Preliminary Study On The Effect Of Wogonin Against Porcine Epidemic Diarrhea Virus Infection In Vitro

Porcine epidemic diarrhea(PED)is an acute highly contact gastrointestinal infection caused by Porcine epidemic diarrhea virus(PEDV).In recent years,the widespread prevalence and high pathogenicity of PEDV have caused huge economic losses to the pig industry in China.Currently,relevant studies have reported that baicalin(Wogonin)exhibits better antiviral activity against coronavirus main protein(Mpro,also known as3 CL protease).Studies have shown that Mpro plays a crucial role in PEDV replication and has become an important target for anti-PEDV drug design.In this experiment,we first determined the anti-PEDV activity of baicalin and further resolved its specific molecular mechanism body.The experimental methods and results are as follows:1.Screening of safe concentration of wogonin: In this experiment,Vero and IPEC-J2 cells were treated with different concentrations of wogonin(6.25～400 μM),and cell samples were collected after 48 h.The effect of wogonin on cell viability was analyzed by detecting CCK-8 content.The results showed that the safe concentrations of wogonin for Vero and IPEC-J2 cells were 6.25～100 μM,and their CC50 values were 475.3 μM and499.4 μM,respectively.2.Study of anti-PEDV activity of wogonin: Vero and IPEC-J2 cells infected with PEDV were treated with wogonin at concentrations ranging from 12.5 to 100 μM,and the antiPEDV activity of wogonin was analyzed.The results of immunofluorescence,RT-PCR and viral load showed that wogonin at concentrations of 25～100 μM all significantly inhibited PEDV infection(P < 0.01)with IC50 values of 54.87 μM and 114.55 μM,respectively.further testing the effect of wogonin on the replication cycle of PEDV showed that wogonin could significantly inhibit the replication of PEDV(P < The results showed that wogonin could significantly inhibit PEDV replication(P < 0.05),highly significantly inhibit PEDV internalization and release(P < 0.001),and directly inactivate PEDV in vitro(P < 0.01).3.Molecular mechanism study of anti-PEDV activity of wogonin: Homology modeling and molecular docking techniques revealed that wogonin can stably embed into the groove of the Mpro protein activity pocket and thus inhibit Mpro binding substrate.Firstly,the stability and moderate affinity between wogonin and PEDV-Mpro were verified by in vitro binding assays of expressed purified PEDV-Mpro protein and wogonin,including differential scanning fluorescence(DSF),microscale thermophoresis(MST)and surface plasmon resonance technique(SPR);secondly,fluorescence resonance energy transfer analysis(FRET)was used to verify that the expression of The enzymatic activity of the purified PEDV-Mpro protein was verified by FRET and the ability of wogonin to inhibit PEDV-Mpro enzymatic activity was confirmed.The results showed that wogonin exhibited antiviral activity against PEDV,significantly inhibited PEDV internalization,replication and release,and directly inactivated PEDV in vitro,and mediated PEDV infection by targeting the PEDV-Mpro protein substrate binding site to inhibit Mpro enzyme activity.This study provides a new direction for understanding the antiviral activity of wogonin and the research of anti-PEDV drugs.