Study On The Anti-HBV Mechanism Of Musca Domestica Antibacterial Peptide Cecropin

Objective:Based on the key events of Hepatitis B virus(HBV)infection,the anti-HBV infection mechanism of Musca domestica Cecropin(MDC)was studied.Exosomes,as a new way of intercellular communication,carrying biological information related to viral infection,play an important role in virus infection.This study was performed in order to explore whether exosome from HepG2.2.15 cell supernatant mediates HBV infection and whether MDC interferes with exosomes-mediated HBV infection.Methods:1.Study on the anti-HBV mechanism of Musca domestica antibacterial peptide Cecropin:(1)Purification of HBV particles from supernatant of HepG2.2.15 cells by Hi-Trap heparin affinity chromatography;Electron microscopy was used to observe the morphology of HBV.(2)MTT assay was used to detect the toxicity of MDC on HepaRG cells;12-day infection experiment:Real-time PCR assay were carried out to detect the definite effect of MDC on Hepatitis B virus' life cycle;Laser confocal microscopy and Flow cytometry were used to test the effect of drug(joining in HBV adhesion phase)on HBV infection rate;MTT assay was used to detect the drug's cytotoxicity in HepG2.2.15 cells,Real-time PCR,ELISA,ICC and Western blotting was used to detect the effect of MDC on HBV replication in HepG2.2.15 cells.(3)Laser confocal microscopy was used to observe the distribution and localization of MDC in cells.Real-time PCR and Western blotting were used to detect the effect of MDC on anti-viral signaling pathway.2.Musca domestica antibacterial peptide Cecropin inhibits exosome-mediated HBV infection:(1)The extraction kit was used to extract the exosomes(HepG2.2.15-EXO)from the supernatant of HepG2.2.15cells;The morphology,size and the protein markers of exosomes was detected by transmission electron microscopy,nanoparticle tracking analyzer,and Western blotting respectively.PCR test was used to detect whether exosomes contain HBV nucleic acid components;(2)Ultra-high resolution microscopic imager of live cell to observe the location of exosomes in cells;flow cytometry was used to detect the spread of exosomes in cells;ELISA,Real-time PCR,ICC,Western blotting was uesd to detect whether HepG2.2.15-EXO mediates HBV infection,and whether MDC interferes with exosome-mediated HBV infection;(3)In order to test the effects of exosomes' concentration,temperature,heparan sulfate and heparinase on the spread of exosomes in cells,flow cytometry was used;Real-time PCR method was used to detect the effect of heparan and heparanase on the expression of HBV-DNA in cells;Results:1.Study on the anti-HBV mechanism of Musca domestica antibacterial peptide Cecropin:(1)Electron microscopy experiments showed that MDC can change the structure of the virus and HBV were almost agglutinate;(2)Drug were added before or after HBV attacking to intervene infection,the results show that drug decreased HBV related indicators(HBV-DNA,HBVcccDNA and pgRNA expression in cells)and it intervened the HBV's life cycle in its infection;MDC(non-toxic concentration range)significantly decreased HBsAg and HBeAg,HBV DNA content,intracellular HBsAg,HBcAg protein expression in HepG2.2.15 cell,having an effect of inhibiting HBV replication.(3)Laser confocal microscopy showed that the MDC was mainly concentrated in the cell membrane,but the cytoplasm and nucleus can also been loaded;Western blotting and Real-time PCR test showed that MDC could activate JAK/STAT antiviral signaling pathway,exerting antiviral effect.2.Musca domestica antibacterial peptide Cecropin inhibits exosome-mediated HBV infection:(1)Related experiments(electron microscopy,NTA,Western blotting)showed that the precipitation extracted from cell supernatant has the particular shape,size and protein maker of exosome.The extraction can be used in subsequent experiments;HBVnucleic acid was found in exosomes in nucleic acid detection;(2)Flow cytometry,ELISA,cellular immunofluorescence assay,Western blotting and Real-time PCR showed that HBV was positively expressed in normal hepatocytes when co-incubated with exosomes.Musca domestica antibacterial peptide Cecropin has a certain inhibitory effect on the infection;(3)Exosome concentration,incubation time,temperature,heparan and heparanase have a certain effect on the spread of exosomes between cells.Conclusion:The Musca domestica antibacterial peptide Cecropin can destroy the structure of HBV virus particles and cause it to agglomerate;it has an inhibitory effect on the adhesion/entry phase and the replication stage of the HBV virus life cycle;MDC activating the intracellular antiviral signaling pathway exerts its antiviral effect;its anti-HBV mechanism is a complex phase,involving multiple targets;and the exosomes from HepG2.2.15 cell supernatant can mediate HBV infection,MDC has a certain inhibitory effect on exosome-mediated HBV infection.