基于转录组学探讨外泌体诱导病毒性哮喘动物模型的作用机制及潜在的中药干预

Mechanism of Exosome-Induced Viral Asthma Model Based on Transcriptomics and Potential Traditional Chinese Medicine Intervention

目的:基于转录组学探究呼吸道合胞病毒(RSV)干预后的外泌体在哮喘发病中的作用及潜在中医药干预机制。方法:利用呼吸道合胞病毒(RSV)干预骨髓间充质干细胞后提取外泌体,并进行透射电镜、粒径(NTA)及蛋白免疫印迹法(Western blot)鉴定外泌体的形态及标志物等;将24只SPF级雄性C57小鼠随机分为正常对照组、呼吸道合胞病毒组、外泌体组,造模3周后进行肺功能、病理染色验证是否存在哮喘病理改变,并对小鼠气道组织进行RNA-seq高通量转录组测序,明确差异基因;通过中药整合药理学方法对关键靶点潜在中药进行预测并汇总。结果:透射电镜显示外泌体呈典型的双层膜茶托样结构,粒径显示其平均直径为133.1 nm;WB法检测到外泌体膜标志蛋白(CD63、CD9和TSG101)的表达。与正常对照组相比,呼吸道合胞病毒组与外泌体组用力肺活量(FVC)明显下降(P<0.05),动态肺顺应性(CDYN)明显下降(P<0.01),呼吸道合胞病毒组、外泌体组最大呼气流量(PEF)明显下降(P<0.05);而呼吸道合胞病毒组和外泌体组肺阻力(RL)明显升高(P<0.01)。在炎症浸润及气道黏液分泌等方面,外泌体组小鼠有炎症细胞浸润及嗜酸性粒细胞浸润,呼吸道合胞病毒组以中性粒细胞、淋巴细胞浸润为主,杯状细胞黏液分泌较外泌体组明显。气道上皮测序结果提示三组间差异基因多富集在免疫、神经及内分泌系统;差异基因共同富集的通路为过氧化物酶体增殖物激活受体、白介素-17、细胞外基质-受体相互作用信号通路。预测的潜在治疗中药以清热药、补虚药及理气活血药为主,其组分能作用多个靶点及通路调节哮喘神经、内分泌及免疫平衡。结论:病毒干预后的外泌体能够诱导哮喘气道炎症、气道重塑等病理改变;中医药从多环节、多靶点、多系统方面发挥了其整体调节作用。

Objective:This study aimed to explore the role of exosomes after respiratory syncytial virus(RSV)intervention in the pathogenesis of asthma and potential mechanisms of traditional Chinese medicine(TCM)intervention based on transcriptomics.Methods:Exosomes were extracted from bone marrow mesenchymal stem cells after RSV intervention,then transmission electron microscopy,nanoparticle tracking analysis(NTA)and Western blot were used to identify the morphology and markers of exosomes.Twenty-four SPF grade male C57 mice were randomly divided into blank control group,RSV group,and exosome group.After modeling for 3 weeks,lung function was measured and pathological staining was performed to verify the presence of pathological changes of asthma.High-throughput transcriptome sequencing(RNA-seq)was conducted on mouse airway tissues to identify differentially expressed genes(DEGs).Finally,potential TCM key targets were predicted and summarized using integrated pharmacological methods.Results:Transmission electron microscopy showed that exosomes exhibited a typical double-layered membrane structure,with an average diameter of 133.1 nm as determined by NTA.Western blot detected the expression of exosomal membrane marker proteins CD63,CD9 and TSG101.Compared with the blank control group,both the RSV group and exosome group showed a significant decrease in forced vital capacity(FVC)(P<0.05)and a marked decrease in dynamic lung compliance(Cdyn)(P<0.01).The peak expiratory flow(PEF)was decreased in the RSV group(P<0.05)and it was significantly decreased in exosome group(P<0.01).Compared with blank control group,the Lung resistance(RL)was significantly increased in the RSV group and exosome group(P<0.01).There was no statistically significant differences in lung function indices between the RSV group and exosome group(P>0.05).In terms of inflammation infiltration and airway mucus secretion,the mice in the exosome group exhibited inflammatory cell infiltration and eosinophil infiltration,while those in RSV group mainly showed neutrophil and lymphocyte infiltration,with significantly more goblet cell mucus secretion than in the exosome group.The sequencing results of airway epithelium suggested that DEGs among the three groups were mainly enriched in the immune,nervous,and endocrine systems,with common enriched pathways including peroxisome proliferator-activated receptor,interleukin 17,and ECM-receptor interaction signaling pathways.The predicted potential TCM were mainly heat-clearing medicinal,tonifying medicinal and qi-regulating and blood-activating medicinal,whose components could act on multiple targets and pathways to regulate asthma neuroendocrine and immune balance.Conclusion:Exosomes after RSV intervention could induce pathological changes of asthma,such as airway inflammation and airway remodeling.TCM exerted an overall regulatory effect from multiple aspects,multiple targets,and multiple systems.