| Background:Seasonal influenza is an acute respiratory infection caused by influenza virus infection.The 1918 Spanish influenza pandemic,the 1957 Asian influenza pandemic,the 1968 Hong Kong influenza pandemic and the 2009 Mexican influenza pandemic have caused serious damage to the social economy and public health.The recent increase activity of IAV in many places in China was caused H1N1 subtype of IAV,posing a major threat to public health.Due to the extremely high variability of influenza viruses,vaccination efficacy is restricted.Anti-influenza medications also face limitations and challenges.Almost all influenza viruses circulating in humans are resistant to alkylamine drugs.The number of commonly used anti-influenza drugs such as neuraminidase inhibitors in clinical are limited.With the widespread use of antiinfluenza drugs in clinical,drug-resistant virus strains continue to emerge,which limits the activity of these existing antiviral drugs.In recent years,a batch of anti-influenza drugs targeting new targets have emerged,such as Xofluza and Favipiravir,but the long-term effect remains to be seen.There is an urgent need to develop more effective antiviral medicines with diverse anti-virus mechanisms since the available antiinfluenza medications still can not fulfill clinical demands and lack variety of antiviral targets.Nanomaterials have unique properties such as water solubility,biocompatibility,low synthesis cost,and low toxicity,which have been widely used in bioimaging,biosensing,biomarkers,photodynamic therapy,and drug delivery.In recent years,more and more researchers have started to discover antiviral drugs from nanomaterials.However,the particle size of some nanomaterials is much smaller than that of the viruses,so the contact area with the viruses is limited,which limits their antiviral activity to some extent.The two-dimensional metal-organic skeleton nanomaterials have the characteristics of large size and high specific surface area,which precisely compensate for the deficiency.The large contact area and the relatively soft,foldable nanosheet structure can greatly improve their interaction with the viruses and thus exhibit better antiviral activity.Copper(Ⅱ)tetracarboxyphenyl porphyrin(Cu-TCPP)is a two-dimensional metal-organic skeleton nanomaterial formed by the coordination of metal ions or clusters with polyhedral organic ligands.Cu-TCPP has the advantages of flexible and adjustable two-dimensional structure,large specific surface area,and porosity,which have been extensively studied in antibacterial and antitumor applications.A highly compatible two-dimensional nanomaterial Cu-TCPP@Mn3O4 was synthesized by the collaborative group.Through the electrostatic adsorption effect,the virus was adsorbed on the surface of two-dimensional MOF,which could effectively reduce the titer of viruses.More importantly,Mn3O4 nanoparticles could effectively clear ROS and down-regulating inflammatory factors to improve the host environment,which may be an attractive therapeutic option in the field of antiviral therapy and is expected to develop into a new generation of antiviral drugs in the future.Objectives:We intend to evaluate the efficacy of Cu-TCPP@Mn3O4 against IAV in vitro and in vivo,analyze the target of Cu-TCPP@Mn3O4 against IAV,and clarify explore the mechanism of Cu-.This research aimed to provide a new therapeutic method for the prevention and treatment of influenza virus through multi-mode synchronous treatment including antiviral,anti-inflammatory and antioxidant.Methods:1.The cytotoxicity of Cu-TCPP@Mn3O4 to the target cells and inhibition effect of CuTCPP@Mn3O4 against differe subtypes of IAV were assessed using the MTT assay;2.The anti-virus activity of Cu-TCPP@Mn3O4 was detected by plaque reduction assay,indirect immunofluorescence assay,western blotting and qRT-PCR;3.Detection the uptake of Cu-TCPP@Mn3O4 by cells using fluorometric method;4.H5N1 pseudovirus neutralization assay was used to detect the effect of CuTCPP@Mn3O4 on virus entry;5.The effect of Cu-TCPP@Mn3O4 on the structure of influenza virus was observed by transmission electron microscopy;6.Time-of-addition,hemagglutination inhibition assay,hemolysis assay,syncyticum inhibition assay,SPR assay,mini-replicon experiment and NA inhibition assay were used to explore the mechanism of Actionof Cu-TCPP@Mn3O4;7.Western blotting and qRT-PCR were used to study the effect of Cu-TCPP@Mn3O4 on IAV or Poly(I:C)-induced inflammatory injury and mechanism of antiinflammation;8.The effect of Cu-TCPP@Mn3O4 on the expression of influenza A virus or LPS induced ROS was detected by immunofluorescence;9.Mice were infected with A/PR/8/34(H1N1)to evaluate the protective effect of CuTCPP@Mn3O4 against influenza virus pneumonia in mice.Results:1.The CC50 of Cu-TCPP@Mn3O4 on MDCK,A549 and Beas-2B cells were more than 50 u2g/mL;2.Cu-TCPP@Mn3O4 effectively inhibited the different subtypes of IAV in vitro,with IC50 of 0.79～1.47 μg/mL;3.A549 cells showed good uptake ability of Cu-TCPP@Mn3O4.4.Cu-TCPP@Mn3O4 could damage the structure of influenza virus;5.Cu-TCPP@Mn3O4 inhibited viral fusion with target cells,but could not affect the IAV RNA polymerase and neuraminidase activities;6.SPR result showed that Cu-TCPP@Mn3O4 could specifically bind to HA2 subunit;7.Cu-TCPP@Mn3O4 had good oxygen production function and could significantly reduce ROS produced by IAV or LPS;8.Cu-TCPP@Mm3O4 could significantly reduce the expression of inflammatory cytokines and chemokines induced by IAV or Poly(I:C);9.Cu-TCPP@Mn3O4 could regulate NF-κB and JAK/STAT signaling pathways;10.Cu-TCPP@Mn3O4 could effectively reduce the viral load in the lung of mice,reduce cytokine levels,reduce lung index in mice with pneumonia,alleviate the lung damage induced by influenza virus,and prolong the survival time of mice with pneumonia.Conclusion:1.Cu-TCPP@Mn3O4 had good anti-IAV activity in vitro with low cytotoxicity;2.Cu-TCPP@Mn3O4 had direct antiviral effects and inhibited influenza virus membrane fusion by acting on IAV hemagglutinin HA2 subunit,thus blocking the entry of IAV;3.Cu-TCPP@Mn3O4 had antiviral,anti-inflammatory and antioxidant effects;4.Cu-TCPP@Mn3O4 had certain anti-IAV effects in vivo;... |
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