基于外泌体转录组学探讨玉蝉卫肺丸治疗变应性鼻炎的机制

Discussion on mechanism of Yuchan Weifei Pills in treating allergic rhinitis based on the exosomal transcriptomics

目的:通过外泌体转录组学探讨玉蝉卫肺丸治疗变应性鼻炎(AR)的机制。方法:将30只SD大鼠随机分为空白组、模型组、玉蝉卫肺丸组,各10只,应用卵清蛋白腹腔注射加鼻黏膜刺激法建立大鼠AR模型。以鼻黏膜HE染色观察鼻黏膜病理改变,免疫组化和Western Blot检测γ干扰素(IFN-γ)、白细胞介素(IL)-4表达水平。采用超速离心法提取大鼠外周血血浆中的外泌体,透射电镜、粒径分析(NTA)检测外泌体形态,qPCR和高通量测序根据P<0.05且|log2(Fold Change)|>1的标准筛选大鼠血浆外泌体中显著差异表达的miRNA,使用miRanda和Targetscan获得差异表达miRNA的下游靶基因,并进行GO、KEGG富集分析。结果:与模型组比较,玉蝉卫肺丸组大鼠鼻黏膜病理改变减轻,鼻黏膜IL-4阳性细胞及蛋白表达显著降低(P<0.05),鼻黏膜IFN-γ阳性细胞及蛋白表达显著升高(P<0.05)。本研究共筛选出10条差异表达的miRNA。靶基因通路分析集中于PI3K-Akt信号通路、TNF信号传导途径、MAPK信号传导途径等。结论:玉蝉卫肺丸治疗AR的机制可能是通过调控血浆外泌体介导的miRNA信号表达,激活MAPK等相关信号通路,抑制Th2细胞因子的表达,调控Th1/Th2平衡。

Objective:To explore the potential mechanisms of Yuchan Weifei Pills(YWP)in the treatment of allergic rhinitis(AR)by using the exosomal transcriptomics.Methods:Thirty SD rats were randomly divided into blank control group,model group and Yuchan Weifei Pills group,10 rats in each group.Mice were subjected to serial intranasal challenge with ovalbumin.HE staining was used to observe the inflammatory reaction of nasal mucosa.Immunohistochemical method and Western Blot was used to analyze IFN-γ,IL-4 of nasal mucosa.The exosomes were extracted from rat peripheral blood plasma by ultracentrifugation,and the morphology of exosomes was detected by transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).qPCR and high-throughput sequencing were used to screen for significantly differentially expressed miRNAs in rat plasma exosomes based on the criteria of P<0.05 and|log2(Fold Change)|>1.The downstream target genes of differentially expressed miRNAs were obtained using miRanda and Targetscan,and GO and KEGG enrichment analyses were performed.Results:Compared with the model group,nasal mucosal pathological changes was reduced,nasal mucosal IL-4 positive cells and protein expression was decreased(P<0.05),and nasal mucosal IFN-γpositive cells and protein expression was increased(P<0.05)in rats of Yuchan Weifei Pills group.A total of 10 miRNA with differential expression were screened.The target gene pathway analysis included PI3K-Akt signaling pathway,TNF signaling pathway and MAPK signaling pathway.MAPK signaling pathway is closely associated with allergic rhinitis.Conclusion:YWP can effectively treat allergic rhinitis in rats,and the mechanism may be through regulation of miRNA signaling expression,activation of MAPK pathway,inhibition of Th2 cellular factors expression and regulation of Th1/Th2 balance.