补肺健脾方对慢性阻塞性肺疾病模型大鼠黏膜屏障的保护作用研究

doi:10.12114/j.issn.1007-9572.2022.0307

中国全科医学 ›› 2022, Vol. 25 ›› Issue (30): 3761-3767.DOI: 10.12114/j.issn.1007-9572.2022.0307

补肺健脾方对慢性阻塞性肺疾病模型大鼠黏膜屏障的保护作用研究

张丛丛¹, 李亚²^,³^,⁴, 李素云²^,³^,⁴, 毛静¹^,^*()

1．450046　河南省郑州市，河南中医药大学药学院
2．450000　河南省郑州市，河南中医药大学第一附属医院中药药理（呼吸）实验室
3．450046　河南省郑州市，河南中医药大学河南省中医药防治呼吸病重点实验室
4．450046　河南省郑州市，河南中医药大学呼吸疾病中医药防治省部共建协同创新中心

收稿日期:2022-03-15 修回日期:2022-05-19 出版日期:2022-10-20 发布日期:2022-06-09
通讯作者: 毛静
张丛丛，李亚，李素云，等.补肺健脾方对慢性阻塞性肺疾病模型大鼠黏膜屏障的保护作用研究[J].中国全科医学，2022，25（30）：3761-3767.[www.chinagp.net]
作者贡献：张丛丛进行实验操作、数据整理和论文撰写；李亚、李素云提出研究思路；毛静制定总体研究目标，设计实验方案并对文章质量进行控制，对文章整体负责。
基金资助:
国家自然科学基金资助项目(81904116); 中医药传承与创新"百千万"人才工程（岐黄工程）项目岐黄学者([2018]284号)

Protective Effect of Bufei Jianpi Formula on Mucosal Barrier in a Rat Model with Chronic Obstructive Pulmonary Disease

Congcong ZHANG¹, Ya LI²^,³^,⁴, Suyun LI²^,³^,⁴, Jing MAO¹^,^*()

1. School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China
2. Chinese Medicine Pharmacology (Respiratory) Laboratory, the First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, China
3. Henan Key Laboratory of Chinese Medicine for Respiratory Disease, Henan University of Chinese Medicine, Zhengzhou 450046, China
4. Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province & Education Ministry of P.R. China, Henan University of Chinese Medicine, Zhengzhou 450046, China

Received:2022-03-15 Revised:2022-05-19 Published:2022-10-20 Online:2022-06-09
Contact: Jing MAO
About author:
ZHANG C C, LI Y, LI S Y, et al. Protective effect of Bufei Jianpi Formula on mucosal barrier in a rat model with chronic obstructive pulmonary disease[J]. Chinese General Practice, 2022, 25 (30) : 3761-3767.

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摘要/Abstract

摘要： 背景人体呼吸道和胃肠消化道具有相似的黏膜结构，黏膜屏障构成机体抵御外邪的第一道防线，当呼吸道感染后刺激黏膜产生局部免疫应答，通过迁移和归巢影响到肠道，导致黏膜局部抗感染能力减弱，诱发黏膜屏障损伤，加重慢性阻塞性肺疾病（COPD）肺部病变。目的基于"肺与大肠相表里"中医药经典脏腑理论，观察补肺健脾方对COPD稳定期模型大鼠肺组织炎性因子表达水平，肺、结肠组织肺肠相关活性肽表达水平的影响。方法 2019年9月至2020年12月选取SPF级SD大鼠50只，采用乱数表法将50只大鼠随机分为对照组、模型组、补肺健脾组、氨茶碱组和益生菌组，每组10只。1~8周采用香烟烟雾暴露联合鼻腔滴注脂多糖（LPS）制作COPD模型（对照组除外）。各组自第9周起灌胃给药，12周结束后取材。在光镜下观察肺、结肠组织病理变化，采用免疫组化法检测肺组织肿瘤坏死因子α（TNF-α）、白介素10（IL-10）表达水平，酶联免疫吸附测定（ELISA）法检测支气管肺泡灌洗液（BALF）分泌型免疫球蛋白A（SIgA）及肺、结肠组织P物质（SP）、血管活性肠肽（VIP）表达水平。结果对照组大鼠肺、结肠组织结构基本完整；模型组大鼠气管周围存在大量炎性细胞，支气管管壁增厚，气管变窄，补肺健脾组改善肺组织病理效果最为显著；模型组大鼠结肠组织出现损伤，表现为大量上皮细胞脱落，隐窝形态大小不一，补肺健脾组改善结肠组织病理效果良好。五组大鼠肺组织TNF-α、IL-10、SP、VIP表达水平比较，差异均有统计学意义（FTNF-α=70.640、FIL-10=8.444、FSP=108.700、FVIP=4.665，P<0.05）；五组大鼠BALF中SIgA表达水平比较，差异有统计学意义（F=26.370，P<0.05）；五组大鼠结肠组织SP、VIP表达水平比较，差异均有统计学意义（FSP=136.600，FVIP=13.980，P<0.05）。与对照组相比，模型组大鼠肺组织TNF-α、SP表达水平与结肠组织SP、VIP表达水平均升高（P<0.05），肺组织IL-10、VIP及BALF中SIgA表达水平均降低（P<0.05）。与模型组相比，补肺健脾组大鼠肺组织TNF-α、SP表达水平与结肠组织SP、VIP表达水平均降低（P<0.05），肺组织IL-10、VIP及BALF中SIgA表达水平均升高（P<0.05）。结论与模型组相比，各治疗组肺肠相关活性肽表达水平均有不同程度改善，补肺健脾组疗效优于其他药物治疗组。补肺健脾方可以减轻肺组织炎性反应，改善肺肠组织病理损伤，其机制与调节SIgA、SP和VIP的表达水平、增强局部黏膜免疫和黏膜屏障功能有关。

关键词: 肺疾病，慢性阻塞性, 免疫，黏膜, 血管活性肠肽, 补肺健脾方, 大鼠

Abstract:

Background

Themucosal structure of the respiratory tract is similar to that of the gastrointestinal tract in humans, which is the body's first line of defense against the invasion of pathogens through the function of mucosal barrier. When the respiratory tract infection stimulates the airway mucosa to produce a local immune response, it affects the intestinal tract through migration and homing, resulting in the weakening of the local mucosal anti-infection ability, inducing mucosal barrier damage, and further aggravating chronic obstructive pulmonary disease (COPD) .

Objective

To explore the effects of Bufei Jianpi Formula on the expression levels of inflammatory factors in lung tissue, and the content of lung-gut related peptides in lung and colon tissues in a rat model of stable COPD based on a typical viscera theory of Traditional Chinese Medicine, namely "the lungs and large intestines are interior-exteriorly related" .

Methods

This experiment was carried out from September 2019 to December 2020. Fifty SPF SD rats were selected, and equally and randomly divided into control, model, Bufei Jianpi, aminophylline and probiotics groups. Except for the control group, the rats in other groups received cigarette smoke exposure combined with intranasal instillation of lipopolysaccharide (LPS) in the first eight weeks of intervention for establishing a COPD model. From the ninth week of intervention, rats in the control and model groups received intragastric administration of 0.9% sodium chloride solution, and those in Bufei Jianpi, aminophylline and probiotics groups received intragastric administration of Bufei Jianpi Formula, aminophylline and probiotics, respectively. The rats were sacrificed after 12 weeks of intervention. The pathological changes of lung and colon tissues were observed under an optical microscope, the expression levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in lung tissue were detected by immunohistochemistry. The contents of secretory immunoglobulin A (SIgA) in bronchoalveolar lavage fluid (BALF) , substance P (SP) and vasoactive intestinal peptide (VIP) in lung and colon tissues were detected by enzyme-linked immunosorbent assay (ELISA) .

Results

In terms of morphology, the lung and colon tissue structures of rats in the control group were basically intact. In the model group, the trachea was narrowed and surrounded by a lot of inflammatory cells with thickened bronchial wall. The improvement in histopathology of the lung tissues was the most obvious in Bufei Jianpi group. The colon tissue of rats in the model group was damaged, which was manifested by numerous shed epithelial cells, and crypts in different shapes and sizes. Bufei Jianpi group demonstrated better improvement in histopathology of the colon tissue. There were statistically significant differences in the expression levels of TNF-α, IL-10, SP and VIP in the lung tissue of the five groups of rats (F_TNF-α=70.640, F_IL-10=8.444, F_SP=108.700, F_VIP=4.665, P<0.05) . The expression level of SIgA in BALF differed significantly across five groups of rats (F=26.370, P<0.05) . The expression levels of SP and VIP in colon tissue also varied significantly across five groups of rats (F_SP=136.600, F_VIP=13.980, P<0.05) . Compared with the control group, the expression levels of TNF-α and SP in the lung tissue and those of SP and VIP in the colon tissue of the model group were increased (P<0.05) ; the expression levels of IL-10 and VIP in the lung tissue, and expression level of SIgA in the BALF of the model group were decreased (P<0.05) . Compared with the model group, the expression levels of TNF-α and SP in the lung tissue and those of SP and VIP in the colon tissue of the Bufei Jianpi group were decreased (P<0.05) ; the expression levels of IL-10 and VIP in the lung tissue, and expression level of SIgA in the BALF of the Bufei Jianpi group were increased (P<0.05) .

Conclusion

Compared with the model group, the improvement in the lung-gut related peptides indices in three treatment groups was better, and the improvement was the best in the Bufei Jianpi group. Bufei Jianpi Formula could improve symptoms of lung inflammation and pathological damage of lung and intestine tissues. The mechanism may be related to the involvement in regulating the content of SIgA, SP and VIP, and enhancing local mucosal immunity and barrier function.

Key words: Pulmonary disease, chronic obstructive, Immunity, mucosal, Vasoactive intestinal peptide, Bufei Jianpi Formula, Rats

张丛丛,李亚,李素云,等. 补肺健脾方对慢性阻塞性肺疾病模型大鼠黏膜屏障的保护作用研究[J]. 中国全科医学, 2022, 25(30): 3761-3767. DOI: 10.12114/j.issn.1007-9572.2022.0307.
Congcong ZHANG,Ya LI,Suyun LI, et al. Protective Effect of Bufei Jianpi Formula on Mucosal Barrier in a Rat Model with Chronic Obstructive Pulmonary Disease[J]. Chinese General Practice, 2022, 25(30): 3761-3767. DOI: 10.12114/j.issn.1007-9572.2022.0307.

图/表 7

图1 各组大鼠肺组织病理变化（HE染色，×200）

Figure 1 Pathological status in lung tissue of rats in five groups

图2 各组大鼠结肠组织病理变化（HE染色，×200）

Figure 2 Pathological status in colon tissue of rats in five groups

图3 各组大鼠肺组织TNF-α、IL-10表达（免疫组化，×200）注：TNF-α=肿瘤坏死因子α，IL-10=白介素10

Figure 3 Expression of TNF-α and IL-10 in lung tissue of rats in five groups

表1 各组大鼠肺组织TNF-α、IL-10表达水平比较（±s）

Table 1 Expression levels of TNF-α and IL-10 in lung tissue of rats in five groups

组别	只数	TNF-α	IL-10
对照组	6	7.39±2.43	71.60±18.61
模型组	6	47.09±8.22^a	34.78±11.90^a
补肺健脾组	6	7.90±2.38^b	70.72±6.07^b
氨茶碱组	6	14.14±3.09^b	58.98±18.12^b
益生菌组	6	19.17±4.98^abc	45.01±8.38^ac
F值		70.640	8.444
P值		<0.001	<0.001

表2 各组大鼠BALF中SIgA表达水平比较（±s，ng/mg）

Table 2 Expression levels of SIgA in BALF of rats in five groups

组别	只数	SIgA
对照组	6	29.00±11.68
模型组	6	1.32±0.88^a
补肺健脾组	6	11.34±3.16^ab
氨茶碱组	6	1.59±0.99^ac
益生菌组	6	5.36±1.60^a
F值		26.370
P值		<0.001

表3 各组大鼠肺组织SP、VIP表达水平比较（±s，pg/mg）

Table 3 Expression levels of SP and VIP in lung tissue of rats in five groups

组别	只数	SP	VIP
对照组	6	1 361.69±45.07	209.81±47.06
模型组	6	1 889.38±58.76^a	101.81±63.92^a
补肺健脾组	6	1 469.96±46.74^ab	191.44±40.75^b
氨茶碱组	6	1 842.52±52.37^ac	172.20±39.61
益生菌组	6	1 640.42±63.66^abcd	145.26±44.11
F值		108.700	4.665
P值		<0.001	0.006

表4 各组大鼠结肠组织SP、VIP表达水平比较（±s，pg/mg）

Table 4 Expression levels of SP and VIP in colon tissue of rats in five groups

组别	只数	SP	VIP
对照组	6	4 786.02±303.29	1.42±0.27
模型组	6	8 180.00±360.98^a	3.16±0.46^a
补肺健脾组	6	6 585.30±168.88^ab	1.96±0.46^b
氨茶碱组	6	7 587.84±422.13^abc	1.63±0.20^b
益生菌组	6	5 018.25±268.37^bcd	2.38±0.66^ab
F值		136.600	13.980
P值		<0.001	<0.001

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补肺健脾方对慢性阻塞性肺疾病模型大鼠黏膜屏障的保护作用研究

Protective Effect of Bufei Jianpi Formula on Mucosal Barrier in a Rat Model with Chronic Obstructive Pulmonary Disease

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