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目的:观察荆芥挥发油及其主要成分胡薄荷酮与薄荷酮体外对甲型流感病毒(H1N1)增殖的影响,并探讨其TLR/IFN信号通路机制。方法:MTT法检测受试药对狗肾传代细胞(MDCK)的半数中毒浓度(TC50)及最大无毒浓度(TC0);血凝法测定甲型流感病毒鼠肺适应株A/PR/8/34(H1N1)对MDCK细胞的感染性;MTT法和细胞病变法(CPE)测定三种药物体外抑制流感病毒增殖的有效浓度(IC50)和治疗指数(TI)。Real-Time PCR法观察药物对H1N1感染MDCK细胞后8h细胞TLR7、IFN-βmRNA表达水平的影响。结果:荆芥挥发油、胡薄荷酮和薄荷酮对MDCK细胞的TC50分别为0.20mg/ml、0.36mg/ml与0.43mg/ml,TC0分别为0.10mg/ml、0.10mg/ml与0.25mg/ml。荆芥挥发油和胡薄荷酮3.1×10-3～0.1mg/ml浓度时能抑制流感病毒增殖,其IC50分别为1.7×10-3mg/ml与1.9×10-3mg/ml,TI分别为120.83、184.54,呈一定量效关系;薄荷酮对流感病毒染毒的MDCK细胞几乎无保护作用。荆芥挥发油3.1×10-3～0.10mg/ml浓度,胡薄荷酮1.3×10-2～0.10mg/ml及薄荷酮7.8×10-3～0.10mg/ml浓度时对流感病毒具有明显直接杀灭作用,其IC50分别为3.1×10-3、7.2×10-3与1.9×10-3mg/ml,TI分别为63.99、50.00和82.88。荆芥挥发油和胡薄荷酮0.1mg/ml浓度时均显著提高TLR7、IFN-βmRNA表达水平,0.25mg/ml薄荷酮显著提高TLR7mRNA表达水平。结论:荆芥挥发油与胡薄荷酮体外明显抑制甲型流感病毒(H1N1)在MDCK细胞中的增殖,薄荷酮作用不明显;三者均对流感病毒有显著直接杀灭作用。其抗病毒作用机制可能与其激活TLR7,诱导IFN-β高表达,激活病毒模式识别系统有关。
Objective: To observe the effects of essential oil of Nepeta and its major components, menthone and menthone, on the proliferation of influenza A virus (H1N1) in vitro and to explore its TLR / IFN signaling pathway. Methods: MTT assay was used to detect the median lethal concentration (TC50) and the maximum nontoxic concentration (TC0) of the test drug on the MDCK cells. The hematocrit assay was used to determine the A / PR / 8 / 34 (H1N1) in MDCK cells. MTT assay and cytopathic assay (CPE) were used to determine the effective concentration (IC50) and therapeutic index (TI) of the three drugs to inhibit influenza virus proliferation in vitro. Real-Time PCR method was used to observe the effects of drugs on the expression of TLR7 and IFN-βmRNA in MDCK cells infected with H1N1 at 8h. Results: The TC50 of volatile oil, menthone and menthone for MDCK cells were 0.20mg / ml, 0.36mg / ml and 0.43mg / ml respectively, TC0 were 0.10mg / ml, 0.10mg / ml and 0.25mg / ml. Nepeta volatile oil and pulegone 3.1 × 10-3 ~ 0.1mg / ml concentration can inhibit the proliferation of influenza virus, the IC50 were 1.7 × 10-3mg / ml and 1.9 × 10-3mg / ml, TI were 120.83, 184.54, showing a certain dose-effect relationship; Menthone has almost no protective effect on influenza virus MDCK cells. Nepeta volatile oil 3.1 × 10-3 ~ 0.10mg / ml concentration, the concentration of 1.3 ~ 10-2 ~ 0.10mg / ml of menthone and 7.8 × 10-3 ~ 0.10mg / ml of menthone have obvious immediate kill With an IC50 of 3.1 × 10-3, 7.2 × 10-3 and 1.9 × 10-3 mg / ml respectively, with TI values of 63.99, 50.00 and 82.88, respectively. Nepeta volatile oil and pulegone 0.1mg / ml concentration significantly increased TLR7, IFN-β mRNA expression levels, 0.25mg / ml menthone significantly increased TLR7mRNA expression levels. CONCLUSION: Volatile oil from Schizonepeta tenuifolia and piperonyl alcohol significantly inhibit the proliferation of influenza virus type 1 (H1N1) in MDCK cells in vitro with no obvious effect on the content of menthone. All three have obvious direct killing effects on influenza virus. Its antiviral mechanism may be related to its activation TLR7, induced IFN-β high expression, activation of the virus pattern recognition system.