基于单细胞测序探讨针刺足三里穴治疗哮喘作用机制的实验研究

An experimental study on mechanism of acupuncture at Zusanli acupoint in treatment of asthma based on single-cell sequencing

中文关键词: 老年  哮喘  细胞衰老  单细胞转录组测序  针刺  足三里

英文关键词: elderly  asthma  cellular senescence  single-cell RNA sequencing  acupuncture  ST36

中文摘要:

      目的 哮喘作为全球性重大公共卫生问题,其防治策略亟待优化.老年哮喘患者因多病共存导致临床管理难度显著增加,而现有药物疗法的不良反应问题促使研究者积极探索新型治疗靶点与干预模式.基于针刺疗法在老年哮喘临床实践中展现的疗效优势,本研究拟通过单细胞转录组测序技术,系统解析针刺干预与细胞衰老调控的潜在关联机制.方法 构建小鼠哮喘模型后,获取肺组织样本进行单细胞RNA测序(scRNA-seq),采用Seurat和Harmony软件包进行数据质控与批次校正.通过整合GWAS数据库的哮喘遗传关联数据和GTEx项目的表达数量性状位点(eQTLs),运用差异表达分析与孟德尔随机化(MR)分析筛选哮喘致病基因.基于GSEA数据库的细胞衰老特征基因集,系统解析针刺干预对细胞衰老相关基因表达谱的动态调控特征.结果 研究可确定,针刺可下调T细胞群比例,尤其是一部分功能性T细胞群CD4+Tem细胞群,从而发挥缓解哮喘2型炎症.而下调该细胞群的趋势与下调细胞衰老相关基因的趋势一致,其中与哮喘致病因果关系最明确的细胞衰老相关基因是FIGNL1在凋亡调控与细胞衰老调控中起到重要作用.结论 针刺ST36通过多途径、多靶点调节免疫反应,缓解老年哮喘患者的2型炎症反应,其中,对细胞衰老的下调进而对T细胞亚群功能的抑制,从而缓解2型炎症的发展,这一机制在单细胞测序层面得到了验证.未来的研究应进一步探索相关基因与通路的作用机制,为针灸在老年哮喘患者治疗中的应用提供更加坚实的理论基础.

英文摘要:

      Objective Asthma is a significant global public health issue,and its prevention and treatment strategies re-quire urgent optimization.The coexistence of multiple diseases in elderly asthma patients significantly increases the difficulty of clinical management,and the side effects of existing drug therapies have prompted researchers to actively explore new treatment targets and intervention models.Based on the therapeutic advantages demonstrated by acupuncture therapy in clinical practice of elderly asthma patients,this study intends to systematically analyze the potential correlation mechanism between acupuncture in-tervention and cell senescence regulation through single-cell transcriptome sequencing technology.Methods After a mouse asthma model was constructed,the lung tissue samples were obtained for single-cell RNA sequencing(scRNA-seq),and the Seurat and Harmony software packages were used for data quality control and batch correction.By integrating asthma genetic association data from GW AS database and expression quantitative trait loci(eQTLs)from GTEx project,the differential ex-pression analysis and Mendelian randomization(MR)analysis were used to identify asthma-related pathogenic genes.Based on the set of cell senescence characteristic genes in the GSEA database,the dynamic regulatory characteristics of acupuncture inter-vention on the expression profiles of cell senescence-related genes were systematically analyzed.Results Acupuncture could down-regulate the proportion of T cell populations,especially a part of the functional T cell subpopulation,such as the CD4+Tem cell subpopulation,thereby exerting a role in alleviating type 2 inflammation of asthma.The trend of downregulating this cell subpopulation was consistent with the trend of downregulating genes related to cell senescence.Among them,the cell se-nescence-related gene with the most definite causal relationship to asthma pathogenesis was FIGNL1 and it played an important role in the regulation of apoptosis and cell senescence.Conclusion Acupuncture at the ST36 acupoint regulates immune re-sponse through multiple pathways and targets and alleviates type 2 inflammatory response in elderly asthma patients.The down-regulation of cell senescence and the subsequent suppression of T cell subpopulation function help reduce the progression of type 2 inflammation.This mechanism has been confirmed through scRNA-seq.Future research should further explore the mechanism of action of related genes and pathways to provide a more solid theoretical basis for the application of acupuncture in the treat-ment of elderly asthma patients.