止咳平喘方对支气管哮喘小鼠气道炎症及TLR4/TRAF6/NF-κB通路的影响

doi:10.11958/20240222

天津医药 ›› 2024, Vol. 52 ›› Issue (9): 924-929.doi: 10.11958/20240222

止咳平喘方对支气管哮喘小鼠气道炎症及TLR4/TRAF6/NF-κB通路的影响

范慧慧(), 任玉梅^△(), 田新磊, 张凯, 李晓丽

河南省中医院（河南中医药大学第二附属医院）儿科（邮编450053）

收稿日期:2024-02-23 修回日期:2024-04-18 出版日期:2024-09-15 发布日期:2024-09-06
通讯作者: ^△E-mail：xiaoxiannv800@163.com
作者简介:范慧慧（1986），女，主治医师，主要从事小儿呼吸、消化系统疾病的诊疗方面研究。E-mail：huihuihzy@163.com
基金资助:
河南省中医药科学研究专项课题(20-21ZY2057)

Effects of Zhike Pingchuan Formula on airway inflammation and TLR4/TRAF6/NF-κB pathway in bronchial asthma mice

FAN Huihui(), REN Yumei^△(), TIAN Xinlei, ZHANG Kai, LI Xiaoli

Department of Pediatrics, Henan Hospital of Traditional Chinese Medicine (the Second Affiliated Hospital of Henan University of Chinese Medicine), Zhengzhou 450053, China

Received:2024-02-23 Revised:2024-04-18 Published:2024-09-15 Online:2024-09-06
Contact: ^△E-mail：xiaoxiannv800@163.com

范慧慧, 任玉梅, 田新磊, 张凯, 李晓丽. 止咳平喘方对支气管哮喘小鼠气道炎症及TLR4/TRAF6/NF-κB通路的影响[J]. 天津医药, 2024, 52(9): 924-929.

FAN Huihui, REN Yumei, TIAN Xinlei, ZHANG Kai, LI Xiaoli. Effects of Zhike Pingchuan Formula on airway inflammation and TLR4/TRAF6/NF-κB pathway in bronchial asthma mice[J]. Tianjin Medical Journal, 2024, 52(9): 924-929.

摘要/Abstract

摘要：

目的探讨止咳平喘方减轻支气管哮喘（BA）小鼠气道炎症的作用及机制。方法 84只BALB/c小鼠随机分为对照组、BA组、止咳平喘方低剂量组、止咳平喘方高剂量组、地塞米松组、脂多糖（LPS）组、止咳平喘方高剂量+LPS组，每组12只。除对照组外，其他组小鼠均构建BA模型，建模成功后进行给药处理，每日1次，持续3周。酶联免疫吸附试验（ELISA）检测血清中免疫球蛋白E（IgE）浓度和肺泡灌洗液中白细胞介素-17（IL-17）、肿瘤坏死因子-α（TNF-α）水平；HE染色检测肺组织病理变化；PAS染色测定支气管上皮杯状细胞增生以及黏液分泌；流式细胞术检测小鼠脾脏组织中辅助性T细胞17（Th17）和调节性T（Treg）细胞水平；Western blot检测肺组织Toll样受体4（TLR4）/肿瘤坏死因子受体相关因子6（TRAF6）/核因子κB（NF-κB）通路相关蛋白表达。结果与对照组比较，BA组肺组织病理损伤严重，支气管上皮杯状细胞增生及黏液分泌增多，IgE含量、TNF-α水平、IL-17水平、Th17细胞比例、Th17/Treg比值及TLR4、TRAF6、p-NF-κB p65蛋白表达升高，Treg细胞比例降低（P<0.05）。与BA组比较，止咳平喘方低剂量组、止咳平喘方高剂量组、地塞米松组支气管上皮杯状细胞增生和黏液分泌减少，肺组织损伤改善，IgE含量、TNF-α水平、IL-17水平、Th17细胞比例、Th17/Treg比值及TLR4、TRAF6、p-NF-κB p65蛋白表达降低，Treg细胞比例升高（P<0.05）；LPS组对应指标变化趋势与上述相反（P<0.05）。与止咳平喘方高剂量组比较，止咳平喘方高剂量+LPS组肺组织病理损伤加剧，支气管上皮杯状细胞增生及黏液分泌增多，IgE含量、TNF-α水平、IL-17水平、Th17细胞比例、Th17/Treg比值及TLR4、TRAF6、p-NF-κB p65蛋白表达升高，Treg细胞比例降低（P<0.05）。结论止咳平喘方可能通过抑制TLR4/TRAF6/NF-κB通路减轻BA小鼠气道炎症反应。

关键词: 哮喘, 止咳平喘方, Toll样受体4/肿瘤坏死因子受体相关因子6/核因子κB通路, 气道炎症

Abstract:

Objective To investigate the effect and mechanism of Zhike Pingchuan Formula on reducing airway inflammation in mice with bronchial asthma (BA). Methods A total of 84 BALB/c mice were randomly separated into the control group, the BA group, the low-dose Zhike Pingchuan Formula group, the high-dose Zhike Pingchuan Formula group, the dexamethasone group, the lipopolysaccharide (LPS) group and the high-dose Zhike Pingchuan Formula+LPS group, with 12 mice in each group. The BA model was constructed in all groups of mice except the control group. After successful modeling, mice were treated with drug administration once a day for 3 weeks. Serum immunoglobulin E (IgE) concentration and levels of interleukin-17 (IL-17) and tumor necrosis factor-α (TNF-α) in alveolar lavage fluid were detected by enzyme linked immunosorbent assay (ELISA). Pathological changes in lung tissue was detected by HE staining. The bronchial epithelium goblet cell proliferation and mucus secretion were determined by PAS staining. Flow cytometry was used to detect levels of T helper cell 17 (Th17) and regulatory T (Treg) cell in mouse spleen tissue. Western blot assay was used to detect toll-like receptor 4 (TLR4)/tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa-B (NF-κB) pathway-related protein expression in lung tissue. Results Compared with the control group, damage of lung tissue was serious, the proliferation of bronchial epithelial goblet cells and the secretion of mucus increased, IgE concentration, TNF-α, IL-17 levels, Th17 cell proportion, Th17/Treg ratio, and TLR4, TRAF6, p-NF-κB p65 protein expression increased, while Treg cell proportion decreased in the BA group (P<0.05). Compared with the BA group, the bronchial epithelial cupular cell proliferation and mucus secretion were reduced in the low-dose Zhike Pingchuan Formula group, the high-dose Zhike Pingchuan Formula group and the dexamethasone group, the lung tissue damage was improved, the concentration of IgE, levels of TNF-α, IL-17, the proportion of Th17 cells, the ratio of Th17/Treg and expression of protein levels of TLR4, TRAF6 and p-NF-κB p65 were reduced, and the proportion of Treg cell was elevated (P<0.05). The trend of corresponding indexes in the LPS group was opposite to the above (P<0.05). Compared with the high-dose Zhike Pingchuan Formula group, the pathological damage of lung tissue increased in the high-dose Zhike Pingchuan Formula+LPS group. The proliferation of bronchial epithelial goblet cells and the secretion of mucus increased, the IgE concentration, TNF-α, IL-17 levels, Th17 cell proportion, Th17/Treg ratio and TLR4, TRAF6, p-NF-κB p65 protein expression increased, while the Treg cell proportion decreased (P<0.05). Conclusion Zhike Pingchuan Formula may reduce airway inflammation in BA mice by inhibiting TLR4/TRAF6/NF-κB pathway.

Key words: asthma, Zhike Pingchuan Formula, toll-like receptor 4/tumor necrosis factor receptor-associated factor 6/nuclear factor kappa-B pathway, airway inflammation

中图分类号:

R256.12

图/表 7

图1 各组小鼠血清中IgE含量变化比较 1：对照组；2：BA组；3：止咳平喘方低剂量组；4：止咳平喘方高剂量组；5：地塞米松组；6：LPS组；7：止咳平喘方高剂量+LPS组。n=12，F=383.078，P<0.01；a与对照组比较，b与BA组比较，c与止咳平喘方低剂量组比较，d与止咳平喘方高剂量组比较，P<0.05。

Fig.1 Comparison of changes in serum IgE concentration of mice between seven groups

表1 各组小鼠肺泡灌洗液中TNF-α、IL-17水平变化比较（n=12，ng/L，$\bar{x}±s$）

Tab.1 Comparison of changes of TNF-α and IL-17 levels in alveolar lavage fluid of mice between seven groups

组别	TNF-α	IL-17
对照组	8.66±0.31	15.52±0.41
BA组	34.95±1.23^a	58.61±2.06^a
止咳平喘方低剂量组	29.51±1.01^b	47.87±1.85^b
止咳平喘方高剂量组	12.88±0.42^bc	20.66±1.31^bc
地塞米松组	12.91±0.43^bc	20.72±1.35^bc
LPS组	43.35±1.87^b	67.68±2.73^b
止咳平喘方高剂量+LPS组	25.54±0.96^d	43.36±2.05^d
F	1 906.643^＊＊	1 535.875^＊＊

图2 肺组织病理变化（HE染色，×200）

Fig.2 Pathological changes in lung tissue (HE staining，×200)

图3 小鼠黏液分泌及支气管上皮杯状细胞增生情况（PAS染色，×400）

Fig.3 Mucus secretion and bronchial epithelial cuprocyte proliferation of mice (PAS staining，×400)

表2 各组小鼠脾脏组织中Th17、Treg比例及Th17/Treg比值变化比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of Th17, Treg cell ratio and Th17/Treg ratio in spleen tissue of mice between seven groups

组别	Th17/%	Treg/%	Th17/Treg比值
对照组	3.19±0.12	33.56±1.64	0.09±0.01
BA组	22.67±1.01^a	12.28±0.56^a	1.85±0.06^a
止咳平喘方低剂量组	16.78±0.73^b	17.73±0.61^b	0.95±0.05^b
止咳平喘方高剂量组	7.54±0.31^bc	29.01±1.15^bc	0.26±0.02^bc
地塞米松组	7.52±0.19^bc	29.22±1.18^bc	0.25±0.02^bc
LPS组	26.67±1.21^b	9.66±0.31^b	2.76±0.13^b
止咳平喘方高剂量+LPS组	14.46±0.68^d	20.23±1.02^d	0.71±0.06^d
F	857.346^＊＊	486.504^＊＊	1 487.178^＊＊

图4 Western blot检测小鼠肺组织中TLR4、TRAF6、p-NF-κB p65蛋白表达 1：对照组；2：BA组；3：止咳平喘方低剂量组；4：止咳平喘方高剂量组；5：地塞米松组；6：LPS组；7：止咳平喘方高剂量+LPS组。

Fig.4 Western blot detection of TLR4, TRAF6 and p-NF-κB p65 protein expression in mouse lung tissue

表3 肺组织中TLR4、TRAF6、p-NF-κB p65蛋白变化比较（n=6，$\bar{x}±s$）

Tab.3 Comparison of changes in TLR4, TRAF6 and p-NF-κB p65 protein in lung tissue between seven groups

组别	TLR4/ GAPDH	TRAF6/ GAPDH	p-NF-κB p65/ NF-κB p65
对照组	0.65±0.04	0.41±0.03	0.23±0.01
BA组	1.84±0.22^a	0.86±0.03^a	0.64±0.05^a
止咳平喘方低剂量组	1.50±0.18^b	0.71±0.05^b	0.50±0.04^b
止咳平喘方高剂量组	1.03±0.11^bc	0.52±0.03^bc	0.31±0.03^bc
地塞米松组	1.01±0.12^bc	0.51±0.04^bc	0.32±0.02^bc
LPS组	2.29±0.23^b	0.94±0.04^b	0.81±0.07^b
止咳平喘方高剂量+LPS组	1.38±0.12^d	0.69±0.05^d	0.47±0.03^d
F	73.344^＊＊	145.358^＊＊	155.540^＊＊

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止咳平喘方对支气管哮喘小鼠气道炎症及TLR4/TRAF6/NF-κB通路的影响

Effects of Zhike Pingchuan Formula on airway inflammation and TLR4/TRAF6/NF-κB pathway in bronchial asthma mice

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