Study On The Mechanism Of Nasturtium Against Influenza Virus H1N1 Based On TLRs Pathwa

Objective:To investigate the effects of crude extract,component fractions(flavonoids,phenolic acids and alkaloids)and representative individual compounds(vitexin,veratric acid and trolline)from the flowers of Trollius chinensis Bunge on the expressions of mRNA and proteins related to the vital genes(TLR3,TBK1,IRF3,TLR4,TAK1,TLR7,NF-κB,IFNβ)in TLR3,TLR4 and TLR7 signaling pathways in H1N1 infected macrophages,as well as the effects on the inflammatory factors such as NO,IL-6,IL-1,TNF-α and IFNβ,so as to ascertain the antiviral mechanism of the flowers:Methods:The RAW264.7 cells model,infected with H1N1,was used for studying the anti-influenza virus activity of the crude extract,the three types of component fractions(flavonoids,phenolic acids,alkaloids)and the three representative individual compounds(vitexin,veratric acid and trolline).The real-time PCR technique was applied to quantitatively determine the mRNA expressions of TLR3,TLR4,TLR7,IRF3,TAK1,TBK1,TRIF,IKKβ,NF-κB,IFNβ in RAW264.7 cells infected with H1N1.The Western blot technique was used to detect the protein expressions of TLR3,TLR4,TLR7,IRF3,TAK1,TBK1,TRIF,IKKα.And the ELISA technique was used to determine the secretion of NO,IL-1,IL-6,TNF-α and IFNβ.Result:The crude extract,the three types of component fractions,and the three representative individual compounds all showed obvious antiviral effects.The anti-viral activity of the flavonoids was the strongest compared to phenolic acids and alkaloids,and the anti-viral activity of the trolline was the strongest among the three representative individual compounds.H1N1 up-regulate the expression of key factors in TLR3 and TLR7 signaling pathways,down-regulate the expression of TLR4,up-regulate the mRNA expression of NF-κB and up-regulate the expression of NO,IL-6,TL-1,TNF-α and IFNp.The crude extract and three types of component fractions can up-regulate TLR3 and TLR4 pathways,up-regulate the expression of inflammatory cytokines IL-1 and IL-6,and down-regulate the expression of IFNβ and TNF-α,and the antiviral effect is consistent with the positive drug Shuanghuanglian injection.Some of the component fractions have dynamic regulation of inflammatory factors,such as flavonoids,and phenolic acids can dynamically regulate NO expression.The effects of the three representative individual components are similar to that of the positive drug ribavirin,which can up-regulate the expression of TLR4 protein,increase the expression of IFNβ,TNF-α and IL-1,and down-regulate the expression of IL-6 and NO.Some individual compounds have a dynamic regulation of inflammatory factors,such as veratric acid,which can dynamicly regulate IL-6 and NO expression.Conclusion:The crude extract,the three types of component fractions and three representative individual compounds all have antiviral activity.The above drugs exert antiviral action by interveneing the TLR3,4 and 7 signaling pathways to dynamicly and comprehensively regulate the expression of inflammatory factors.The crude extract and three types of component fractions exert their antivirus effect through regulating the expression of various cytokines and immunity.Their antiviral mechanism is the result of the multi-component and multi-pathway comprehensive action similar to that of a Chinese medicine compound Shuanghuanglian injection.The mechanism of the three representative individual compounds,which is relatively simpler and mainly up-regulate the expression of IFNβ,is similar to that of a western medicine ribavirin.