The Mechanism Research Of Matrine Activating Autophagy To Inhibit Enterovirus Proliferation

Human enterovirus is one of the most common pathogenic factors.EVA71 and CVA6 of Enterovirus type A cause typical and atypical hand foot mouth disease respectively,and EVD68 of enterovirus type D causes respiratory disease with different symptoms,threatening human health.For the treatment of Enterovirus,there is still a lack of targeted drugs in clinic,so it is urgent to develop effective anti-enterovirus drugs.In this study,EVA71,EVD68 and CVA6 viruses were used as the research objects,and RD(human rhabdomyoma cells)and HeLa(cervical cancer cells)were used as host cells.Using CCK8 assay,cell morphology analysis,protein imprinting,RT-q PCR,TCID50,MDC autophagy staining,co-immunoprecipitation,construction of plasmid and transfection and c0-immunofluorescence localization and Si RNA transfection experiment methods,such as,To investigate the antiviral effects of matrine,lanatoside C and lupeol,and to screen drugs with broad antiviral spectrum to study the mechanism.The study is divided into 3 parts:(1)Matrine inhibits enterovirus replicationFirst,in RD(human rhabdomyoma cells)and HeLa(cervical cancer cells),whether matrine,lanatoside C and lupeol had anti-enterovirus EVA71,CVA6 and EVD68 effects,it was found that only matrine could significantly inhibit the content of enterovirus EVA71,EVD68 and CVA6 VP1 protein.Subsequently,experiments confirmed that matrine could significantly reduce the lesion effect of infected virus cells,reduce the m RNA level of three enteroviruses,and reduce the content of viral VP1 protein and virions.After determining that matrine has an anti-enteroviral effect,the specific links of its effect were analyzed.After pretreatment with matrine,RD cells were infected with three kinds of enterovirus at 4 ℃ and 37 ℃ respectively,and then the m RNA levels of the three kinds of enterovirus were detected.There was no difference between the matrine treated group and the virus infected group,indicating that matrine could not prevent the three kinds of enterovirus from adsorbing and entering the host cells.Then matrine was added at 0,2,5,8 and 12 hours after enterovirus infection.It was found that matrine could reduce the content of virus VP1 protein,indicating that matrine inhibited the replication phase of enterovirus EVA71,EVD68 and CVA6,and the earlier matrine treatment,the more obvious the inhibition of virus replication.(2)Matrine inhibits enterovirus replication by activating autophagyFirstly,Proteasome inhibitor MG132,Lysosome inhibitor chloroquine and autophagy inhibitor 3-MA were used to explore whether the mechanism of matrine inhibiting enterovirus replication involves Proteasome or Autophagosome pathway.The results showed that the autophagy inhibitor 3-MA not only upregulated the content of viral VP1 protein,but also upregulated the level of viral m RNA and the content of viral particles,while antagonizing the antiviral effect of matrine.It suggests that matrine may inhibit enterovirus replication by activating autophagy pathway.Later,it was found that the levels of LC3-Ⅱ and P62 proteins were significantly higher after administration of matrine than those in the control group and virus treatment group(LC3-Ⅱ protein content>200 times,P62 protein content>20 times).LC3-Ⅱ protein was a marker protein for autophagy,confirming that matrine did indeed cause autophagy in host cells and excessive autophagy far beyond physiological levels.At the same time,matrine also significantly reduced the VP1 protein content of various viral groups representing virus replication levels in host cells,it indicates that matrine inhibits enterovirus replication by activating autophagy pathway.(3)Study on mechanism of matrine activation of autophagy and inhibition of enterovirus replicationThe experimental results showed that matrine only upregulated the content of P62 and LC3 in autophagy related proteins;In the viral protein particle transfection and expression system,matrine only reduced the content of VP3 protein in nine Viral protein components.Immunoprecipitation and immunofluorescence co-localization experiments revealed that VP3 binds to P62 but not LC3.After si RNA transfection interfered with the expression of P62,the ability of matrine to inhibit virus replication was significantly weakened,and the ability of matrine to directly reduce the content of Viral protein VP3 was also significantly weakened.This indicates that the mechanism of matrine inhibiting enterovirus replication through autophagy is closely related to P62.Matrine binds VP3 protein through P62 protein,reduces its content,and then inhibits virus replication and expansion.The research concludes as follows:1.Matrine can inhibit the replication phase of Enterovirus EVA71,EVD68 and CVA6 in RD and HeLa cells,and has a broad-spectrum anti-enterovirus effect.2.Matrine inhibits replication of Enterovirus EVA71,EVD68 and CVA6 by activating host cell excessive autophagy.3.Matrine activates the autophagy pathway to inhibit the replication of enterovirus,which is realized through the targeted binding of the host cell P62 protein to the viral structural protein VP3,reducing its content and thus inhibiting viral replication.