The Inhibitory Effect And Mechanism Of Japonicone V Against Hepatitis E Virus Replication

Hepatitis E virus(HEV)is one of the major causes of acute hepatitis in humans worldwide.HEV infections have been considered to be transmitted via the consumption of contaminated water and food such as undercooked pork,and HEV is responsible for at least 20 million infections and 70,000 deaths every year,especially among the pregnant women,a high fatality rate of up to 25% has been reported.Emerging data indicate that autophagy is involved in regulating the replication of many viruses,including hepatitis B virus(HBV)and hepatitis C virus(HCV),and the autophagy machinery is required for virus replication.But it remains unclear whether autophagy is involved in the replication of HEV or not.Therefore,we intend to investigate the effect of autophagy on HEV replication,and the intervention of natural autophagy inhibitors on HEV replication and the possible involvement of autophagic pathway in this process.In this study,the used methods and important results are listed as follows:1.The cells model of human Hepatoma Huh7 cells and HEV stably transfected p6 cells were used to study the regulation of autophagy on HEV replication.Through fluorescence microscope,flow cytometer and Western blot,the number of autophagosomes and acid bodies,the fluorescence intensity of AO,MDC and the expression of autophagy-related proteins Beclin-1,LC3-II,and P62 in p6 cells(HEV transfected Huh7 cell)were investigated and compared with Huh7 cell.To clarify the role of autophagy on HEV replication,the HEV levels in p6 cells in the presence of 3-methyladenine(3-MA)or chloroquine(CQ),two autophagy inhibitors were analyzed.As expected,3-MA and CQ suppressed autophagy significantly,as indicated by a corresponding decreased beclin-1 expression,reduced LC3-II accumulation,and increased p62 accumulation in p6 cells.Importantly,the inhibition of autophagy by 3-MA and CQ significantly repressed HEV replication.Collectively,these results indicated that HEV infection induced autophagy,which was required for its replication.2.Among a group of natural products,we selectively examined the anti-HEV effects of three natural autophagy regulators that showed autophagy inhibition activities in cancer cells,including baicalin,a flavonoid from Scutellaria radix,and Japonicone U and Japonicone V,two sesquiterpene lactone derivatives from the flowers of Inula japonica in p6 cells.The cytotoxicity of these natural autophagy inhibitors in p6 cells was firstly measured using the CCK-8 assay.The results shown that baicalin,japonicone U and japonicone V at the concentrations of 50 μM,10 μM,and 1.5 μM,respectively have low cytotoxicity on p6 cells.Moreover,baicalin,japonicone U,and japonicone V at the concentrations of 50 μM,10 μM,and 1.5 μM,could efficiently decrease HEV cellular genomic RNA level.The inhibitory rate against [88]HEV replication by baicalin and Japonicone U was about 40%,and the inhibitory rate against HEV replication by Japonicone V was as high as 80%;the optimal time of Japonicone V to inhibit HEV replication was 24 h,and the optimum concentration was 1.5μM.3.To further investigate the effect of Japonicone V on autophagy and the role of Japonicone V on HEV replication,flow cytometry and Western blot were adopted to observe the number of autophagosomes and acidic bodies and the expression of autophagy marker proteins in p6 cells after treatment with Japonicone V.Furthermore,high-throughput transcriptomics was used to analyze the possible mechanism for Japonicone V to inhibit HEV replication.The result showed that Japonicone V treatment decreased the AO and MDC fluorescence intensity of p6 cells in a dosedependent manner.Moreover,Japonicone V treatment significantly decreased the accumulation of LC3-II and increased the accumulation of P62 in the HEV infectious p6 cells,indicating that Japonicone V inhibited autophagy in the HEV infected cell model.When the effects of CQ(an autophagy inhibitor)and rapamycin(an autophagy inducer)on Japonicone V induced HEV replication were measured in p6 cells,CQ exerted a synergistic anti-HEV effect with Japonicone V,Rapamycin attenuated Japonicone V-induced autophagy inhibition.High-throughput transcriptomics analysis suggested that Japonicone V might inhibit HEV replication by regulating some signal pathways related to autophagy,immune and energy metabolism.Western blot shown that Japonicone V increased the expression of PI3 K,AKT,m TOR,P38,JNK proteins.Collectively,Japonicone V restricted HEV replication by inhibiting autophagy induced by HEV infection through activating PI3 K / AKT / m TOR and MAPK signaling pathways.