| Porcine Hemagglutinating Encephalomyelitis is a highly contagious disease caused by Porcine Hemagglutinating Encephalomyelitis Virus(PHEV).It has caused great economic losses to pig industry in many countries.At present,there are no specific anti-virus drugs and treatment methods.Previous studies showed that PHEV infection could induce autophagy in nerve cells,and the replication and proliferation of PHEV was negatively correlated with autophagy.In addition,PHEV infection can down-regulate the expression of PGRN and induce nerve injury.Studies have shown that trehalose(TRE)is a non-toxic disaccharide and can pass through the blood-brain barrier.Trehalose can induce autophagy and up-regulate the expression of PGRN in vivo and vitro,which has broad prospects in the treatment of neurodegenerative diseases including Huntington’s disease and Parkinson’s disease.Whether trehalose plays an anti PHEV role by regulating autophagy or PGRN expression remains unclear.In order to determine the anti PHEV effect of trehalose,we used Neuro-2a(N2a)of mouse infected with PHEV as cell model.Firstly,we determined the concentration of trehalose as 100 m M according to the results of cell activity test.Secondly,cells were treated with three different ways,trehalose before inoculation of virus,trehalose after inoculation of virus,or trehalose and virus at the same time.Western Blot,q RTPCR and immunofluorescence methods were used to detect trehalose.It was found that trehalose could significantly reduce the content of virus in cells,indicating that trehalose had a significant antiviral replication effect in vitro.In addition,taking mice infected with PHEV as the model,the total amount of PHEV in brain tissue of trehalose treated mice was significantly lower than that of control mice,indicating that trehalose also has significant antiviral effect in vivo.Trehalose can induce autophagy in N2 a cells through m TOR independent pathway.In order to elucidate the role of autophagy in trehalose anti PHEV,we treated N2 a cells with autophagy inhibitor 3-methyladenine(3-MA)and autophagy inducer rapamycin(RAPA)respectively,and then inoculated them with virus.Western Blot analysis showed that inhibition of autophagy could weaken the effect of trehalose on PHEV replication,and induction of autophagy via m TOR pathway could enhance the effect of trehalose on PHEV replication.The results showed that autophagy pathway played an important role in the process of trehalose inhibiting PHEV replication.Previous studies found that PHEV infection significantly down-regulated the expression of PGRN and CTSD,which are closely related to lysosomal injury,and led to the increase of lysosomal volume and other damaging changes.In this study,the cells were treated with trehalose first,and then treated with trehalose.Western Blot and indirect immunofluorescence detection showed that trehalose could alleviate the down-regulation of PGRN expression induced by PHEV,restore lysosomal morphology,and increase CTSD expression.It suggested that trehalose could repair lysosomal injury induced by PHEV by up regulating PGRN.In addition,in order to clarify the role of PGRN in trehalose antiviral process,trehalose was added to overexpressed or PGRN knockout N2 a cells infected by PHEV.Western Blot was used to detect the change of virus content.It was found that overexpression of PGRN did not significantly affect the antiviral effect of trehalose,but the effect of trehalose on PHEV disappeared after PGRN knockout,these results indicate that PGRN plays an important regulatory role in trehalose anti PHEV.In conclusion,this study confirmed that trehalose has a significant anti PHEV replication effect in vivo and in vitro,and it has a good prevention and treatment effect on PHEV infected mouse model,and preliminarily revealed that PGRN plays an active role in trehalose anti PHEV process.The results can provide an important theoretical basis for the development of anti PHEV drugs in veterinary clinic. |
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