小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究

陈恂; 燕晓茹; 李敏; 万通; 蒋艳文; 冀晓华

doi:10.3969/j.issn.1006-2157.2023.09.012

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小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究

实验研究 | 更新时间：2023-10-09

- 小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究
- Experimental study of Xiao’er Jianpi Yifei Formula improving airway hyperresponsiveness in asthmatic mice
- 北京中医药大学学报 2023年46卷第9期页码：1267-1279
- 作者机构：
  
  中国中医科学院西苑医院　北京　100091
- 作者简介：
  
  陈恂，男，博士，主治医师
  # 冀晓华，教授，主任医师，主要研究方向：中医药防治儿童呼吸系统过敏性疾病，E-mail：13611199408@163.com
- 基金信息：
  
  国家自然科学基金青年项目(82104934);中国中医科学院科技创新工程项目(CI2021A02502;CI2021A02508);中国中医科学院优秀青年科技人才培养专项(ZZ15-YQ-007)
- DOI：10.3969/j.issn.1006-2157.2023.09.012
  中图分类号： R285.5
- 收稿：2023-03-29，
  
  网络首发：2023-07-27，
  
  纸质出版：2023-09-30
- 稿件说明：
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陈恂, 燕晓茹, 李敏, 等. 小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究[J]. 北京中医药大学学报, 2023,46(9):1267-1279.

CHEN Xun, YAN Xiaoru, LI Min, et al. Experimental study of Xiao’er Jianpi Yifei Formula improving airway hyperresponsiveness in asthmatic mice[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023, 46(9): 1267-1279. DOI: 10.3969/j.issn.1006-2157.2023.09.012.
陈恂, 燕晓茹, 李敏, 等. 小儿健脾益肺方改善哮喘小鼠气道高反应的实验研究[J]. 北京中医药大学学报, 2023,46(9):1267-1279. DOI： 10.3969/j.issn.1006-2157.2023.09.012.

CHEN Xun, YAN Xiaoru, LI Min, et al. Experimental study of Xiao’er Jianpi Yifei Formula improving airway hyperresponsiveness in asthmatic mice[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023, 46(9): 1267-1279. DOI: 10.3969/j.issn.1006-2157.2023.09.012. DOI：

摘要

目的

探讨小儿健脾益肺方通过高迁移率组蛋白B1(HMGB1)/Toll样受体4(TLR4)/核转录因子-κB(NF-κB)炎症信号通路改善哮喘小鼠气道高反应的作用机制。

方法

60只SPF级Balb/c小鼠随机数字表法分为空白组、模型组、布地奈德组、小儿健脾益肺方组、布地奈德＋小儿健脾益肺方组，用卵清白蛋白(OVA)致敏、激发建立小鼠急性哮喘模型，分别予布地奈德(0.2 g/L)、小儿健脾益肺方(1.875 g/kg)干预。肺功能检测气道阻力，支气管肺泡灌洗液吉姆萨染色检测小鼠各种细胞占比情况，肺组织切片HE及PAS染色观察，ELISA检测血清及支气管肺泡灌洗液中OVA-IgE、白细胞介素(IL)-6、IL-1β、γ干扰素(IFN-γ)、肿瘤坏死因子-α(TNF-α)含量，蛋白质印迹法及免疫荧光检测HMGB1、髓样分化因子88(MyD88)、TLR4、p-NF-κB/NF-κB等通路蛋白表达情况。

结果

与空白组比较，模型组气道阻力明显升高(

0.01)，支气管肺泡灌洗液中嗜酸性粒细胞比例明显升高(

0.01)，组织病理学染色较空白组出现明显炎性细胞浸润、杯状细胞增生、气道上皮水肿、管腔狭窄，血清及支气管肺泡灌洗液中OVA-IgE、IL-6、IL-1β、IFN-γ、TNF-α表达升高(

0.01)，HMGB1、MyD88、TLR4、NF-κB等通路蛋白表达升高(

0.05)，NF-κB磷酸化程度升高(

0.05)。与模型组比较，布地奈德组、小儿健脾益肺方组、布地奈德＋小儿健脾益肺方组气道阻力降低，支气管肺泡灌洗液中巨噬细胞占比升高、嗜酸性粒细胞降低(

0.05)，组织病理学染色结果显示炎症细胞浸润改善、平滑肌层增厚缓解、上皮完整性增加，杯状细胞化生减少，HE评分、PAS评分与模型组比较差异具有统计学意义(

0.05)。血清和支气管肺泡灌洗液中，小儿健脾益肺方组、布地奈德组、布地奈德＋小儿健脾益肺方组与模型组比较均能明显降低各项指标的含量(

0.01)；与布地奈德组比较，小儿健脾益肺方组血清OVA-IgE含量明显升高(

0.05)，小儿健脾益肺方组和布地奈德＋小儿健脾益肺方组HMGB1含量明显降低(

0.05)。蛋白质印迹法检测结果显示，与模型组比较，小儿健脾益肺方组、布地奈德组、布地奈德＋小儿健脾益肺方组均能有效降低HMGB1、MyD88和TLR4蛋白的表达和NF-κB的磷酸化程度，差异具有统计学意义(

0.01)；与布地奈德组相比，小儿健脾益肺方组HMGB1的表达明显下降，而TLR4、MyD88的表达升高，布地奈德＋小儿健脾益肺方组HMGB1、TLR4、MyD88的表达均明显下降，差异均具有统计学意义(

0.05)。免疫荧光检测结果显示，小儿健脾益肺方组、布地奈德组、布地奈德＋小儿健脾益肺方组HMGB1与模型组比较明显降低(

0.01)，布地奈德＋小儿健脾益肺方组的HMGB1和TLR4较布地奈德组均明显降低(

0.05)。

结论

小儿健脾益肺方和布地奈德均能有效缓解哮喘相关病理改变，降低炎症因子表达，抑制HMGB1/TLR4/NF-κB炎症信号通路，改善气道高反应。小儿健脾益肺方和布地奈德联合应用优于布地奈德单独应用。HMGB1/TLR4/NF-κB炎症信号通路是健脾益肺方改善儿童哮喘气道高反应性的潜在作用机制。

Abstract

Objective

We sought to explore the mechanism of

Xiao

’

er Jianpi Yifei

Formula in improving airway hyperresponsiveness in asthmatic mice signaling pathway through the HMGB1/TLR4/NF-κB inflammatory signaling pathway.

Methods

Sixty SPF Balb/c mice were randomly divided into the control group

the model group

the budesonide group

the

Xiao

’

er Jianpi Yifei

Formula group

and the budesonide +

Xiao

’

er Jianpi Yifei

Formula group. The acute asthma model was established by sensitization and stimulation with ovalbumin(OVA). Budesonide(0.2 g/L) and

Xiao

’

er Jianpi Yifei

Formula(1.875 g/kg) were used for intervention. The degree of airway hyperresponsiveness was evaluated by measuring airway resistance. The proportion of different types of cells in bronchoalveolar lavage fluid(BALF) was detected by Giemsa staining. Pathological changes were observed by HE staining and PAS staining in lung tissue sections. The expression levels of OVA-IgE

IL-6

IL-1β

IFN-γ

and TNF-α in serum and BALF were evaluated by ELISA

and the expression of HMGB1

MyD88

TLR4

and p-NF-κB/NF-κB were detected by Western blotting and immunofluorescence.

Results

Compared to the control group

the model group showed a significant increase in airway resistance(

0.01)

an obvious increase in the proportion of eosinophils in bronchoalveolar lavage fluid(

0.01)

significant inflammatory cell infiltration

goblet cell hyperplasia

epithelial swelling

luminal stenosis in tissue pathology staining

and a significant increase in the expression levels of OVA-IgE

IL-6

IL-1β

IFN-γ

and TNF-α in serum and BALF(

0.01). The expression levels of HMGB1

MyD88

TLR4

and NF-κB were also elevated(

0.05). The phosphorylation of NF-κB was increased(

0.05). Compared to the model group

the budesonide group

the

Xiao

’

er Jianpi Yifei

Formula group

and the budesonide+

Xiao

’

er Jianpi Yifei

Formula group exhibited reduced airway resistance

an increased proportion of macrophages in BALF

and reduced eosinophil counts(

0.05). The histopathological staining result showed that the infiltration of inflammatory cells was improved

the smooth muscle layer thickening reduced

the epithelial integrity was increased

and the metaplasia of Goblet cell was reduced. HE scores and PAS scores compared to the model group were statistical significant(

0.05). In the serum and BALF

the contents of various indexes in the

Xiao

’

er Jianpi Yifei

Formula group

the budesonide group

and the budesonide+

Xiao

’

er Jianpi Yifei

Formula group were lower than those in the model group(

0.01); Compared to the budesonide group

the serum OVA-IgE content in the

Xiao

’

er Jianpi Yifei

Formula group was greater (

0.05)

while the HMGB1 content in the

Xiao

’

er Jianpi Yifei

Formula group and the budesonide+

Xiao

’

er Jianpi Yifei

Formula group was lower(

0.05). Western blotting result showed that

compared to the model group

the expression of HMGB1

MyD88

and TLR4 proteins

and the phosphorylation degree of NF-κB could be effectively reduced (

0.01) in the

Xiao

’

er Jianpi Yifei

Formula group

the budesonide group

and the budesonide+

Xiao

’

er Jianpi Yifei

Formula group . Compared to the budesonide group

the expression of HMGB1 in the

Xiao

’

er Jianpi Yifei

Formula group was decreased significantly

while the expression of both TLR4 and MyD88 was increased

and the expressions of HMGB1

TLR4

and MyD88 in the budesonide+

Xiao

’

er Jianpi Yifei

Formula group were decreased significantly

with statistically significant differences(

0.05). The IF result showed that HMGB1 expression levels in the

Xiao

’

er Jianpi Yifei

Formula group

budesonide group

and budesonide+

Xiao

’

er Jianpi Yifei

Formula group were significantly lower than that in the model group(

0.01)

and the HMGB1 and TLR4 expression levels in the budesonide+

Xiao

’

er Jianpi Yifei

Formula group were significantly lower than those in the budesonide group(

0.05).

Conclusion

Xiao

’

er Jianpi Yifei

Formula and budesonide could effectively alleviate asthma-related pathological changes

reduce the expression of inflammatory cytokines

inhibit the HMGB1/TLR4/NF-κB inflammatory signaling pathway

and improve airway hyperresponsiveness. The combination of

Xiao

’

er Jianpi Yifei

Formula and budesonide was superior to budesonide alone. The HMGB1/TLR4/NF-κB inflammatory signaling pathway may be a potential mechanism for

Xiao

’

er Jianpi

Yifei Formula to improve airway hyperresponsiveness in children with asthma.

关键词

Keywords

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