【摘要】 目的 基于转录组学研究金银花醇提物在流感病毒感染巨噬细胞中的抗病毒、抗炎作用及其分子机制。方法 以PR8(100 TCID₅₀)毒株感染J774A. 1巨噬细胞，设对照组、模型组和金银花组（6.25μg/ml）。免疫荧光、Western blot法及RTq PCR检测病毒核蛋白（nucleoprotein,NP）水平; RT-q PCR及ELISA测定促炎性细胞因子IL-6、TNF-α和IL-1β水平; RNA高通量测序筛选差异基因并进行GO、KEGG富集; RT-q PCR验证通路关键基因表达;免疫荧光检测NF-κB p65表达及核转位。结果与模型组相比，金银花醇提物显著下调病毒NP的mRNA和蛋白表达（P<0.001）; IL-6、TNF-α mRNA水平及血清IL-6、TNF-α、IL-1β水平显著下降（P<0.01或P<0.001）。转录组学分析共鉴定出262个交集差异基因，显著富集于TLR、RLR、NLR及胞质DNA感应等模式识别受体（pattern recognition receptor,PRR）通路; RT-q PCR证实TLR3、RIG-Ⅰ、NLRC5、IFI202等关键基因表达被显著抑制（P<0.01或P<0.001）。免疫荧光证实金银花醇提物抑制NF-κB p65的表达（P<0.001）及核转位（P<0.01）。结论 金银花醇提物通过抑制PRR-NF-κB通路过度激活及其下游级联效应，同时阻断病毒复制与炎症风暴，为流感重症的双重干预作用提供新策略。更多还原

【Abstract】 Objective To study the antiviral and anti-inflammatory effects of Lonicera japonica Thunb ethanol extract in influenza A virus-infected macrophages and its mechanisms base on transcriptomics.Methods J774A.1macrophages were infected with PR8strain(100 TCID₅₀) and divided into control,model,and Lonicera japonica Thunb（6.25μg/ml） groups.Viral nucleoprotein（NP）levels were detected by immunofluorescence,Western blot,and RT-q PCR.The levels of pro-inflammatory cytokines IL-6,TNF-αand IL-1β were determined by RT-q PCR and ELISA.RNA high-throughput sequencing was used to screen differentially expressed genes,followed by GO and KEGG enrichment analyses.The expression of key pathway genes were verified by RT-q PCR.The expression and nuclear translocation of NF-κB p65 were visualized by immunofluorescence.Results Compared with the model group,Lonicera japonica Thunb ethanol extract significantly downregulated the mRNA and protein expression of viral NP（P<0.001）; the mRNA levels of IL-6 and TNF-α and the serum levels of IL-6,TNF-α and IL-1β were significantly decreased（P<0.01 or P<0.001）.A total of 262 intersection differential genes were identified by transcriptomics analysis,which were significantly enriched in pattern recognition receptor（PRR） pathways such as TLR,RLR,NLR and cytoplasmic DNA sensing; RT-q PCR confirmed that the expressions of key genes such as TLR3,RIG-Ⅰ,NLRC5 and IFI202 were significantly inhibited（P<0.01 or P<0.001）.Immunofluorescence assay verified that Lonicera japonica Thunb ethanol extract inhibited the expression of NF-κB p65（P<0.001） and its nuclear translocation（P<0.01）.Conclusion Lonicera japonica Thunb ethanol extract exerts a dual intervention effect on influenza severe cases by inhibiting the overactivation of the PRR-NF-κB pathway and its downstream cascade effects,and blocking viral replication and inflammatory storm,providing a new strategy for the treatment of severe influenza.更多还原