Study On The Activity And Mechanism Of Theaflavin-3’-Galate(TF2b) Against Influenza A (H1N1) Virus

Objective:Influenza A virus is highly mutable,spreads widely,and can easily cause seasonal epidemics,which can have a serious impact on social and economic development as well as public health security.Currently,antiviral drug research is an important aspect of influenza virus prevention and control.This study aims to analyze the anti-influenza virus activity of four theaflavins and their derivatives（TF1,TF2a,TF2b,and TF3）,as well as their potential antiviral mechanisms.Initially,the effectiveness and timeliness of theaflavins and their derivatives against influenza virus were analyzed in vitro.Subsequently,the therapeutic effect of these compounds was evaluated in influenza virus-infected animals（BALB/c mice）to explore the relationship between host immune response and inflammation,elucidate the potential targets and molecular signals of theaflavins and their derivatives in combating viruses,and provide directions and strategies for the development of new natural anti-influenza virus drugs.Methods:1.The safety and antiviral effectiveness of four different structures of theaflavins and their derivatives were assessed using MDCK and A549 cell lines as cell carriers through the MTT assay and cytopathic effect（CPE）assay.The Reed-Muench method was used to calculate their half maximal inhibitory concentration(IC₅₀)on cell growth,half maximal effective concentration(EC₅₀)on influenza A virus,and therapeutic index SI(CC₅₀/EC₅₀).Different treatment modes（pre-treatment,co-treatment,and post-treatment）were utilized to analyze the inhibitory effect of theaflavins and their derivatives on the life cycle of influenza virus.Following this,the expression of viral nucleoprotein（vNP）in the nucleus was detected by indirect immunofluorescence,and the number of virus-infected positive cells was analyzed.Finally,the anti-influenza virus potential of theaflavins and their derivatives was further explored through viral titer detection of different subtypes of influenza virus strains.2.Theaflavins and their derivatives with anti-influenza virus activity were evaluated in vitro based on an animal infection model of influenza A virus.The body weight changes and death of influenza virus-infected mice were monitored within 14 days to evaluate their protective effect against influenza virus-infected mice.In addition,on day 3（3 dpi）and day5（5 dpi）after infection,infected mice were randomly euthanized,and their lung tissue and blood samples were collected.Among them,lung tissue was used for lung index measurement,tissue viral titer detection,H&E staining,and immunohistochemical analysis,while blood samples were used for ELISA and blood biochemical tests.3.The mRNA expression relative levels of representative cytokines and chemokines in lung tissue homogenates were quantified using RT-qPCR.The transcriptome sequencing results were analyzed for KEGG enrichment used to explore the potential biological metabolic pathways of theaflavins and their derivatives against influenza virus.Finally,Western blot（WB）was used to detect the expression of proteins in its anti-influenza virus-related pathways to determine the regulation that theaflavins and their derivatives are likely to participate in influenza virus infection,as well as potential targets in the inflammatory response.Results:1.Among the four theaflavins and their derivatives,only TF1 and TF2b exhibited inhibitory effects against influenza virus H1N1-UI182 at the cellular level.Meanwhile,TF2a and TF3 showed no significant inhibitory effect against influenza virus.The former showed a higher inhibition rate in a dose-dependent manner（at a safe concentration）in the case of post-treatment,indicating that TF1 and TF2b mainly exert an inhibitory effect after the virus infiltrates the cell.Furthermore,using different subtypes of influenza A virus strains（H1N1-PR8,H3N2,and H5N1）to infect cells separately,TF1 and TF2b also showed strong inhibitory activity.By analyzing the expression rate of v NP positive cells（number of positive cells/total number of cells）,compared with the strong inhibitory effects of TF1 and TF2b,the antiviral effects of TF2a and TF3 were not evident.2.By analyzing the body weight changes and death of influenza virus-infected mice within 14 days,we found that TF1 could not effectively improve the weight loss trend of mice,and could not improve its survival rate.However,the administration of TF2b can inhibit the fatal pneumonia caused by influenza virus infection,reduce the viral load in the lungs of influenza-infected mice,alleviate the weight loss of infected mice,and reduce the mortality of infected mice.Histopathological analysis showed that after influenza virus infection,there were hemorrhage,massive and obvious cell debris,expansion of alveolar wall,and significant lymphocyte infiltration in the alveoli of mice.However,the administration of TF2b perfectly protected the pathological damage caused by the virus to the above-mentioned tissues.Compared with mice infected with the virus alone,the TF2b-treated mice showed increased lymphocyte（Lymph）counts,decreased neutrophil（Gran）counts,and monocyte（Mon）counts in the blood.In addition,TF2b reduced the total protein content of secreted inflammatory cytokines IL-6,IL-10,and IL-1βin the serum of virus model mice and significantly reduced the m RNA expression levels of cytokines（IL-6,TNF-α,and IL-1β）,interferon（IFN-αand IFN-γ）,and chemokines（CXCL-2 and CCL-3）.3.Transcriptome sequencing analysis revealed a significant enrichment of the Toll-like receptor signaling pathway following TF2b treatment of influenza virus infection.Through molecular detection and analysis,it was discovered that the cascade signaling pathway（MAPK/p38）mediated by Toll-like receptor 4（TLR4）played an important role in TF2b’s fight against influenza virus infection.Influenza virus infection leads to the upregulation of TLR4 expression,which promotes the phosphorylation of ERK and P38 proteins.However,TF2b’s treatment could significantly inhibit the activation of TLR4/MAPK/p38 signaling pathways and reduce the expression of cytokines（TNF-α,IL-6,IL-10,and IL-1β）induced by influenza virus infection.In summary,this study explored the anti-IAV aspects of theaflavins and their derivatives.This makes it possible for further development and utilization in the future and provides a feasible reference for the prevention and control of influenza virus epidemics.It also expands the scope of application of natural products in the field of anti-influenza viruses.