南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (10): 1492-1500.doi: 10.12122/j.issn.1673-4254.2021.10.07

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COPD患者免疫细胞亚群特征及六味补气胶囊干预的分子机制

王小乐，朱 洁，高雅婷，吴 凡，杨勤军，童佳兵，张星星，王传博，吴 迪，李泽庚   

  1. 安徽中医药大学研究生院，中西医结合学院，安徽 合肥 230012；第一附属医院，安徽 合肥 230031；安徽省中医药科学院中医呼吸病防治研究所，安徽 合肥 230031；安徽省教育厅重点实验室中医药防治肺系重大疾病重点实验室，安徽 合肥 230031; 安徽医科大学第二附属医院中医科，安徽 合肥 230601
  • 出版日期:2021-10-20 发布日期:2021-11-11

Liuwei Buqi capsule modulates immune function by targeting multiple immune cell subsets in lung tissue of patients with COPD

WANG Xiaole, ZHU Jie, GAO Yating, WU Fan, YANG Qinjun, TONG Jiabing, ZHANG Xingxing, WANG Chuanbo, WU Di, LI Zegeng   

  • Online:2021-10-20 Published:2021-11-11

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摘要： 目的 探讨慢性阻塞性肺疾病（COPD）肺组织免疫细胞亚群特征及六味补气胶囊改善其免疫炎症失衡的机制。方法 基于基因表达汇编（GEO）数据库，下载COPD相关研究的单细胞测序数据，使用R软件中Seurat包分析获取COPD免疫细胞亚群特征，以构建COPD的各免疫细胞亚群的“免疫细胞亚群-差异基因”关系；通过中药系统药理学数据库与分析平台（TCMSP）平台获取六味补气胶囊的主要活性成分及靶基因，将靶基因映射到COPD各免疫细胞亚群的差异基因，构建“六味补气胶囊-免疫细胞亚群-靶基因”网络，通过京都基因与基因组百科全书（KEGG）富集分析，研究该网络中靶基因的显著富集的通路,并获取显著富集通路间关联的靶基因。经六味补气胶囊干预COPD大鼠模型实验研究，分析其对COPD大鼠的肺功能和肺组织病理改变，血清IL1B、NF-κB、TNF-α的表达情况，及肺组织IKBα、JNK、c-JUN、c-FOS的蛋白表达。结果 COPD和正常对照中均存在巨噬细胞、肺泡巨噬细胞等18种免疫相关细胞亚群。与正常对照相比，COPD患者肺组织中巨噬细胞、cDC1、pDC、肥大细胞、T细胞、成熟树突状细胞占总细胞数比的差异均具有统计学意义（P<0.05）。六味补气胶囊靶向多个免疫细胞亚群，且靶基因主要富集于脂质和动脉粥样硬化、IL-17 信号通路、Toll样受体信号通路、TNF 信号通路等免疫炎症相关信号通路，其中CXCL8、IL1B、JUN、NFKBIA、MAPK8、FOS等可能是显著富集通路间的关联基因，动物实验研究表明六味补气胶囊可有效改善COPD大鼠肺功能和肺组织病理改变，并降低COPD大鼠血清IL1B、TNF-α、NF-κB（P<0.05）和肺组织肺组织JNK、c-JUN、c-FOS（P<0.01）蛋白的表达，升高肺组织IκBα蛋白表达（P<0.01）。结论 六味补气胶囊可能通过靶向COPD患者肺组织中多种免疫细胞亚群发挥免疫调节作用。

关键词: 慢性阻塞性肺疾病, 六味补气胶囊, 免疫炎症, 单细胞转录组测序

Abstract: Objective To investigate the characteristics of immune cell subsets in the lung tissues of patients with chronic obstructive pulmonary disease (COPD) and the mechanism of Liuwei Buqi capsule in modulating immune and inflammatory imbalance in COPD. Methods We downloaded COPD-related single-cell RNA sequencing data from Gene Expression Omnibus (GEO) and identified COPD immune cell subsets using the Seurat package in the R software to construct an immune cell subsets-differential genes network. The target genes and active ingredients of Liuwei Buqi capsule were obtained from the Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), and the Liuwei Buqi capsule-immune cell subsets-target genes network was constructed by mapping the target genes to the differentially expressed genes in each immune cell subset. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to analyze significantly enriched pathways of the target genes, and the key genes involved in the top 20 pathways were identified. In a rat model of COPD, we investigated the effects of Liuwei Buqi capsule on pulmonary function, lung tissue pathology, serum levels of IL-1β, NF-κB, and TNF-α, and expressions of IKBα, JNK, c-JUN, and c-FOS proteins in the lung tissue. Results A total of 18 immune-related cell subsets, including macrophages and alveolar macrophages, were identified in both COPD patients and healthy control subjects, and the patients with COPD showed significant changes the percentages of macrophages, cDC1, pDC, mast cells, T cells, and mature dendritic cells (P<0.05). Liuwei Buqi capsules targeted multiple immune cell subsets, and the identified target genes were enriched mostly in such immune and inflammation-related signaling pathways as lipids and atherosclerosis, IL-17 signaling pathway, Toll-like receptor signaling pathway, and TNF signaling pathway; the genes CXCL8, IL1B, JUN, NFKBIA, MAPK8, and FOS were the key genes involved in the significantly enriched pathways. In the rat models of COPD, treatment with Liuwei Buqi capsule significantly improved pulmonary function, alleviated lung pathologies, reduced serum levels of IL-1β, TNF-α, and NF-κB (P<0.05) and pulmonary expressions of JNK, c-JUN, and c-FOS (P<0.01) protein, and increased pulmonary expression of IκBα (P<0.01). Conclusion Liuwei Buqi capsule may play an immunomodulatory role by targeting multiple immune cell subsets in the lung tissue of COPD patients.

Key words: chronic obstructive pulmonary disease, Liuwei Buqi capsule, immune and inflammation, single-cell RNA sequencing