Study On Anti-influenza A Virus Effect Of Cepharanthine In Vitro And Vivo

Background:Influenza A virus(IAV)is a common influenza virus that can cause severe acute respiratory disease.It mainly attacks alveolar epithelial cells and respiratory epithelial cells,causing alveolar epithelial damage and hindering gas exchange,which leads to pneumonia and development of ARDS.Influenza A virus can easily cause seasonal influenza or trigger a global pandemic.Due to wide spread of influenza A virus,the rapid mutation rate of antigens,and the limited speed of vaccine development,antiinfluenza drug is still one of the important measures and treatment to prevent influenza virus infection.Anti-influenza virus drugs that mainly inhibit viral neuraminidase have been widely used clinically,but the emergence of corresponding drug-resistant strains brings great difficulties to the prevention and treatment of influenza.Therefore,there is an urgent need for breakthroughs in the mechanism of action of new antiviral drugs.Among them,the research and development of antiviral drugs focus on the polymerase of virus replication and/or host immunity has become the current development direction in the field of innovative drug research and development.In response to influenza virus polymerase,the successful launch of a new fast-acting anti-influenza drug baloxavir(Xofluza)has broadened the traditional concept of development of antiviral drugs.The research on antiviral drugs that regulate host immunity has become a new direction of research and development of antiviral drugs.If the two strategies are combined,it may constitute a new opportunity for the development of new antiviral drugs.In recent years,the research and development of natural products has attracted much attention,which has the important characteristics of low toxicity and multiple targets.Cepharanthine(CEP),a bisbenzylisoquinoline alkaloid isolated from the plants of the tetragoniaceae,has anti-tumor,anti-malarial,antibacterial,and immune function regulation effects etc.The Chinese patent medicine Qianjintiengsu tablet has been used clinically for decades,and it has been marketed both abroad and domestically.Recent studies have shown that CEP has an inhibitory effect on a variety of coronaviruses,including SARS-CoV2 and OC43.These results suggest that CEP is likely to have broad-spectrum antiviral effects.However,the application of CEP in anti-influenza A virus has not been reported yet.Objectives:O ur study intends to investigate anti-influenza A virus activity of CEP in vitro and vivo,reveal the anti-influenza A virus mechanism of CEP from both the virus and host.Our results can be helpful to explore the target of CEP against influenza A virus and clarify the anti-inflammation mechanisms of CEP.Our study provides a reference for exploring new uses of old drugs,provides scientific basis for the development of CEP as a potential drug against influenza A virus infection,and provides potential new solutions for clinical treatment.Methods:1.MTT method was used to determine toxicity of CEP to the target cells,and cytopathic method and plaque reduction method were used to evaluate the antiviral activity of CEP against multiple of influenza A viruse strains;2.Indirect immunofluorescence,Western blotting and qRT-PCR were performed to detect the inhibitory effect of CEP on influenza A virus replication;3.MTT method was employed to measure the combined effect of CEP and neuraminidase inhibitor zanamivir;4.Mini-replicon experiment was used to measure the activity of influenza virus polymerase;5.Western blotting and qRT-PCR were used to investigate the anti-inflammatory effect of CEP on IAV-induced inflammatory response and its mechanism;6.STAT1 or STAT2 knockdown experiment was employed to test the effect of CEP on JAK/STAT signaling pathway;7.Western blotting and immunoconfocal experiments were performed to investigate the effect of CEP on NF-κB signaling pathway;8.The antivrial activity of CEP in vivo was evaluated by mouse model of influenza A virus infection.Results:1.CEP displayed an obvious inhibitory effect against the different subtypes of influenza A virus,including A/WSN/1933(H1N1),A/Aichi/2/68(H3N2),A/Puerto Rico/8/34(H1N1),A/FM-1/1/47(H1N1).IC50 were 2.34±0.86,5.50±2.22,1.08±0.72 and 3.51±0.47μM,respectively;2.The CEP played an important role in the early replication stage of influenza A virus;3.CEP significantly reduced the activity of influenza virus polymerase in a concentration-dependent manner;4.The combination index of CEP and NA inhibitor zanamivir is less than 0.1,suggested the combination CEP and zanamivir exerted synergistic anti-influenza effects in vitro;5.CEP could inhibit the expression of related proteins of JAK/STAT and NF-κB signaling pathway including p-JAK1,p-STAT1,p-STAT2 and p-NF-κB p65,and markedly reduce the overexpression of related cytokines;6.CEP could alleviate influenza virus-induced lung injury,reduce viral load and improve the survival rate of H1N1-infected mice in vivo.Conclusion:1.CEP inhibited the infection of different subtypes of influenza A virus with low cytotoxicity in vitro;2.CEP disrupted the early replication of influenza A virus;3.CEP exhibited a direct antiviral effect by blocking the polymerase activity of influenza A virus;4.The combination of CEP and zanamivir has a synergistic anti-influenza virus effect;5.The anti-inflammatory effect of CEP might be related to JAK/STAT and NF-κB signaling pathways;6.CEP exerted certain anti-influenza A virus effect in vivo.