固本防哮饮对哮喘缓解期小鼠气道上皮紧密连接蛋白及CCSP的影响

姜越; 张宇林; 董盈妹; 严花; 赵霞

doi:10.14148/j.issn.1672-0482.2022.0483

固本防哮饮对哮喘缓解期小鼠气道上皮紧密连接蛋白及CCSP的影响

doi: 10.14148/j.issn.1672-0482.2022.0483

姜越^{1, 2,},
张宇林^{1, 2},
董盈妹^{1, 2},
严花^{1, 2},
赵霞^{1, 2, ,}

1.
南京中医药大学附属医院, 江苏南京 210029
2.
南京中医药大学中医儿科学研究所，江苏省儿童呼吸疾病(中医药)重点实验室，江苏南京 210023

基金项目:

国家自然科学基金面上项目 81774367

江苏省领军人才项目 SLJ0224

江苏省大学生创新创业训练计划项目 202010315046

江苏省研究生科研与实践创新计划 KYCX22_1909

详细信息

作者简介:
姜越, 女, 硕士研究生, E-mail: jiangyue2009@126.com

通讯作者:
赵霞, 女, 教授, 主要从事中医药防治小儿肺脾系疾病、儿童体质、中医药标准化研究, E-mail: zhaoxiahy@126.com

中图分类号: R285.5
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出版历程
- 收稿日期: 2022-01-19
- 网络出版日期: 2022-06-07
- 发布日期: 2022-06-10

Effects of Guben Fangxiao Decoction on Airway Epithelial Tight Junction Protein and CCSP in Mice of Asthma in Remission Stage

JIANG Yue^{1, 2
,},
ZHANG Yu-lin^{1, 2},
DONG Ying-mei^{1, 2},
YAN Hua^{1, 2},
ZHAO Xia^{1, 2
, ,}

1.
The Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing 210029, China
2.
Institute of Pediatrics, Nanjing University of Chinese Medicine, Jiangsu Key Laboratory of Pediatric Respiratory Disease, Nanjing 210023, China

摘要

摘要: 目的研究固本防哮饮对哮喘缓解期小鼠气道上皮紧密连接蛋白及Clara细胞分泌蛋白(CCSP)的影响, 探讨其防治缓解期哮喘的可能机制。方法联合使用卵清蛋白(OVA)及呼吸道合胞病毒(Respiratory syncytial virus, RSV)建立哮喘缓解期小鼠模型, 将36只Balb/c小鼠随机分为正常组、模型组、固本防哮饮低剂量组、固本防哮饮中剂量组、固本防哮饮高剂量组和孟鲁司特钠组, 每组6只。模型建立完成后, 固本防哮饮各剂量组分别予不同剂量(12、24、36 g·kg^-1)固本防哮饮, 孟鲁司特钠组予2.6 mg·kg^-1孟鲁司特钠，各组均灌胃28 d。末次给药后24 h留取样本。使用苏木精-伊红(HE)染色法观察各组小鼠肺组织病理; Western blot、qPCR及免疫组化检测各组小鼠肺组织中闭锁小带蛋白-1(ZO-1)等气道上皮紧密连接蛋白表达; ELISA法检测肺组织及肺泡灌洗液(BALF)中CCSP的表达。结果 HE染色结果显示, 与正常组相比, 模型组小鼠肺组织中可见明显的炎性细胞浸润(P < 0.01), 固本防哮饮中、高剂量组小鼠肺组织中炎性细胞浸润减少(P < 0.05，P < 0.01);与正常组相比，模型组中ZO-1、闭合蛋白(Occludin)和桥粒芯胶蛋白(Desmocollin)的蛋白表达水平显著降低(P < 0.05，P < 0.01)，免疫组化结果显示上述指标蛋白在模型组中表达减少(P < 0.01, P < 0.001)且排列松散, 固本防哮饮各剂量组对上述蛋白表达有不同程度地回调; 同时，模型组中ZO-1、ZO-2、纽带蛋白(Vinculin)、连环蛋白(Catenin) mRNA的表达水平显著降低(P < 0.05，P < 0.01)，固本防哮饮高剂量组ZO-1、ZO-2、Vinculin、Catenin mRNA表达水平显著升高(P < 0.05);与正常组相比, 模型组小鼠肺组织及BALF中CCSP的表达显著下降(P < 0.01), 低、高剂量固本防哮饮显著增加肺组织及BALF中CCSP的表达(P < 0.05, P < 0.01)。结论固本防哮饮能够上调气道上皮紧密连接蛋白以及CCSP的表达水平, 修复上皮损伤, 恢复上皮屏障功能, 从而防治哮喘。
- 固本防哮饮 /
- 哮喘缓解期 /
- 上皮屏障 /
- 紧密连接蛋白 /
- CCSP
Abstract: OBJECTIVE To observe the effects of Guben Fangxiao Decoction (GBFXD) on airway tight junction protein and CCSP, and to explore the possible mechanism of GBFXD on airway inflammation in remission stage of asthma. METHODS Mouse model of asthma in remission stage was established by sensitization and stimulation of OVA combined with respiratory syncytial virus (RSV) infection. Mice were randomLy divided into six groups (n=6): control group, model group, GBFXD low-dose group (GBFXD-L), GBFXD medium-dose group (GBFXD-M), and GBFXD high-dose group (GBFXD-H) and montelukast group.The mice were developed asthma model and given different concentrations of GBFXD (12, 24, 36 g·kg^-1)and montelukast sodium (2.6 mg·kg^-1) by gavage according to groups for 28 days. The mice were sacrificed and samples were obtained after the intervention. Hematoxylin-eosin (HE) staining was used to observe the pathological features of lung tissues in each group. The expression of CCSP in lung tissues and alveolar lavage fluid (BALF) was determined by ELISA. Western blot, qPCR and immunohistochemical assay were used to detect the expression of ZO-1 and other airway epithelial tight junction proteins in lung tissues of mice in each group. RESULTS Compared with the control group, the model mice in remission stage are mainly infiltrated by inflammatory cells in the lung tissue (P < 0.01), and the infiltration of inflammatory cells in the lung tissues of mice was alleviated after the treatment (P < 0.01). The protein expression levels of ZO-1, Occludin and Desmocollin in the model group were significantly lower than those in the normal group (P < 0.05, P < 0.01), immunohistochemical results showed that the expression of the above indexes was reduced and the proteins were loosely arranged in the model group. Meanwhile, the mRNA expression levels of ZO-1, ZO-2, Vinculin and Catenin in the model group significantly decreased (P < 0.05, P < 0.01). Compared with the normal group, the expression of CCSP in the model group significantly decreased in lung tissue and BALF (P < 0.01), while the expression of CCSP in BALF and lung tissue was significantly increased by the intervention of low and high doses of GBFXD (P < 0.05, P < 0.01). CONCLUSION GBFXD can repair the epithelial injury and restore the epithelial barrier function by up-regulating the expression of epithelial tight junction protein and CCSP, an indicator of epithelial cell permeability change, so as to reduce the impact of various external pathogenic factors on the airway and preventing and treating asthma.
- Guben Fangxiao Decoction /
- asthma remission stage /
- epithelial barrier /
- tight junction protein /
- CCSP

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图 1 各组小鼠肺组织病理代表图(HE, ×200)

注: 与正常组相比, ^**P < 0.01;与模型组相比, ^#P < 0.05, ^##P < 0.01。x±s, n=3。

Figure 1. Representative photograph of lung tissue pathology of mice in each group(HE, ×200)

下载: 全尺寸图片幻灯片

图 2 肺组织ZO-1、Occludin、Desmocollin蛋白表达

注: 与正常组相比, ^*P < 0.05, ^**P < 0.01;与模型组相比, ^#P < 0.05, ^###P < 0.001。x±s, n=3。

Figure 2. Protein expression of ZO-1, Occludin and Desmocollin in lung tissue

下载: 全尺寸图片幻灯片

图 3 肺组织ZO-1、Occludin、Desmocollin蛋白表达(IHC, ×200)

注: 与正常组相比, ^**P < 0.01, ^***P < 0.001;与模型组相比, ^#P < 0.05, ^###P < 0.001。x±s, n=3。

Figure 3. Protein expression of ZO-1, Occludin and Desmocollin in lung tissue(IHC, ×200)

下载: 全尺寸图片幻灯片

图 4 肺组织及BALF中CCSP蛋白表达

注: 与正常组相比, ^**P < 0.01;与模型组相比, ^#P < 0.05, ^###P < 0.001。x±s, n=3。

Figure 4. Expression of CCSP protein in lung tissue and BALF

下载: 全尺寸图片幻灯片

表 1 qPCR引物序列

Table 1. The qPCR primer sequences

基因	引物序列(5'→3')
GAPDH	F: CGTGTTCCTACCCCCAATGT R: TGTCATACTTGGCAGGTTTCT
ZO-1	F: AAAGAGAAAGGTGAAACACTGC R: TTTTAGAGCAAAAGACCAACCG
ZO-2	F: CTGGGCAGAGTGAAAATATTCG R: CTTGATTGGAACCGCATAGATG
Catenin	F: GATACATCCGCTCTGCTCTTCGTC R: CTACTCGCTCATGCTCACTCGTAAG
Vinculin	F: GCATTGCAAAGAAGATTGATGC R: TAATTCTGCAATCTTCCGAGCT

下载: 导出CSV

表 2 肺组织中ZO-1、Vincullin、Catenin、ZO-2 mRNA表达(x±s, n=3)

Table 2. Expression of ZO-1, Vincullin, Catenin, ZO-2 mRNA in lung tissue(x±s, n=3)

组别	ZO-1	Vinculin	Catenin	ZO-2
正常组	1	1	1	1
模型组	0.50±0.06^**	0.25±0.07^**	0.50±0.04^*	0.33±0.05^**
固本防哮饮低剂量组	0.91±0.16^#	0.40±0.14	0.92±0.11^#	0.71±0.09
固本防哮饮中剂量组	0.71±0.11	0.74±0.08^#	0.71±0.05	0.66±0.21
固本防哮饮高剂量组	0.93±0.08^#	0.79±0.13^#	0.94±0.18^#	0.77±0.12^#
孟鲁司特钠组	0.72±0.12	0.47±0.07	0.87±0.11	0.50±0.08
注: 与正常组相比, ^P < 0.05, ^*P < 0.01;与模型组相比, ^#P < 0.05。

下载: 导出CSV

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