Antiviral Mechanism Research Of Magnolol And Honokiol Against Grass Carp Reovirus

Grass carp is the largest farming freshwater fish in China in recent years.With the high density of the breeding industry,the pathogenic microorganisms cause frequent outbreaks of diseases,which bring serious economic losses to the breeding industry.The hemorrhagic disease caused by Grass Carp Reovirus(GCRV)is the most serious viral disease in grass carp.The disease is susceptible to infection and has high mortality,which greatly affects and endangers the health and survival of grass carp.At present,pesticides and veterinary drugs are mainly used to control the occurrence of diseases in the aquaculture industry.However,effective and broad-spectrum drugs are still scarce,of which the available antiviral drugs are limited,and the effectiveness of the drug is highly susceptible to be affected by viral variation and drug resistance.Chinese herbal medicines have the advantages of low toxicity,easy degradation,rich resources and long usage history,which is an ideal safe and harmless aquatic drug.In this study,medicinal plants with anti-GCRV activity were screened,and then separated and analyzed to find the main active compounds to obtain the monomer compound with effective anti-GCRV activity.The anti-GCRV activity of the monomer compounds was evaluated by infection experiments in vitro and in vivo.Moreover,the mechanism of anti-apoptosis by monomer compounds and the interaction between monomer compounds and host cells were explored,aiming to provide the theoretical basis for the development of antiviral drugs in aquaculture.The results obtained in this work were as follows: 1.The screening of Chinese medicinal herb and monomeric compound against GCRVThirty kinds of medicinal plants were selected to prepare plant extracts,and the toxicity of extracts to CIK cells was determined by MTT.And then plant extracts with the maximum safety concentration were used to screen the activity of anti-GCRV.Reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)screening results showed that Trichosanthes Kirilowii,Magnolia officinalis and Citrus medica showed inhibitory effect on GCRV replication,and the corresponding concentration were 90 mg/L,34 mg/L and 240 mg/L,respectively.Among those,Trichosanthes Kirilowii inhibited the GCRV structural protein VP3(0.53-fold,P < 0.05),and Citrus medica inhibited GCRV VP5 mRNA expression(0.36-fold,P < 0.05).Magnolia officinalis exhibited good antiviral activity,significantly reducing GCRV VP1(0.48-fold,P < 0.05),GCRV VP3(0.52-fold,P < 0.05)and GCRV VP5(0.38-fold,P < 0.05)mRNA expression levels.Ribavirin is a recognized potent broad-spectrum antiviral drugs,and has good inhibition effect on GCRV replication,significantly inhibiting the GCRV VP1(0.20-fold,P < 0.05),GCRV VP3(0.26-fold,P < 0.05)and GCRV VP5(0.39-fold,P < 0.05).Therefore,ribavirin is used as positive control drug in this study.Further,the two main active components of Magnolia officinalis were separated and analyzed to be magnolol and honokiol.In the anti-GCRV activity screening of monomer compound,magnolol and honokiol had better effect on virus expression.The safe concentration of magnolol on CIK cells was 2.51±0.51 mg/L,and honokiol was 3.18±0.61 mg/L.Under the maximum safety concentration,the inhibition rate of honokiol was over 70%(P < 0.05),while the inhibitory rate of magnolol was over 60%(P < 0.05),and both the two compounds showed the inhibitory activity to multiple fragments of the virus.2.Study on the anti-GCRV activity of the magnolol and honokiolThe metabolisms of magnolol and honokiol in CIK cells were detected by high performance liquid chromatography(HPLC).The content increased along with time,reached a peak at 48 h,followed by a decrease and almost completely metabolized at 96 h.In the three groups,including compounds administrated to the cells before GCRV challenge,compounds administrated to the cells after GCRV infection and incubation of GCRV and compounds,RT-qPCR and Western blot assays showed that magnolol and honokiol inhibited GCRV expression,playing roles not in the process of virus adsorption and invasion of cells,but in the process of virus replication.Therewith,GCRV-infected grass carp summer flowers were treated with magnolol and honokiol by injection manner,which revealed that magnolol and honokiol reduced viral expression and mortality in vivo,and improved the body's antioxidant capacity.Thus,magnolol and honokiol can be used as potentially alternative drugs against GCRV infection.3.Study on the mechanism of anti-apoptosis of magnolol and honokiolGCRV infection induced CIK cells to show typical characteristics of apoptosis by scanning electron microscopy and fluorescence staining,including cell swelling,the formation of apoptotic bodies,cell morphology disappeared,nuclear fragmentation and cytoplasmic degradation.Flow cytometry analysis showed that 21.54% cells appeared apoptosis at 48 h of the virus infection,and the increased S and G2/M phase cells leading the cell cycle arrest phenomenon.However,magnolol and honokiol made infected cells maintain normal cell morphology,and apoptosis rate were respectively decreased by 11.47% and 13.13% and S and G2/M arrest was released.RT-qPCR and Western blot assay showed that magnolol and honokiol inhibited the expression of the pro-apoptotic proteins(P < 0.05).In order to better understand the mechanism of small molecular compounds by bioinformatics analysis,online STITCH software was used to predict the interaction between compounds and protein.Prediction and experiments results showed that magnolol and honokiol downregulated the expression level of caspase 3 in absence of viral infection.The prokaryotic recombinant technology was used to construct prokaryotic expression plasmid of caspase 3 protein(pET28a-caspase3),and caspase 3 protein(35.8 KDa)was obtained by the transformation,induction and purification technology.In vitro developing tests found that magnolol and honokiol prevented the combination of caspase 3 and its substrate.Therefore,magnolol and honokiol inhibited the apoptosis induced by virus,which was likely due to direct effect of compounds and caspase 3.4.Effect on the host antiviral immune responses of honokiolThe RT-qPCR results showed that magnolol and honokiol enhanced antiviral immune responses in host cells.After viral infection,magnolol promoted the expression of I-IFN and the downstream effectors of Mx1,and honokiol enhanced cytokine expression of IL-1?,TNF? and NF-?B.By using NF-?B inhibitor and the eukaryotic expression vector of IL-1? and TNF?,NF-?B-mediated cytokine response pathway played a positive antiviral role in GCRV infection.In addition,as the results of previous studies,the activation of NF-?B in CIK cells may inhibit caspase 8 activity.Such compounds by enhancing the host antiviral response to inhibit virus replication,to a certain extent,break through the limitations of the drugs and have a broad-spectrum resistance.In summary,the results revealed that Magnolia officinalis inhibited GCRV replication in vitro,and the main active ingredient of Magnolia officinalis,namely magnolol and honokiol,effectively inhibited viral replication in vitro and in vivo,enhanced host antioxidant capacity,reduced the mortality rate and improved the survival rate of cells and fish.Magnolol and honokiol can keep the normal cell morphology and function by inhibition on cell apoptosis induced by virus,which were likely due to the interaction with caspase 3 by the degradation or inhibiting effect.In addition,magnolol and honokiol can enhance the antiviral immune response of host cells and show the broad-spectrum antiviral significance,which has broad application prospects in prevention and control of diseases in grass carp.