The Effect And Related Mechanism Of Honokiol On Prevention And Treatment Of Porcine Hemagglutinating Encephalomyelitis Virus Infection

Porcine hemagglutinating encephalomyelitis,an acute and highly contagious disease in pigs,is caused by porcine hemagglutinating encephalomyelitis virus（PHEV）.It has a high mortality rate for piglets,and its harm cannot be ignored.However,to date,there are no safe and effective commercial vaccines and treatments for PHEV infection.Honokiol（HNK）is a dihydroxybiphenyl compound,which is one of the main medicinal components in Magnolia officinalis.It can pass the blood-brain barrier and has significant effects in the treatment of various neurological diseases.It is not clear whether honokiol has a protective effect on the nervous system damage caused by PHEV infection.Based on this,this paper carries out the following research work.In this study,PHEV-infected N2 a cells were used as an in vitro infection model to determine the inhibitory effect of honokiol on PHEV replication in vitro.Then,the in vivo infection model of PHEV-infected C57BL/6 mice was used to study the effect of honokiol on PHEV infection in vivo.In vitro experiments,cell activity assay（CCK-8）and Western blot（WB）were used to confirm that honokiol could effectively inhibit PHEV replication.The optimal concentration of honokiol was 25μM/L.The results showed that honokiol could significantly inhibit the adsorption and entry of PHEV on N2 a cells.On this basis,three modes were designed: adding honokiol first and then inoculation with PHEV（simulated prevention mode）,adding honokiol and inoculation with PHEV（simulated direct disinfection mode）,and inoculation with PHEV and then inoculation with honokiol（simulated treatment mode）.Western blotting assay（WB）,Quantitative Real-time PCR（q PCR）and indirect immunofluorescence assay（IFA）were used to detect PHEV content.The results showed that compared with the control group,the m RNA and protein levels of PHEV in the three drug treatment modes were significantly decreased,and THE IFA test further confirmed that honokiol could significantly inhibit THE replication of PHEV in vitro.After considering the PHEV infection caused by nerve damage,is small,the clinical significance of the use of drug treated in vivo experiments,this research USES honokiol medication after 7 days,then through intranasal approach to mice vaccinated PHEV,the results found that honokiol can alleviate the neurologic symptoms of PHEV infected mice,prolong the survival time,has certain preventive effect.Compared with the control group,honokiol significantly inhibited PHEV expression at m RNA and protein levels,and antigen distribution was also significantly reduced.In conclusion,honokiol inhibits PHEV replication in vivo and in vitro.In order to reveal the mechanism of honokiol’s anti-PHEV action,we found that honokiol up-regulated the expression of progranulin（PGRN）in normal N2 a cells by drug treatment.After knockout of PGRN in cells,PHEV infection and drug treatment showed that the anti-PHEV effect of honokiol was significantly reduced compared with the control group,indicating that the anti-PHEV effect of honokiol depended on the expression of PGRN.It has been confirmed that lysosome dysfunction occurs when PGRN is deficient.In this study,IFA detection showed that PHEV infection caused the increase of lysosome volume,and honokiol could repair the abnormal accumulation of lysosome caused by PHEV infection,but its influence on lysosome function still needs to be further explored.Previous studies of our group found that PHEV replication was correlated with autophagy to some extent.To further confirm that the anti-PHEV effect of honokiol may depend on the expression of autophagy or the autophagy-related kinase phosphatidylinositol 3-kinase（PI3K）,normal N2 a cells treated with honokiol were detected in this study.Honokiol can induce autophagy in nerve cells.After treating cells with autophagy inhibitor 3-MA and interfering PI3 K expression in cells,the contents of LC3 and PHEV were measured respectively,and it was found that the anti-PHEV effect of honokiol depended on autophagy induced by PI3 K pathway.In conclusion,this study confirmed that honokiol has significant anti-PHEV effects in vivo and in vitro,especially when used to prevent PHEV infection,which lays a foundation for the later clinical application.At the same time,this study analyzed that the antiviral effect of honokiol was closely related to the expression of PGRN and autophagy induced by THE PI3 K pathway,providing a new idea and research basis for the further development of specific targeted drugs against PHEV infection.