Study On The Anti-influenza Virus Effect And Immunologic Mechanism Of Yuye Detoxification Particle And Mussaenda Pubescens

Influenza A virus is the causative agent of the most common and highly infectious human disease.This cosmopolitan pathogen is responsible for a significant morbidity and mortality in humans as well as animals.Despite extensive clinical and pathological studies on influenza,our understanding of the mechanisms of disease development is still incomplete.The mammalian innate immune system recognizes the presence of invading pathogens by a family of receptors belonging to the Toll-like receptors(TLRs).Viral infection triggers an array of immune responses that effectively limit the replication and spread of viral infection.Recent studies have demonstrated that influenza viral infection up-regulates the expression of TLR7.In human immune cells,the expression of TLR7 in a given cell type is mostly to plasmacytoid dendritic cells(pDC)and monocytes.The plasmacytoid dendritic cells(pDC)and monocytes recognize ssRNA of virus mediated by MyD88 dependence pathway.The host cleared the virus by the innate immune response,and trigger the subsequent specific immuneresponse.The recognition of these viruses by plasmacytoid dendritic cells and monocytes through TLR7 results in their activation of costimulatory molecules and production of cytokines.If the pathway lost their normal control,the inflammatory hurt will be happened.The pathogenesis of influenza belongs to the theory"toxin hurts collaterals" of our Chinese medicine,and the toxin often result in the infectious lung hurt.The development of the influenza is process of the war between the inflammatory and anti-inflammatory.Yuye detoxification particle includes Mussaenda pubescens,honeysuckle screwtree root,flos chrysanthemi indici,chrysanthemum,Centella asiatic,holly root,which can clear the heat and relieve the toxic substance.Mussaenda pubescens is the monarch drug of Yuye detoxification particle,which has the effect of removing dampness and Detoxification.Previous results showed that,the drug has good inhibiting effect against a variety of respiratory viruses in vitro,such as rhinovirus,adenovirus,respiratory syncytial virus,at the same time,has the effect of anti influenza virus in vivo significantly.In this work,we investigated the antiviral ways and effect of the drug and the possible recognization mechenism that TLR7 recognize ssRNA;We try to discover the antiviral molecular target of the Yuye detoxification particle and Mussaenda pubescens combine with the Chinese medical theory.ObjectivesTo study the anti-influenza A virus(H1N1)activity of the Yuye detoxification particle and Mussaenda pubescens both in vitro and in vivo,and reveal the molecular mechanism that Mussaenda pubescens he drug inhibits the influenza virus via the MyD88-dependent pathway mediated the Toll-like receptor(TLR)7 and finding the antiviral molecular targets of the Chinese medicine.Methods1.The median toxic concentration(TC50)of Yuye detoxification particle and its herbal composition on MDCK cells were determined by MTT assay.Anti-influenza virus activity of Yuye detoxification particle and its herbal composition determined by hemagglutination test and cytopathic effect assay.2.Anti-influenza virus activity in chick embryos:the embryos were treated with different dosage of drugs,which were injected into the allantoic cavity in preventive and therapeutic administration way.Allantoic fluid was collected after incubating for 48h,and the allantoic fluid HA on the chick embryo level was used as a measurement indicator.3.Anti-influenza virus activity in mice:the mice which be infected with influenza virus were treated respectively with drugs in therapeutic administration way,and the death protection rate,lung index and pathomorphological change were adapted to evaluate the antiviral activity in mice.4.To survey the influence of the drug on the serum level of IL-1β、TNF-α、IL-6 in the mice’s lung tissue by the method of-ELISA test.5.To survey the influence of the drug on the level of TLR7、MyD88、NF-κB、AP-1mRNA by the method of real-time PCR assay and the protein expression of TLR7、MyD88、NF-κB、AP-1 by the method of western blot assay.Results1.The TC50 of Yuye detoxification particle and its herbal composition on MDCK cells:Yuye detoxification particle>5 mg/ml、Mussaenda pubescens>5mg/ml、honeysuckle>10mg/ml、screwtree root<1.25mg/ml、flos chrysanthemi indici>10mg/ml、chrysanthemum>5mg/ml、Centella asiatica>20mg/ml、holly root>20mg/ml。Detoxification particle,Honeysuckle,Mussaenda pubescens,holly root showed an obvious inhibitive effect against adsorption of the virus and inhibition on its prolongation inside MDCK,and the effective concentrations were 5mg/ml,5mg/ml,5mg/ml,20mg/ml respectively.2.In the therapeutic administration way,the high,medium and low dose group of Yuye detoxification particle and Mussaenda pubescens had good inhibitory effect by the allantoic cavity injection on A/FM1/47(H1N1)strains(P<0.01),High dose group of Yuye detoxification particle and Mussaenda pubescens,medium dose group of Mussaenda pubescens had significant inhibitory effect on H3N2 virus(P<0.05);in the preventive administration way,all group of Yuye detoxification particle and Mussaenda pubescens had obivious inhibitory effect on H1N1 strains(P<0.01)while high dose group of Yuye detoxification particle and Mussaenda pubescens had significant inhibitory effect on H3N2 virus(P<0.05).3.All group of Yuye detoxification particle and high,medium dose group of Mussaenda pubescens has a significant effect on increasing death protection rate in A/FM1/47(H1N1)strain infected mice(P<0.01).In addition,the high,medium and low dose group of Yuye detoxification particle and Mussaenda pubescens has a significant effect on decreasing lung index in therapeutic administration compared to controlled groub(P<0.01).Both drug improved the inflammatory lesions in the lung tissue of the infected mice.4.The high,medium and low dose group of Mussaenda pubescens can down-regulate the amount of IL-1β、TNF-α、IL-6,and shows dose dependent relationship significantly.5.The high,medium and low dose group of Mussaenda pubescens can down-regulate the expression of mRNA of the signal proteins such as TLR7、MyD88 and NF-κB in the signal transduction pathway mediated by the TLR7.The down-regulation is significantly and shows dose dependent relationship.The high and medium dose group of Mussaenda pubescens can down-regulate the expression of the TLR7 protein while all groub of that can down-regulate the expression of the TLR7 protein MyD88 and NF-κB detected by western blot.ConclusionYuye detoxification particle and Mussaenda pubescens exerted anti-virus effects in the therapy of influenza virus infection,and Mussaenda pubescens inhibited the transcripion and expression of IL-1β、TNF-α、IL-6 by repressing the TLR7 mediated MyD88-dependent signaling pathway and the nuclear expression of NF-κB.