不同方法建立大鼠慢性阻塞性肺疾病模型的比较研究

doi:10.12300/j.issn.1674-5817.2021.118

摘要/Abstract

摘要：

目的比较烟草烟雾暴露（cigarette smoke exposure，CSE）及其联合细菌脂多糖（lipopolysaccharide，LPS）气道内滴注两种方法建立大鼠慢性阻塞性肺疾病（chronic obstructive pulmonary disease，COPD）模型的效果及特点。方法将SD大鼠随机分为对照组、CSE组和CSE+LPS组，每组10只，分别给予正常环境饲养、熏烟箱CSE、熏烟箱CSE联合LPS气道内滴注。建立大鼠COPD模型后24周检测大鼠肺功能，HE染色观察气道和肺组织病理变化，ELISA检测血清白细胞介素-8（interleukin-8，IL-8）和肿瘤坏死因子-ɑ（tumor necrosis factor ɑ，TNF-ɑ）含量。结果 CSE+LPS组和CSE组大鼠肺功能结果显示，气道阻力、静态肺顺应性、功能残气量均高于对照组，而潮气量、每分钟通气量、第50 ms用力呼气容积与用力呼气容积比均低于对照组（均P＜0.05），而CSE+LPS组比CSE组大鼠的肺功能明显下降（均P＜0.05）。CSE组和CSE+LPS组肺泡腔均呈代偿性扩张，LPS+CSE组可见部分间隔变薄、断裂，形成肺大泡。CSE+LPS组和CSE组大鼠血清IL-8和TNF-ɑ含量均明显高于对照组（均P＜0.05），而且CSE+LPS组大鼠TNF-ɑ表达水平明显高于CSE组（P＜0.05）。结论应用CSE联合气管滴注LPS的方法建立的COPD大鼠模型更接近于人类疾病的临床表现，CSE+LPS联合方法优于单纯CSE的方法。

关键词: 慢性阻塞性肺疾病, 脂多糖, 烟草烟雾, 肺功能, 大鼠模型

Abstract:

Objective To compare the effects and characteristics of cigarette smoke exposure (CSE) alone and CSE combined with airway instillation of bacterial lipopolysaccharide (LPS) in chronic obstructive pulmonary disease (COPD).Methods Male SD rats were randomly divided into control, CSE, and CSE+LPS groups, with 10 rats in each group. After 24 weeks, the models were established and the lung function of the rats was measured. Hematoxylin-eosin (HE) staining was performed to observe the pathological changes in the airway and lung tissue. The ELISA method was used to detect the level of serum interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) in peripheral blood.Results Airway resistance (RI), functional residual capacity (FRC), and chord compliance (Cchord) of the CSE and LPS+CSE groups were higher than those of the control group (all P < 0.05), while the tidal volume (TV), minute volume (MV), and forced expiratory volume in 50 ms (FEV50) / forced vital capacity (FVC) of the CSE and LPS+CSE groups were lower than those of the control group (all P < 0.05). HE staining of lung tissue showed that the average alveolar intercept and thickness of the small airway walls were higher in the CSE and LPS+CSE groups than those in the control group. Compensatory enlargement was evident in the alveolar cavity of the CSE and CSE+LPS groups, and the alveolar septum widened, with a fusion of pulmonary alveoli in the CSE+LPS group. The levels of IL-8 and TNF-α in serum of the CSE and CSE+LPS groups were higher than those of the control group (all P < 0.05). The level of TNF-α in serum of the CSE+LPS group was higher than that of the CSE group (P < 0.05).Conclusion The CSE combined with LPS method is superior to CSE alone for establishing the COPD rat model, and the combined model is closer to clinical manifestations.

Key words: Chronic obstructive pulmonary disease, Lipopolysaccharide, Cigarette smoke, Lung function, Rat models

中图分类号:

Q95-33

卢心鹏, 刘蓉, 黄文博, 赵瑾, 李洪涛. 不同方法建立大鼠慢性阻塞性肺疾病模型的比较研究[J]. 实验动物与比较医学, 2022, 42(3): 201-206.

Xinpeng LU, Rong LIU, Wenbo Huang, Jin ZHAO, Hongtao LI. A Comparative Study of Chronic Obstructive Pulmonary Disease Rat Models Established by Different Methods[J]. Laboratory Animal and Comparative Medicine, 2022, 42(3): 201-206.

图/表 5

图1 造模后各组大鼠的体质量生长曲线图注：Control指正常环境下饲养的对照组，CSE指烟草烟雾暴露组，CSE+LPS指烟草烟雾暴露联合细菌脂多糖气道内滴注组。每组10只大鼠。

Figure 1 Body weight growth curve of rats in each group after modelingNote： Control， rats in the control group raised in a normal environment； CSE， rats in the cigarette smoke exposure group； CSE+LPS， rats in the intratracheal LPS instillation combined with CSE group. There were 10 rats in each group.

表1 造模后各组大鼠的肺功能指标 (xˉ±s, n=10)

Table 1 Lung functions of rats in each group after modeling

Group	RI/(cmH₂O·s^－1·mL^－1)	TV/mL	Cchord/(mL·cmH₂O^－1)	FRC/mL	MV/mL	FEV50/ (FVC/%)
Control	0.250±0.026	3.90±0.19	1.210±0.042	5.29±0.51	233.98±11.40	0.155±0.041
CSE	0.315±0.018^*	3.64±0.10^*	1.310±0.071^*	5.88±0.59^*	218.72±5.18	0.118±0.030^*
CSE+LPS	0.348±0.025^*#	3.57±0.14^*	1.395±0.120^*	6.58±0.44^*#	214.12±8.60^*	0.099±0.035^*#

图2 各组大鼠肺组织病理形态变化（HE染色， ×100）注：Control指正常环境下饲养的对照组，CSE指烟草烟雾暴露组，CSE+LPS指烟草烟雾暴露联合细菌脂多糖气道内滴注组。

Figure 2 Pathological changes of lung tissues of rats in each group （HE staining， ×100）Note： Control， rats in the control group raised in a normal environment； CSE， rats in the cigarette smoke exposure group； CSE+LPS， rats in the intratracheal LPS instillation combined with CSE group.

表2 各组大鼠血清中IL-8和TNF-ɑ含量 (xˉ±s, n=10)

Table 2 Serum contents of IL-8 and TNF-ɑ in different groups of rats

CSE

CSE+LPS

162.87±17.95^*

160.72±33.04^*

109.35±13.78^*

141.10±18.82^*#

《实验动物与比较医学》有关实验动物福利伦理内容的说明

参考文献 18

1	World Health Organization. Projection of mortality and cause of death, 2016 to 2060 [EB/OL]. [2019-10-14]. .
2	WANG C, XU J Y, YANG L, et al. Prevalence and risk factors of chronic obstructive pulmonary disease in China (the China Pulmonary Health[CPH]study): a national cross-sectional study[J]. Lancet, 2018, 391(10131):1706-1717. DOI:10.1016/S0140-6736(18)30841-9 .
3	李征途, 徐小明, 巩雪芳, 等. 应用熏烟联合脂多糖方法建立慢性阻塞性肺疾病相关肺动脉高压小鼠模型[J]. 国际呼吸杂志, 2014, 34(1):27-30, 封3.
	LI Z T, XU X M, GONG X F, et al. Cigarette smoking plus airway LPS inhalation induced COPD-PAH in mouse[J]. Int J Respir, 2014, 34 (1):27-30. DOI:10.3760/cma.j.issn.1673-436X.2014.01.005 .
4	LI D F, WANG J, SUN D J, et al. Tanshinone IIA sulfonate protects against cigarette smoke-induced COPD and down-regulation of CFTR in mice[J]. Sci Rep, 2018, 8(1):376. DOI:10.1038/s41598-017-18745-5 .
5	LU W J, LI D F, HU J Y, et al. Hydrogen gas inhalation protects against cigarette smoke-induced COPD development in mice[J]. J Thorac Dis, 2018, 10(6):3232-3243. DOI:10.21037/jtd.2018. 05.93 .
6	LI Y, LI S Y, LI J S, et al. A rat model for stable chronic obstructive pulmonary disease induced by cigarette smoke inhalation and repetitive bacterial infection[J]. Biol Pharm Bull, 2012, 35(10):1752-1760. DOI:10.1248/bpb.b12-00407 .
7	周凤, 李德富, 袁良, 等. 两种不同方法建立的小鼠慢性阻塞性肺疾病模型的比较研究[J]. 中华结核和呼吸杂志, 2019, 42(5):367-371.
	ZHOU F, LI D F, YUAN L, et, al. A comparative study of two thronic obstructive pulmonary disease mouse models established by different methods[J]. Chin J Tuberc Respir Dis, 2019, 42(5):367-371. DOI:10.3760/cma.j.issn.1001-0939.2019.05.010 .
8	SHU J Z, LI D F, OUYANG H P, et al. Comparison and evaluation of two different methods to establish the cigarette smoke exposure mouse model of COPD[J]. Sci Rep, 2017, 7(1):15454. DOI:10.1038/s41598-017-15685-y .
9	WRIGHT J L, CHURG A. A model of tobacco smoke-induced airflow obstruction in the Guinea pig[J]. Chest, 2002, 121(5 Suppl):188S-191S. DOI:10.1378/chest.121.5_suppl.188s .
10	LIU X Y, MA C Q, WANG X Y, et al. Hydrogen coadministration slows the development of COPD-like lung disease in a cigarette smoke-induced rat model[J]. Int J Chron Obstruct Pulmon Dis, 2017, 12:1309-1324. DOI:10.2147/COPD.S124547 .
11	HE F, LIAO B L, PU J D, et al. Exposure to ambient particulate matter induced COPD in a rat model and a description of the underlying mechanism[J]. Sci Rep, 2017, 7:45666. DOI:10.1038/srep45666 .
12	王光艳, 刘菁, 刘学东. 烟熏PM_2.5颗粒建立COPD大鼠模型[J]. 国际呼吸杂志, 2017, 37(21):1610-1613. DOI:10.3760/cma.j.issn.1673-436X.2017.21.002 .
	WANG G Y, LIU J, LIU X D. Rat model of COPD established by inhalation with PM_2.5 [J]. Int J Respir, 2017, 37(21):1610-1613. DOI:10.3760/cma.j.issn.1673-436X.2017.21.002 .
13	VERNOOY J H J, DENTENER M A, VAN SUYLEN R J, et al. Long-term intratracheal lipopolysaccharide exposure in mice results in chronic lung inflammation and persistent pathology[J]. Am J Respir Cell Mol Biol, 2002, 26(1):152-159. DOI:10.1165/ajrcmb.26.1.4652 .
14	HU Y X, CUI H, FAN L, et al. Resveratrol attenuates left ventricular remodeling in old rats with COPD induced by cigarette smoke exposure and LPS instillation[J]. Can J Physiol Pharmacol, 2013, 91(12):1044-1054. DOI:10.1139/cjpp-2012-0464 .
15	庞宝森, 李远红, 张洪玉. 慢性阻塞性肺疾病动物模型的研究进展[J]. 心肺血管病杂志, 2005, 24(1):45-46. DOI:10.3969/j.issn.1007-5062.2005.01.016 .
	PANG B S, LI Y H, ZHANG H Y. Research progresseson animal models of emphysema[J]. Journal of Cardiovascular & Pulmonary Diseases, 2005, 24(1):45-46. DOI:10.3969/j.issn.1007-5062.2005.01.016 .
16	UNG T T, NGUYEN T T, LIAN S, et al. Nicotine stimulates IL-6 expression by activating the AP-1 and STAT-3 pathways in human endothelial EA.hy926 cells[J]. J Cell Biochem, 2019, 120(4):5531-5541. DOI:10.1002/jcb.27837 .
17	王妍, 朱虹江, 和春芳, 等. 清肺化痰颗粒对慢性阻塞性肺疾病急性加重期大鼠相关炎症因子及氧化应激反应的影响研究[J]. 现代中西医结合杂志, 2021, 30(12):1271-1276, 1281. DOI:10.3969/j.issn.1008-8849.2021.12.004 .
	WANG Y, ZHU H J, HE C F, et al. Effects of Qingfei Huatan granules on related inflammatory factors and oxidative stress reaction in rats with acute exacerbation of chronic obstructive pulmonary disease[J]. Modern Journal of Integrated Traditional Chinese and Western Medicine, 2021, 30(12): 1271-1276, 1281. DOI:10.3969/j.issn.1008-8849.2021.12.004 .
18	肖长栓, 刘娅平, 杨景哲. 雾化吸入和腹腔注射依达拉奉对烟雾吸入性损伤肺模型大鼠保护作用的比较研究[J]. 中国药房, 2021, 32(1):70-76. DOI:10.6039/j.issn.1001-0408.2021.01.13 .
	XIAO C S, LIU Y P, YANG J Z. Comparative study of protective effects of atomization inhalation and intra-peritoneal injection of edaravone on smoke inhalation lung injury model rats[J]. China Pharmacy, 2021, 32(1):70-76. DOI:10.6039/j.issn.1001-0408.2021.01.13 .

[1]	杨丽雅, 宋涛, 何佳璘, 郭一鸣, 亓明康, 王含必, 王慧萍. 阴道萎缩大鼠模型的建立及在药效评价中的应用[J]. 实验动物与比较医学, 2022, 42(6): 531-540.
[2]	李思迪, 付彬, 郭中坤, 林颖杰, 张振宇, 米传靓, 王可洲. 利用CRISPR/Cas9技术构建脂多糖结合蛋白基因敲除小鼠[J]. 实验动物与比较医学, 2022, 42(4): 294-300.
[3]	赵斯佳, 何新宇, 景泉, 马林, 郭春岚, 万阔. 大鼠急性牙髓炎痛敏模型的疼痛效应评价[J]. 实验动物与比较医学, 2022, 42(4): 333-341.
[4]	张成财, 刘尹航, 陈超, 江文正. 脂多糖对裸鼹鼠肺脏组织影响的初探[J]. 实验动物与比较医学, 2020, 40(6): 523-527.
[5]	安鑫, 刘兰静, 步建平. 眼缺血综合征大鼠视网膜形态及RhoA和ROCK-2表达的变化[J]. 实验动物与比较医学, 2020, 40(5): 397-.
[6]	刘乾, 管思彬, 韩锋锋, 郭雪君. 人参皂甙Rg1慢性阻塞性肺疾病大鼠肺组织上皮间充质转化的影响[J]. 实验动物与比较医学, 2018, 38(6): 422-427.
[7]	黎民, 常学辉, 张良芝, 雷震, 罗申. 不同部位注射6-羟基多巴胺建立帕金森病大鼠模型比较分析[J]. 实验动物与比较医学, 2018, 38(4): 261-266.
[8]	赵玮, 俞冰, 张水娟, 贾永良, 谢强敏. 应用无约束全身体积描记系统检测小鼠肺功能[J]. 实验动物与比较医学, 2017, 37(5): 383-389.
[9]	熊英, 张金娟, 李玲, 陈燕, 吴艳俊, 黄惠妍. 香烟烟雾对慢性阻塞性肺疾病大鼠肺功能及血清细胞因子水平影响[J]. 实验动物与比较医学, 2017, 37(4): 315-319.
[10]	刘乾, 刘松, 徐卫国, 管思彬, 郭雪君. 泛素特异性蛋白酶19对烟熏诱导慢性阻塞性肺疾病大鼠模型骨骼肌萎缩的作用及机制[J]. 实验动物与比较医学, 2017, 37(3): 185-190.
[11]	高峰, 张士更, 张楠, 张布衣, 戴玉樑, 李绍江. 慢性膀胱炎相关膀胱过度活动症大鼠模型的建立[J]. 实验动物与比较医学, 2016, 36(5): 340-344.
[12]	卫振, 娄绘芳, 吴凯, 陈雁虹, 谢敏, 刘迪文, 谢强敏. 两个豚鼠品系速发型哮喘模型中组胺受体的表达及对肺功能的影响[J]. 实验动物与比较医学, 2016, 36(2): 101-106.
[13]	冯佳, 向阳, 夏燕, 陈安平, 杨年安, 龚书识, 袁林. 三种大鼠骨质疏松模型的比较研究[J]. 实验动物与比较医学, 2015, 35(1): 52-55.
[14]	乔明, 刘兰英, 王和生. 慢性过敏性哮喘大鼠模型的建立与评价[J]. 实验动物与比较医学, 2014, 34(1): 79-82.
[15]	田小芸, 董敏, 尤金炜, 赵长霖, 许龙祥, 恽时锋. 先天性白内障大鼠培育初报[J]. 实验动物与比较医学, 2013, 33(5): 358-360.