| Influenza virus is a respiratory pathogen which causes widespread infections globally.It is very easy to infect and spread between humans and animals,causing influenza pandemics.Influenza A virus is prone to antigenic variation,which makes the virus often appear unpredictable new subtypes.The emergence of new viral variants continues to pose a challenge to public health and serious damage to the development of livestock and poultry breeding,and causes huge economic loss around the world.At present,vaccines and drugs are the main means to effectively prevent and treat influenza.However,due to the continuous emergence of new influenza viruses,influenza vaccines have problems such as short validity period,low potency,low protection rate,and no protective effect on new subtypes,and the commonly used anti-influenza drugs in clinical practice are also constantly showing high toxicity and side effects and are prone to drug resistance.The disadvantages such as sexiness limit the prevention and treatment effect of anti-influenza drugs.Therefore,it is imminent to develop new anti-influenza agents.In recent years,some endogenous small molecules from the host have been found to be able to effectively inhibit influenza virus infection.They participate in the replication or pathogenesis of the virus in the host,and play an active therapeutic role in the process of IAV infection without obvious side effects.It has extensive application prospects in the development of new anti-influenza virus preparations.Sodium taurocholate is the sodium salt of the primary bile acid taurocholic acid that mainly exists in the body.It is synthesized by the liver and widely exists in the bile of cattle,sheep,snake and other animals.It is used as the main component of bile drugs.Studies have shown that sodium taurocholate has pharmacological effects.However,there are few reports about the anti-influenza virus effect of sodium taurocholate.This study reveals the potential anti-influenza virus activity of Sodium taurocholate hydrate in vitro and in vivo respectively,and explores its mechanism of action,which provides a theoretical basis for the development of new anti-influenza agents.The main findings of the present research are as follows:1.The evaluation of anti-influenza virus activity of sodium taurocholate hydrate at the cellular level.The inhibitory effect of different concentrations of sodium taurocholate hydrate on the infection of A549 cells by recombinant H5N6 virus（JX-GFP）carrying GFP fluorescent protein was detected by fluorescence microscope observation,western blotting,flow cytometry analysis,and TCID50.The results showed that sodium taurocholate hydrate could significantly reduce the fluorescence density of JX-GFP infected A549 cells,inhibit the expression of NP protein,and significantly inhibit the content of virus particles released into the cell supernatant in a dose-dependent manner.In addition,sodium taurocholate hydrate can significantly inhibit the proliferation of H5N6（JX）,H1N1（PR8）,H3N2（HB）,and H9N2 strains on A549,MDCK and Caco-2 cells,and has broad Spectrum anti-influenza effect.2.The anti-PR8 mechanism of sodium taurocholate hydrate at the cellular level.The effect of sodium taurocholate hydrate incubation after virus infection was preliminarily explored through the time course inhibition test,and the time-of-addition analysis test found that the incubation inhibition effect of it was most obvious at 0-4 hours after virus infection.The adsorption,endocytosis and nuclear import experiments found that it had no effect on the above processes;neuraminidase activity test found that it did not affect the release of the virus either.RT-PCR was used to explore its effect on the production of influenza virus RNA,and it was found that it could obviously inhibit the v RNA,m RNA and c RNA expression of influenza virus NP protein.In addition,it did not affect the activity of IFN-βafter virus-infected cells by dual-luciferase reporter and RT-PCR experiments,but significantly inhibited the inflammatory response caused by viral infection.3.The evaluation of anti-influenza virus activity of sodium taurocholate hydrate at the cellular level.The effect of sodium taurocholate hydrate on the survival rate and weight change rate of influenza-infected mice was detected,and it was found that sodium taurocholate hydrate could significantly increase the survival rate of influenza-infected mice,reduce the weight loss of mice,and more regain weight early.In addition,sodium taurocholate hydrate could significantly reduce post-infection lung index,lung virus titer,and NP m RNA expression in trachea and lung tissue,and improve pathological changes in lung tissue.4.The study on the inflammatory inhibitory effect of sodium taurocholate hydrate on influenza infected mice.In order to detect the effect of sodium taurocholate hydrate on the inflammatory response of infected mice,the expression of some inflammatory factors in lung tissue and peripheral blood were respectively detected by RT-q PCR and ELISA.It was found that TNF-α,IL-1βand IL-6 expression in mice lung tissue on the 3rd day after infection with sodium taurocholate hydrate was significantly reduced.These results indicate that the antiviral activity of sodium taurocholate in vivo depends on its anti-inflammatory mechanism.5.The effect of sodium taurocholate hydrate on TLR4/NF-κB signaling pathway after influenza infection.In each group,TLR4,NF-κB,and IκBαexpression in mice lung tissue was determined by RT-q PCR.Our results showed that sodium taurocholate hydrate treatment decreases the TLR4 and NF-κB and increases the IκBαm RNA expression.The expression levels of TLR4/NF-κB pathway-related proteins in mice lung tissue and A549 cells infected with influenza virus by western blot assay.The results showed that it could inhibit TLR4,NF-κB,p-NF-κB,and p-IκBαprotein expression in vivo and in vitro.It shows that sodium taurocholate hydrate plays the role of anti-influenza virus activity by inhibiting the activation of TLR4/NF-κB signal pathway to resist the inflammatory damage caused by influenza infection. |
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