The Mechanism(s) Of Deguelin Inhibiting HCV Replication Through Suppressing Cellular Autophagy

Objective Previous studies have shown that deguelin,a natural compound derived from Mundulea sericea(Leguminosae)and some other plants has exhibited an activity to inhibit autophagy,a cellular machinery required for hepatitis C virus(HCV)replication.This study aims to illuminate the impact of deguelin on HCV replication and mechanism(s)involved.We expect to provide some clues for the development of novel anti-HCV drugs based on the perspective of cellular autophagy.Methods This study was mainly carried out by in vitro research,using HCV JFH-1 virus to infect Huh7 hepatoma cell line to simulate HCV infection in vivo.Firstly,to investigate the effect of deguelin on the HCV replication,we used different concentrations of deguelin to treat the HCV-infected Huh7 cells for different times.The levels of HCV RNA and HCV core protein were detected by RT-PCR and Western blot respectively.Next,to investigate the effect of deguelin on cellular autophagy,under the condition of autophagosomal inhibitor CQ pretreatment or not,we used deguelin to treat the HCV-infected or uninfected Huh7 cells,RT-PCR and Western blot were used to detect the expression levels of LC3 mRNA and LC3B-I,LC3B-II proteins.Then,investigating the effect of deguelin on tunicamycin(autophagy inducer)-induced atuophagy,under the condition of autophagosomal inhibitor CQ pretreatment or not,we used deguelin to treat the Huh7 cells with or without tunicamycin treatment,RT-PCR and Western blot were used to detect the expression levels of LC3 mRNA and LC3B-I,LC3B-II proteins.Furthemore,we also used GFP-LC3 lentivirus to transfect Huh7 cells to investigate the effect of deguelin on cellular autophagy,the numbers of GFP-LC3-positive dots were detected by fluorescent microscopy.Finally,RT-PCR and WB were used to investigate the effect of deguelin treatment on the important autophagy-related factors,under the condition of silencing or over-expressing the selected autophagy factors,the expression levels of HCV RNA and HCV core protein,LC3B-I and LC3B-II proteins were detected by using RT-PCR and Western blot,to elucidate the relationship between deguelin,autophagy and HCV replication.Results1.Deguelin inhibits HCV replication in Huh7 cells.We used different concentrations of deguelin to treat the HCV-infected Huh7 cells for different times.The levels of intracellular HCV RNA and HCV core protein in deguelin-treated cells were significantly lower than the control group(p<0.05),and we observed that the inhibitory effect of deguelin on HCV replication was in a dose-and time-dependent manner,which indicated that deguelin treatment could inhibit HCV replication in Huh7 cells.2.Deguelin treatment inhibits cellular autophagy in Huh7 cells.In the absence or presence of CQ,HCV-infected or uninfected,there was no significant effect(p>0.05)at LC3 mRNA level in Huh7 cells after deguelin treatment.But the protein levels of LC3B-II as well as conversion of LC3B-I to LC3B-II were significantly decreased(p<0.05).The results indicated that deguelin had the activity to inhibit cellular autophagy,and it inhibitd the upstream event(s)at early stage of autophagy.3.Deguelin treatment inhibits cellular autophagy induced by tunicamycin treatment in Huh7 cells.Under the condition of CQ treatment or untreatment,deguelin treatment had no significant effect(p>0.05)at LC3 mRNA level in tunicamycin-treated Huh7 cells.But the protein levels of LC3B-II as well as conversion of LC3B-I to LC3B-II were significantly decreased(p<0.05).The results indicated that deguelin had the activity to inhibit tunicamycin-induced cellular autophagy,and it inhibitd the upstream event(s)at early stage of autophagy.4.Deguelin treatment inhibits the expression of autophagy-related factor Beclin1.Under the condition of deguelin treatment,the RNA and protein levels of Beclin1 in HCV-infected or uninfected Huh7 cells were both significantly decreased(p<0.05),which indicated that deguelin could inhibit the expression of Beclin1.5.Deguelin treatment inhibits HCV replication through suppressing cellular autophagy.Under the conditions of deguelin treatment,the expression of intracellular HCV core,LC3B-II protein and LC3B-I/II were significantly up-regulated in the over-expressing group compared with the blank plasmid-transfected cells(p<0.05),which indicated that overexpression of Beclin1 could promote autophagy and attenuate the inhibitory effect of deguelin on HCV.The expression of HCV core,LC3B-II protein and LC3B-I/II in silencing group were significantly decreased(P<0.05),which indicated that silencing Beclin1 could inhibit autophagy and enhance the inhibition of deguelin on HCV replication.Conclusions These result indicated that deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells.This study identified a new natural compound with anti-HCV activity,deguelin,and elucidated its partial mechanism of anti-HCV replication,providing a theoretical basis for the development of novel anti-HCV drugs from the perspective of autophagy.