Effect of fangchinoline against H1N1 infection by blocking autophagy
Objective To investigate the antiviral effect of fangchinoline against the H1N1 virus and elucidate its molecular mechanism in regulating cellular autophagy.Methods CCK-8 method was used to detect the effect of tetrandrine of 0.312 5,0.625 0,1.250 0,2.500 0,5.000 0 0,10.000 0 0,20.000 0,40.000 0,and 60.000 0 0 μmol·L-1 on viability of MDCK cells.MDCK cells were divided into control group,model group,and tetrandrine(2.5,5.0,and 10.0 μmol·L-1)groups.Except for control group,MDCK cells were infected with H1N1 virus with a multiple infection index(MOI)of 0.1,and co-incubated with tetrandrine for 12 h.At the same time,tetrandrine(10.0 μmol·L-1)was set up incubated with the virus for 4 and 8 h,and detected the inhibitory effect of tetrandrine on virus replication through real-time fluorescence quantitative PCR(qRT-PCR,detecting HIN1 mRNA expression)and Western blotting[H1N1 virus nucleoprotein(NP)].PI/Hoechst 33342 staining was used for detection of tetrandrine on MDCK cell death.QRT-PCR method was used to detect the effects of pre-treatment with tetrandrine,early drug treatment for viral infection,late drug treatment for viral infection,as well as adsorption and entry stage administration on virus replication.MDCK cells were transfected with EGFP-LC3 or EGFP-mCherry-LC3 dual fluorescent plasmids to observe the fluorescence intensity of EGFP-LC3 and EGFP-mCherry-LC3 co-localization.MDCK cells transfected with EGFP-LC3 plasmid were infected with H1N1 virus,and immunofluorescence was used to detect the effect of tetrandrine on LC3 and co-localization with intracellular viral NP protein.A549 cells were cultured in vitro and infected with H1N1 virus with a MOI of 0.1.Western blotting was performed to detect the effect of tetrandrine on expression of LC3Ⅱ/LC3Ⅰ protein.Results The half inhibitory concentration(IC50)of tetrandrine on MDCK cells is 40.19 μmol·L-1.Compared with model group,tetrandrine inhibited HIN1 mRNA expression in a concentration dependent manner and NP protein expression in a concentration and time dependent manner(P＜0.01,0.001),significantly reduced H1N1 induced cell death(P＜0.001),and inhibited the entry process of H1N1 virus.Compared with control group,treatment with tetrandrine induced LC3 fluorescence aggregation and significantly increased co-localization of EGFP-LC3 and mCherry-LC3 dual fluorescence(P＜0.001).Compared with model group,treatment with tetrandrine significantly reduced the fluorescence quantity of NP(P＜0.001),while causing the accumulation of LC3 fluorescence and co-localization with intracellular viral NP proteins,causing accumulation of LC3Ⅱ(P＜0.01,0.001).Conclusion Fangchinoline is consistent with bafilomycin Al,blocking the autophagy pathway,causing viral particles to remain in the autophagy,and destroying the life cycle of the virus.
fangchinolineinfluenza A virus subtype H1N1autophagychloroquinebafilomycin A1
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