张贝贝, 曾梦楠, 张钦钦, 王茹, 郭彭莉, 卢婉晴, 贾菊芳, 刘萌, 张宇涵, 冯卫生, 郑晓珂. 葶苈大枣泻肺汤对哮喘大鼠免疫炎症和肠道菌群的影响J. 药学学报, 2022, 57(8): 2364-2377. DOI: 10.16438/j.0513-4870.2022-0116
引用本文: 张贝贝, 曾梦楠, 张钦钦, 王茹, 郭彭莉, 卢婉晴, 贾菊芳, 刘萌, 张宇涵, 冯卫生, 郑晓珂. 葶苈大枣泻肺汤对哮喘大鼠免疫炎症和肠道菌群的影响J. 药学学报, 2022, 57(8): 2364-2377. DOI: 10.16438/j.0513-4870.2022-0116
ZHANG Bei-bei, ZENG Meng-nan, ZHANG Qin-qin, WANG Ru, GUO Peng-li, LU Wan-qing, JIA Ju-fang, LIU Meng, ZHANG Yu-han, FENG Wei-sheng, ZHENG Xiao-ke. Effects of Tingli Dazao Xiefei Decoction on the immune inflammation and intestinal flora in asthmatic ratsJ. Acta Pharmaceutica Sinica, 2022, 57(8): 2364-2377. DOI: 10.16438/j.0513-4870.2022-0116
Citation: ZHANG Bei-bei, ZENG Meng-nan, ZHANG Qin-qin, WANG Ru, GUO Peng-li, LU Wan-qing, JIA Ju-fang, LIU Meng, ZHANG Yu-han, FENG Wei-sheng, ZHENG Xiao-ke. Effects of Tingli Dazao Xiefei Decoction on the immune inflammation and intestinal flora in asthmatic ratsJ. Acta Pharmaceutica Sinica, 2022, 57(8): 2364-2377. DOI: 10.16438/j.0513-4870.2022-0116

葶苈大枣泻肺汤对哮喘大鼠免疫炎症和肠道菌群的影响

Effects of Tingli Dazao Xiefei Decoction on the immune inflammation and intestinal flora in asthmatic rats

  • 摘要: 从免疫炎症和肠道菌群角度探究葶苈大枣泻肺汤对哮喘的干预作用机制, 为指导临床合理用药提供理论依据。通过腹腔注射卵清蛋白(ovalbumin, OVA) 致敏液和雾化激发构建哮喘大鼠模型, 分为正常组(control, CON)、模型组(model, M)、地塞米松组(dexamethasone, DEX, 0.075 mg·kg-1) 和葶苈大枣泻肺汤组(Tingli Dazao Xiefei Decoction, TLDZ, 3.5 g·kg-1)。首先通过咳嗽、喘息、酚红排泌、苏木精-伊红染色(hematoxylin-eosin staining, H & E)、马松染色(Masson) 和过碘酸雪夫氏染色(periodic acid Schiff, PAS) 观察葶苈大枣泻肺汤对大鼠哮喘症状、肺脏和气管病理变化的影响; ELISA检测大鼠血清中转化生长因子β1 (transforming growth factor β1, TGF-β1)、白细胞介素(interleukin, IL) 6和IL-10水平及肺泡灌洗液中γ干扰素(interferon γ, IFN-γ)、免疫球蛋白E (immunoglobulin E, IgE)、IL-4、IL-17A和肿瘤坏死因子α (tumor necrosis factor α, TNF-α) 水平; qRT-PCR检测肺组织中IL-5、IL-13和IL-33的mRNA水平; 然后采用流式细胞术结合免疫组化检测脾脏中巨噬细胞和中性粒细胞水平和外周血中自然杀伤细胞(natural killer cell, NK)、辅助性T细胞(helper T cell, Thc)、树突状细胞(dendritic cell, DC)、调节性T细胞(regulatory T cell, Treg) 和辅助性T细胞17 (T helper cell 17, Th17) 水平; 接着通过16S rDNA高通量测序分析哮喘大鼠肠道菌群。病理学和炎症因子结果显示, 葶苈大枣泻肺汤可有效减轻大鼠哮喘症状, 改善肺部组织病理变化, 减少杯状细胞和胶原纤维产生, 抑制哮喘大鼠炎症反应; 免疫细胞结果显示, 葶苈大枣泻肺汤可有效增加哮喘大鼠NK、Thc、DC和Treg细胞水平并降低巨噬细胞、中性粒细胞和Th17细胞水平; 肠道菌群结果显示, 葶苈大枣泻肺汤可增加哮喘大鼠乳酸杆菌、瘤胃球菌、克里斯滕森菌、双歧杆菌和真杆菌属水平, 降低厚壁菌、MucispirillumRomboutsia和脱硫弧菌属水平。因此, 推测葶苈大枣泻肺汤可通过调节哮喘大鼠体内免疫炎症反应和肠道菌群从而改善大鼠哮喘症状。本文中所有动物实验都获得河南中医药大学伦理学委员会批准。

     

    Abstract: The study aims to explore the intervention mechanism of Tingli Dazao Xiefei Decoction on asthma from the perspective of immune inflammation and intestinal flora, providing a theoretical basis for guiding clinical medication. The ovalbumin (OVA) asthmatic rat model was established by intraperitoneal injection of OVA sensitization solution and aerosol challenge, and divided into control (CON), model (M), dexamethasone group (DEX, 0.075 mg·kg-1) and Tingli Dazao Xiefei Decoction (TLDZ, 3.5 g·kg-1). Firstly, the effects of Tingli Dazao Xiefei Decoction on asthma symptoms of rats, lung and trachea pathological changes of asthmatic rats were observed by inducing cough and asthma experiment, phenol red excretion, hematoxylin-eosin staining (H & E), Masson and periodic acid Schiff (PAS) staining; the levels of transforming growth factor β1 (TGF-β1), interleukin (IL) 6 and IL-10 in rat serum and the levels of interferon γ (IFN-γ), immunoglobulin E (IgE), IL-4, IL-17A and tumor necrosis factor α (TNF-α) in bronchoalveolar lavage fluid (BALF) were detected by ELISA; the mRNA levels of IL-5, IL-13 and IL-33 in the lung were determined by qRT-PCR; the levels of macrophages and neutrophils in the spleen and the levels of natural killer cell (NK), helper T cell (Thc), dendritic cell (DC), regulatory T cell (Treg) and T helper cell 17 (Th17) in the peripheral blood were measured by flow cytometry combined with immunohistochemistry; the intestinal flora of asthmatic rats were analyzed by 16S rDNA high-throughput sequencing. Pathology and inflammatory results showed that Tingli Dazao Xiefei Decoction could effectively alleviate the asthma symptoms in rats, improve the pathological changes of lung tissue, reduce the production of goblet cells and collagen fibers, and reduce the inflammatory response in asthmatic rats; the results of immune cells showed that Tingli Dazao Xiefei Decoction could effectively increase the levels of NK, Thc, DC and Treg cells and reduce the levels of macrophages, neutrophils and Th17 cells in asthmatic rats; the results of intestinal flora showed that Tingli Dazao Xiefei Decoction could increase the levels of Lactobacillus, Ruminococcus, Christensenellaceae, Bifidobacterium and Eubacterium_xylanophilum-group, and decrease the levels of Firmicutes, Desulfovibrio, Mucispirillum and Romboutsia in asthmatic rats. Therefore, it is speculated that Tingli Dazao Xiefei Decoction can improve the symptom of asthmatic rats by regulating the immune inflammation and intestinal flora in the asthmatic rats. All animal experiments in this article were approved by the Ethics Committee of Henan University of Chinese Medicine.

     

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