桔梗元参汤对过敏性哮喘小鼠气道炎症和黏液分泌的影响及其机制

doi:10.13481/j.1671-587X.20240102

摘要/Abstract

摘要：

目的探讨桔梗元参汤（JGYST）对过敏性哮喘小鼠气道组织炎症和黏液分泌的影响，并阐明其相关作用机制。方法 40只雄性C57BL/J小鼠随机分为对照组、JGYST组、卵清蛋白（OVA）组及OVA+JGYST组。OVA组和OVA+JGYST组小鼠用50 μg OVA腹腔注射致敏，每周2次，致敏后连续7 d用OVA 20 μg滴鼻激发哮喘；OVA+JGYST组小鼠每次OVA激发前1 h用JGYST 200 μL灌胃，共灌胃7次；对照组用相同剂量的PBS缓冲液腹腔注射、滴鼻和灌胃；JGYST组小鼠用相同剂量PBS缓冲液腹腔注射和滴鼻，用相同剂量JGYST灌胃。采用HE染色和过碘酸-雪夫（PAS）染色观察各组小鼠肺组织病理形态表现并计算炎症评分和PAS评分，流式细胞术检测各组小鼠肺组织中嗜酸性粒细胞、中性粒细胞、辅助性T淋巴细胞1（Th1）、辅助性T淋巴细胞2（Th2）和树突状细胞（DCs）数及成熟DCs百分率和活性氧（ROS）水平，实时荧光定量PCR（RT-qPCR）法检测各组小鼠肺组织中白细胞介素4（IL-4）、白细胞介素10（IL-10）和肿瘤坏死因子α（TNF-α）mRNA表达水平。结果 HE染色和PAS染色，对照组小鼠气道和肺泡结构完整、无炎症细胞浸润，无黏液分泌；与对照组比较，OVA组小鼠气道组织周围有大量炎症细胞浸润，炎症评分和PAS评分明显升高（P<0.01）；与OVA组比较，JGYST组和OVA+JGYST组小鼠气道组织炎症细胞浸润减少，炎症评分和PAS评分明显降低（P<0.01）。流式细胞术检测，与对照组比较，OVA组小鼠肺组织中嗜酸性粒细胞、Th2和DCs数均明显增加（P<0.05或P<0.01），成熟DCs百分率和ROS水平均明显升高（P<0.01）；与OVA组比较，JGYST组和OVA+JGYST组小鼠肺组织中嗜酸性粒细胞、Th2和DCs数均明显减少（P<0.01），成熟DCs百分率和ROS水平均明显降低（P<0.01）。RT-qPCR法检测，与对照组比较，OVA组小鼠肺组织中IL-4、IL-10和TNF-α mRNA表达水平升高（P<0.01）；与OVA组比较，JGYST组和OVA+JGYST组小鼠肺组织中IL-4和TNF-α mRNA表达水平降低（P<0.01），IL-10 mRNA表达水平升高（P<0.01）。结论 JGYST可以减轻过敏性哮喘小鼠气道组织炎症和黏液分泌，其作用机制可能与其减少Th2细胞和DCs数量，降低ROS水平和促炎细胞因子表达水平同时升高抗炎细胞因子表达水平有关。

关键词: 桔梗元参汤, 哮喘, 气道炎症, 活性氧, 细胞因子

Abstract:

Objective To discuss the effect of Jiegeng Yuanshen Tang（JGYST） on airway tissue inflammation and mucus secretion in the mice with allergic asthma， and to clarify the related mechanism. Methods Forty male C57BL/J mice were randomly divided into control group， JGYST group， ovalbumin （OVA） group， and OVA + JGYST group. The mice in OVA group and OVA +JGYST group were sensitized with 50 μg OVA via intraperitoneal injection twice weekly， followed by 20 μg OVA nasal drops daily for 7 d to induce asthma；the mice in OVA +JGYST group were gavaged with 200 μL JGYST 1 h before each OVA challenge， and the administration lasted for 7 d； the mice in control group were given equivalent dose of PBS via intraperitoneal injection， nasal drops， and gavage； the mice in JGYST group were given the same dose of PBS for intraperitoneal and nasal administration and gavaged with the same dose of JGYST. The pathomorphology of lung tissue of the mice in various groups was observed by HE staining and periodic acid-Schiff （PAS） staining， and the inflammation and PAS scores were calculated； flow cytometry method was used to detect the numbers of eosinophils， neutrophils， helper T lymphocyte 1 （Th1） cells， helper T lymphocyte 2 （Th2） cells， and dendritic cells （DCs）， as well as the percentage of mature DCs and level of reactive oxygen species （ROS） in lung tissue of the mice in various groups；real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of interleukin-4 （IL-4）， interleukin-10 （IL-10）， and tumor necrosis factor-α （TNF-α） mRNA in lung tissue of the mice in various groups. Results The HE and PAS staining results showed that the mice in control group had intact airway and alveolar structure，without infiltration of inflammatory cells or mucus secretion； compared with control group， there was a large number of infiltrating inflammatory cells in airway tissue of the mice in OVA group，and the inflammation and PAS scores were increased （P<0.01）； compared with OVA group， the infiltration of inflammatory cells in airway tissue of the mice in JGYST group and OVA + JGYST group was decreased， and the inflammation and PAS scores were significantly decreased （P<0.01）. The flow cytometry results showed that compared with control group， the numbers of eosinophils， Th2 cells， and DCs in lung tissue of the mice in OVA group were increased （P<0.05 or P<0.01）， and the percentage of mature DCs and level of ROS were significantly increased （P<0.01）； compared with OVA group， the numbers of eosinophils， Th2 cells， and DCs in lung tissue of the mice in JGYST group and OVA + JGYST group were decreased （P<0.01）， and the percentage of mature DCs and level of ROS were significantly decreased （P<0.01）. The RT-qPCR results showed that compared with control group， the expression levels of IL-4， IL-10， and TNF-α mRNA in lung tissue of the mice in OVA group were increased （P<0.01）； compared with OVA group， the expression levels of IL-4 and TNF-α mRNA in lung tissue of the mice in JGYST group and OVA + JGYST group were decreased （P<0.01）， while the expression level of IL-10 mRNA was increased （P<0.01）. Conclusion JGYST can alleviate the airway tissue inflammation and mucus secretion in the mice with allergic asthma，and its mechanism may be related to reducing the number of Th2 cells and DCs， decreasing the ROS level and expression level of proinflammatory cytokine， and increasing the expression level of anti-inflammatory cytokine.

Key words: Jiegeng Yuanshen Tang, Asthma, Airway inflammation, Reactive oxygen species, Cytokine

中图分类号:

R562

唐红梅,李月蛟,王星,王志彬,袁谢芳,王孝芸. 桔梗元参汤对过敏性哮喘小鼠气道炎症和黏液分泌的影响及其机制[J]. 吉林大学学报(医学版), 2024, 50(1): 10-17.

Hongmei TANG,Yuejiao LI,Xing WANG,Zhibin WANG,Xiefang YUAN,Xiaoyun WANG. Effect of Jiegeng Yuanshen Tang on airway inflammation and mucus secretion in allergic asthmatic mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2024, 50(1): 10-17.

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Primer	Sequence（5'－3'）
IL-4	Forward: ATCATCGGCATTTTGAACGAGGTC
	Reverse: ACCTTGGAAGCCCTACAGACGA
IL-10	Forward: CGGGAAGACAATAACTGCACCC
	Reverse: CGGTTAGCAGTATGTTGTCCAGC
TNF-α	Forward: GGTGCCTATGTCTCAGCCTCTT
	Reverse: GCCATAGAACTGATGAGAGGGAG
GAPDH	Forward: CATCACTGCCACCCAGAAGACTG
	Reverse: ATGCCAGTGAGCTTCCCGTTCAG

Group	Inflammation score	PAS score
Control	2.17±0.82	0.67±0.52
OVA	7.75±0.82^*	1.83±0.41^*
JGYST	2.83±0.61^△	0.50±0.55^△
OVA+JGYST	4.50±0.84^△	0.83±0.41^△

Group	Number of eosinophils	Number of neutrophils
Control	603.33±293.62	857.50±326.55
OVA	9 957.67±5 692.08^*	3 261.50±3 144.02
JGYST	382.33±140.36^△	673.50±195.18
OVA+JGYST	3 222.00±1 843.63^△	3 569.00±2 212.35

Group	Number of Th1	Number of Th2
Control	664.00±707.45	167.33±51.76
OVA	377.67±410.03	1 234.33±456.00^*
JGYST	323.83±113.95	204.83±84.02^△
OVA+JGYST	401.67±264.29	496.67±275.74^△

Group	Number of total DCs	Number of mature DCs	Percentage of mature DCs (η/%)
Control	1 7291.83±3 202.22	2 324.17±527.28	13.54±2.28
OVA	36 178.33±16 558.82^*	28 040.50±13 712.93^**	76.85±7.08^**
JGYST	13 857.67±3 931.81^△	1 977.83±848.21^△	13.78±3.07^△
OVA+JGYST	16 398.33±6 992.92^△	8 022.67±5 424.33^△	45.00±13.49^△