祛风止咳方对咳嗽敏感性增高豚鼠模型TRPV1通路的影响

薛珊珊; 柏璐; 孙昕宸; 吴拥军

doi:10.14148/j.issn.1672-0482.2023.0131

祛风止咳方对咳嗽敏感性增高豚鼠模型TRPV1通路的影响

doi: 10.14148/j.issn.1672-0482.2023.0131

1.
南京中医药大学附属医院, 江苏南京 210029
2.
南京中医药大学第一临床医学院, 江苏南京 210023

基金项目:

国家自然科学基金青年科学基金项目 81804152

南京中医药大学与企业合作横向研究课题 2020

南京中医药大学附属医院国自然培育研究项目 2022

详细信息

作者简介:
薛珊珊, 女, 主治中医师, E-mail: 390429607@qq.com

通讯作者:
吴拥军, 男, 教授, 博士生导师，主要从事中医耳鼻咽喉科学相关研究, E-mail: wooyongjun@163.com

中图分类号: R285.5
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出版历程
- 收稿日期: 2022-11-22
- 网络出版日期: 2023-02-17
- 发布日期: 2023-02-10

Effect of Qufeng Zhike Decoction on TRPV1 Pathway in Guinea Pig Model with Increased Cough Sensitivity

1.
The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China
2.
The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的观察祛风止咳方对咳嗽敏感性增高豚鼠模型TRPV1通路表达的影响, 探讨其干预喉源性咳嗽的作用机制。方法采用卵蛋白腹腔注射致敏, 结合烟熏的方法制备喉源性咳嗽(咳嗽敏感性增高豚鼠) 模型, 随机分为空白组、模型组、激动剂组(祛风止咳方+辣椒素)、中药组(祛风止咳方)、对照组(美敏伪麻溶液)。观察各组给药前后咳嗽次数变化, HE染色观察咽喉黏膜、气管黏膜、肺组织形态结构变化, ELISA法检测血清中白细胞介素(IL)-1β、IL-10、IgE表达, 咽喉、气管黏膜中瞬时受体电位香草酸亚型1(TRPV1)、降钙素基因相关肽(CGRP)、神经激肽A(NKA)、P物质(SP) 表达水平, Western blot法检测延髓咳嗽中枢IL-1β、TRPV1、胶质纤维酸性蛋白(GFAP)、神经生长因子(NGF) 的表达。结果与空白组比较, 模型组咳嗽敏感性显著增高(P < 0.01), 咽喉、气管黏膜、肺组织可见黏膜下水肿、炎细胞浸润, 嗜酸性粒细胞计数增加, 血清IL-1β、IgE表达升高(P < 0.01), IL-10表达降低(P < 0.01), 咽喉、气管黏膜的TRPV1、NKA、CGRP、SP含量增加(P < 0.01), 延髓咳嗽中枢IL-1β、TRPV1、GFAP、NGF蛋白表达升高(P < 0.01);与模型组比较, 中药组咳嗽次数明显减少(P < 0.01), 黏膜下水肿及炎细胞浸润明显改善, 血清嗜酸粒细胞计数、嗜酸性粒细胞百分率明显下降(P < 0.05, P < 0.01), 血清IL-1β、IgE表达明显降低(P < 0.01), IL-10表达明显升高(P < 0.01), 咽喉、气管黏膜的TRPV1、NKA、CGRP、SP含量降低(P < 0.05, P < 0.01), 延髓咳嗽中枢IL-1β、TRPV1、GFAP、NGF蛋白表达降低(P < 0.01);与对照组比较, 中药组干预后, 血清IgE含量、气管黏膜CGRP含量、延髓咳嗽中枢TRPV1、GFAP、NGF蛋白表达方面有统计学差异(P < 0.05, P < 0.01), 其他无明显差异; 与激动剂组比较, 两组间差异有统计学意义(P < 0.05, P < 0.01)。结论祛风止咳方治疗喉源性咳嗽可能是通过调控TRPV1, 抑制气道神经源性炎症, 降低外周气道和咳嗽中枢高敏感性, 减少咳嗽及复发。
- 祛风止咳方 /
- 喉源性咳嗽 /
- TRPV1 /
- 咳嗽敏感性增高
Abstract: OBJECTIVE To observe the effect of Qufeng Zhike Decoction on the expression of the TRPV1 pathway in a guinea pig model with increased cough sensitivity, and to explore its mechanism of intervention on the laryngeal cough. METHODS The laryngeal cough (guinea pig with increased cough sensitivity) model was prepared by intraperitoneal injection of egg protein sensitization combined with smoking and randomly divided into the blank group, model group, agonist group (Qufeng Zhike Decoction+capsaicin), Chinese medicine group (Qufeng Zhike Formula) and control group (Meimin Pseudoephedrine Solution). The changes in cough times before and after treatment were observed in each group. The morphology of throat mucosa, tracheal mucosa, and lung tissue was observed by H&E staining. The expressions of serum interleukin (IL)-1β, IL-10, and IgE were detected by ELISA. The expression levels of TRPV1, CGRP, NKA, and SP in the throat and tracheal mucosa were also detected by ELISA. The expressions of IL-1β, TRPV1, GFAP, and NGF in the cough center of medulla oblongata were detected by Western blot. RESULTS Compared with the blank control group, the cough sensitivity of the model group was significantly increased (P < 0.01). Submucosal edema, inflammatory cell infiltration, and eosinophil count were observed in the throat, tracheal mucosa, and lung tissue. The expressions of serum IL-1β and IgE were increased (P < 0.01), IL-10 was decreased (P < 0.01), the contents of TRPV1, NKA, CGRP, and SP in the throat and tracheal mucosa were increased (P < 0.01), and the protein expressions of IL-1β, TRPV1, GFAP and NGF in the medullary cough center were increased (P < 0.01). Compared with the model group, the Qufeng Zhike Decoction had significantly reduced cough times (P < 0.01), significantly improved submucosal edema and inflammatory cell infiltration, significantly decreased serum eosinophil count and eosinophil percentage (P < 0.05, P < 0.01), significantly decreased serum IL-1β and IgE expressions (P < 0.01), and significantly increased IL-10 expression (P < 0.01). The contents of TRPV1, NKA, CGRP, and SP in the throat and tracheal mucosa were decreased (P < 0.05, P < 0.01), and the protein expressions of IL-1β, TRPV1, GFAP, and NGF in the cough center of medulla oblongata were decreased (P < 0.01). After treatment with Qufeng Zhike Decoction, compared with the Meimin Pseudoephedrine Solution, there were significant differences in serum IgE content, CGRP content in tracheal mucosa, and protein expressions of TRPV1, GFAP and NGF in cough center of the medulla oblongata (P < 0.05, P < 0.01), but no other significant differences; compared with the Qufeng Zhike Decoction+capsaicin, the difference between the two groups was statistically significant (P < 0.05, P < 0.01). CONCLUSION Qufeng Zhike Decoction in treating laryngeal cough may regulate the expression of inflammatory factors through TRPV 1 pathway, inhibit neurogenic airway inflammation, reduce the high sensitivity of the peripheral airway and cough center, and reduce the recurrence.
- Qufeng Zhike Decoction /
- laryngeal cough /
- TRPV1 /
- cough sensitivity

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图 1 各组豚鼠咳嗽敏感性比较

Figure 1. Comparison of cough sensitivity of guinea pigs in each group

下载: 全尺寸图片幻灯片

图 2 各组豚鼠咽喉黏膜、气管黏膜、肺组织HE染色图(×200)

注: A.空白组; B.模型组; C.祛风止咳方+辣椒素组; D.祛风止咳方组; E.美敏伪麻溶液组

Figure 2. Hematoxylin-eosin(HE) staining of lung tissues of guinea pigs in each group (×200)

下载: 全尺寸图片幻灯片

图 3 各组豚鼠延髓咳嗽中枢相关蛋白的表达水平

Figure 3. Expression of cough center related proteins in medulla bulbar of guinea pigs in each group

下载: 全尺寸图片幻灯片

表 1 各组豚鼠血细胞分析比较

Table 1. Comparison of blood cell analysis of guinea pigs in each group

组别	WBC×10⁹/L	NEU×10⁹/L	LYM×10⁹/L	EOS×10⁹/L	EOS/%
空白组	8.51±3.37	4.03±2.02	3.78±1.62	0.07±0.01	0.82±0.12
模型组	9.84±3.35	3.65±1.39	5.53±1.99	0.08±0.03	0.70±0.23
祛风止咳方+辣椒素组	9.37±2.93	4.43±1.56	4.45±1.85	0.05±0.02	0.48±0.23^*#
祛风止咳方组	10.61±2.95	4.48±1.22	5.65±2.53	0.04±0.03^#	0.37±0.15^*##
美敏伪麻溶液组	11.32±6.31	6.05±5.86	5.78±1.44	0.05±0.04	0.38±0.19^*##
注: 与空白组比较, ^*P < 0.01;与模型组比较, ^#P < 0.05，^##P < 0.01。

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表 2 各组豚鼠血清IL-1β、IL-10、IgE的含量(x±s)

Table 2. Serum IL-1β, IL-10 and IgE contents of guinea pigs in each group(x±s)

组别	IL-1β/(pg·mL^-1)	IL-10/(pg·mL^-1)	IgE/(ng·mL^-1)
空白组	413.47±14.11	1 516.56±42.66	332.61±10.32
模型组	1 188.35±38.11^**	315.40±10.39^**	1544.48±37.87^**
祛风止咳方+辣椒素组	951.75±55.13^**##	551.78±47.03^**	1 162.46±62.23^**##
祛风止咳方组	813.61±62.78^**##△	1 023.22±43.45^**##△△	830.43±68.61^**##△△
美敏伪麻溶液组	788.18±53.48^**##△△	996.83±41.40^**##△△	746.30±78.58^{**##△△▲}
注: 与空白组相比, ^**P < 0.01;与模型组相比, ^##P < 0.01；与祛风止咳方+辣椒素组相比, ^△P < 0.05, ^△△P < 0.01;与祛风止咳方组相比, ^▲P < 0.05。

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表 3 各组豚鼠咽喉黏膜TRPV1、NKA、CGRP、SP的水平(x±s)

Table 3. Expression of TRPV1, NKA, CGRP and SP in throat mucosa of guinea pigs in each group(x±s)

组别	TRPV1/(ng·mL^-1)	NKA/(pg·mL^-1)	CGRP/(pg·mL^-1)	SP/(pg·mL^-1)
空白组	1.38±0.04	50.18±2.17	11.46±0.25	42.13±1.62
模型组	8.90±0.74^**	192.92±19.03^**	82.06±6.62^**	262.61±17.28^**
祛风止咳方+辣椒素组	6.05±0.45^**##	153.61±19.93^**	43.62±3.65^**##	185.30±11.59^**##
祛风止咳方组	3.33±0.34^**##△△	93.17±5.94^**##△	19.50±0.69^**##△△	84.97±4.44^**##△△
美敏伪麻溶液组	3.04±0.43^**##△△	95.22±7.29^**##	20.11±1.12^**##△△	78.76±5.96^**##△△
注: 与空白组相比, ^**P < 0.01;与模型组相比, ^##P < 0.01;与祛风止咳方+辣椒素组相比, ^△P < 0.05，^△△P < 0.01。

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表 4 各组豚鼠气管黏膜TRPV1、NKA、CGRP、SP的水平(x±s)

Table 4. Expression of TRPV1, NKA, CGRP and SP in trachea mucosa of guinea pigs in each group(x±s)

组别	TRPV1/(ng·mL^-1)	NKA/(pg·mL^-1)	CGRP/(pg·mL^-1)	SP/(pg·mL^-1)
空白组	0.55±0.03	74.72±1.40	14.47±0.49	56.24±1.84
模型组	7.04±0.48^**	234.14±27.51^**	24.56±1.48^**	165.64±32.49^**
祛风止咳方+辣椒素组	5.36±0.44^**##	192.96±21.93^**	20.14±0.40^**##	130.60±6.41^**
祛风止咳方组	3.55±0.42^**##△△	88.40±3.01^**##△△	16.07±0.67^**##△△	87.36±5.64^**#△△
美敏伪麻溶液组	3.10±0.57^**##△△	93.70±5.41^**##△△	17.90±0.68^{**##△△▲▲}	75.07±9.38^*##△△
注: 与空白组比较, ^P < 0.05, ^*P < 0.01;与模型组比较, ^#P < 0.05, ^##P < 0.01;与祛风止咳方+辣椒素组比较, ^△△P < 0.01;与祛风止咳方组比较, ^▲▲P < 0.01。

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