基于外泌体miRNA探讨针刺“内迎香”穴治疗变应性鼻炎的作用机制

Mechanism of Needling EX-HN9 in Treating AR Based on Exosomal miRNA

目的:筛选针刺内迎香穴治疗变应性鼻炎(AR)大鼠血浆外泌体中差异表达的miRNA,并对其进行生物信息学分析,探讨针刺内迎香穴治疗AR的可能作用机制。方法:将30只SPF级SD大鼠随机分为空白组(Ko组)、模型组(Mu组)和针刺组(Zh组),每组各10只。模型组和针刺组采用卵清蛋白腹腔注射及鼻黏膜刺激法建立变应性鼻炎大鼠模型,造模后对针刺组大鼠予针刺内迎香穴进行干预,每周2次,连续3周。HE染色观察鼻黏膜病理形态改变;超速离心法提取大鼠血浆外泌体,透射电镜(TEM)和粒径分析(NTA)检测外泌体形态和粒径浓度;高通量测序以P<0.05的标准筛选大鼠血浆外泌体中显著差异表达的miRNA;利用miRanda和Targetscan得出差异表达miRNA靶基因,进行基因本体论(GO)和京都基因与基因组百科全书(KEGG)富集分析。结果:针刺作用关键miRNA共有10个,其中5个上调,5个下调,共得出7 408个靶基因,涉及1 130个生物过程、158个细胞组分、166个分子功能,KEGG信号通路有31条(P<0.05)。结论:针刺内迎香穴可明显改善AR大鼠鼻黏膜组织炎性细胞浸润情况,其机制可能与通过调节rno-miR-92a-3p、rno-let-7d-3p、rno-miR-328a-3p、rno-miR-223-3p、rno-miR-23a-3p、rno-miR-451-5p、novel_314、rno-miR-144-5p、rno-miR-23b-3p及rno-miR-144-3p的表达,进而调控MAPK信号通路有关。

Objective:To screen the differentially expressed miRNAs in plasma exosomes of rats with allergic rhinitis(AR) after needling Neiyingxiang(EX-HN9),to analyze bioinformatics and explore the potential action mechanism of needling EX-HN9 in treatment of AR.Methods:30 SPF-grade SD rats were randomly divided into the blank group,the model group and the acupuncture group,with 10 rats in each group.The model of AR was established by intraperitoneal injection of ovalbumin and nasal mucosa stimulation.After successful modeling,the acupuncture group was treated with needling EX-HN9,twice a week for three consecutive weeks.HE staining was used to observe the pathological changes in the nasal mucosa.Ultracentrifugation was used to extract plasma exosomes,and transmission electron microscope(TEM) and nanoparticle tracking analysis(NTA) were used to detect the morphology and particle size concentration of the extracellular vesicles.High-throughput sequencing was used to screen significantly differentially expressed miRNAs in rat plasma extracellular vesicles with a significance threshold of P<0.05.miRanda and Targetscan were used to identify target genes of the differentially expressed miRNAs,and gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis were performed.Results:There were a total of 10 key miRNAs for acupuncture,including 5 up-regulated and 5 down-regulated,yielding a total of 7 408 target genes,which involved 1 130 biological processes,158 cell components,166 molecular functions,and 31 KEGG signaling pathways(P<0.05).Conclusion:Needling EX-HN9 remarkably improve the infiltration of inflammatory cells in nasal mucosal tissues in AR rats,which is possibly related to the regulation of the expressions of rno-miR-92a-3p,rno-let-7d-3p,rno-miR-328a-3p,rno-miR-223-3p,rno-miR-23a-3p,rno-miR-451-5p,novel_314,rno-miR-144-5p,rno-miR-23b-3p and rno-miR-144-3p and further to regulate the MAPK signaling pathway.