挑出50只建立慢阻肺“肺脾气虚证”模型，余下10只作为对照组。通过烟熏加脂多糖法并配合番泻叶浸液建立大鼠慢阻肺脾肺气虚证模型。将建模成功的大鼠随机分为5组，地塞米松组给予1.95 mg/kg的地塞米松，党参多糖高、中、低剂量组分别给予200、100、50 mg/kg的党参多糖，模型组和对照组均按照大鼠体质量1 mL/kg灌胃生理盐水。每日1次，连续治疗30 d。流式细胞仪检测大鼠外周血中CD3⁺、CD4⁺、CD4⁺/CD8⁺水平；瑞氏-吉姆萨染色（Giemsa）后对肺组织灌洗液中炎性细胞计数；苏木素-伊红（HE）染色检测大鼠肺组织病变；实时定量聚合酶链式反应（qRT-PCR）检测大鼠肺组织NF-κB、核因子κB抑制蛋白α（IκBα）信使核糖核酸（mRNA）相对表达量；通过免疫共沉淀法检测肺组织中NF-κB和IκBα结合水平；通过凝胶迁移实验（EMSA）检测大鼠肺组织中NF-κB与DNA结合情况；通过蛋白免疫印记（WB）检测大鼠肺组织核NF-κB、胞浆NF-κB、IκBα蛋白表达水平及磷酸化核因子κB抑制蛋白α（p-IκBα）水平。[结果] 治疗后，地塞米松组和党参多糖高、中、低剂量组大鼠外周血CD3⁺、CD4⁺、CD4⁺/CD8⁺均高于模型组（P<0.05）；模型组巨噬细胞、淋巴细胞和中性粒细胞数量均高于对照组（P<0.05），地塞米松组和党参多糖高、中、低剂量组均低于模型组（P<0.05）；与对照组相比，模型组大鼠肺组织出现严重炎性细胞浸润，气管壁变形增厚，地塞米松组和党参多糖高、中、低剂量组均出现好转；免疫共沉淀结果显示，IκBα与NF-κB可结合，且模型组IκBα和NF-κB结合最少，党参多糖低剂量组稍多，地塞米松组和中剂量组较多，党参多糖高剂量组更多，对照组最多。IκBα mRNA、胞浆NF-κB蛋白和IκBα蛋白相对表达水平结果显示：模型组均低于对照组（P<0.05），地塞米松组和党参多糖高、中、低剂量组均高于模型组（P<0.05）。[结论] 党参多糖可抑制慢阻肺肺脾气虚证大鼠T细胞免疫紊乱，减轻气道炎症反应和肺组织病变，推测可能与抑制NF-κB信号传导，下调NF-κB mRNA和核NF-κB蛋白表达水平，上调IκBα mRNA和蛋白表达水平，上调胞浆NF-κB蛋白表达水平，抑制NF-κB核位移，抑制p-IκBα有关。

Abstract:

[Objective] To study the Codonopsis pilosula polysaccharides by regulating NF-κB signaling pathway to alleviate T cell immune disorder and airway inflammation in rats with Chronic obstructive pulmonary disease (COPD).[Methods] The 50 SD rats were randomly selected from 60 SD rats to establish the COPD lung spleen qi deficiency syndrome model,and the remaining 10 were recorded as the control group. The COPD lung and spleen qi deficiency syndrome rat model was established by lipopolysaccharide (LPS) and smoke with the infusion of sennata leaves. The successfully modeled rats were randomly divided into 5 groups. In the dexamethasone group,1.95 mg/kg dexamethasone was given normal saline according to the body weight 1 mL/100 g by gavage. Codonopsis pilosula polysaccharides high,medium and low dose groups were given 200,100 and 50 mg/kg of Codonopsis pilosula polysaccharides respectively,which was dissolved in normal saline according to the body weight 1mL/kg by gavage. The rats in the model group and the control group were given 1 mL/kg normal saline. Once a day for 30 days,the levels of CD3⁺,CD4⁺,CD4⁺/CD8⁺ in peripheral blood of rats were detected by flow cytometry. Inflammatory cell count in lavage fluid of lung tissue was detected by Giemsa staining (Giemsa). Lung lesions in rats was detected by HE staining. The mRNA relative expression levels of nuclear transcription factor κB (NF-κB) and nuclear factor κB inhibitory protein α (IκBα) in rat lung tissues were measured by real-time quantitative polymerase chain reaction (qRT-PCR). NF-κB and IκBα binding levels in lung tissue were detected by immunoprecipitation. NF-κB and DNA in rat lung tissue were detected by electrophoretic mobility shift assay (EMSA). The protein expression levels of nuclear NF-κB,cytoplasmic NF-κB,IκBα and p-IκBα in rat lung tissues were detected by Western Blot (WB).[Results] After treatment,the peripheral blood CD3⁺,CD4⁺,CD4⁺/CD8⁺ levels of the dexamethasone group and the Codonopsis pilosula polysaccharides high,medium and low dose groups were higher than that in the model group (P<0.05). The number of macrophages,lymphocytes and neutrophils in the model group were higher than that in the control group (P<0.05),the dexamethasone group and Codonopsis pilosula polysaccharides high,medium and low dose groups were lower than the model group (P<0.05). In the model group,the lung tissues showed severe inflammatory cell infiltration,and the air tube wall was deformed and thickened,the dexamethasone group and Codonopsis pilosula polysaccharides high,medium and low dose groups all improved. The results of immunocoprecipitation showed that I kappa B α and NF-kappa B could be combined,and the model group had the least binding rate of IκBα and NF-κB,a little more in the low dose group of Codonopsis polysaccharide,more dexamethasone group and medium dose group,more in the high dose group of dangshen polysaccharide,and the most in the control group.,IκBα mRNA,cytoplasmic NF-κB protein and IκBα protein relative expression levels showed:the model group is lower than the control group (P<0.05),the dexamethasone group and the Codonopsis pilosula polysaccharides high,medium and low dose groups were higher than the model group (P<0.05).[Conclusion] Codonopsis pilosula polysaccharides can inhibit T-cell immune disorder in rats with COPD lung and spleen qi deficiency syndrome,reduce airway inflammation and lung tissue lesions. It may be related to the inhibition of NF-κB signaling,down-regulation of NF-κB mRNA and nuclear NF-κB protein expression levels,up-regulation of IκBα mRNA and protein expression levels,up-regulation of cytoplasmic NF-κB protein expression level,and inhibit NF-κB nuclear displacement,inhibition of p-IκBα.