Inhibition Effects of Ethyl Acetate Extracts from Inonotus obliquus on Prion Replication

To investigate the effect and potential mechanism of ethyl acetate extracts from Inonotus obliquus in inhibiting the replicaton of prion, multiple methods were utilized. Using CCK-8 assay to evaluate the cytotoxicity of dichloromethane extracts, ethyl acetate extracts, n-butanol extracts and obtain the maximal concentrations of various extracts. Prion-infected cell model SMB-S15 were treated with three extracts at safe concentrations (0.1×2⁻¹⁴~0.1×2⁻⁸ mg/mL) and the inhibitory effects of different extracts on prion replication were analyzed by PrP-specific Western blots. The expression of reactive oxygen species, hydrogen peroxide and related antioxidant factors were measured in SMB-S15 cells treated with ethyl acetate extracts. The results showed that the maximum safe concentrations of dichloromethane extracts, ethyl acetate extracts and n-butanol extracts was 0.1×2⁻⁸ mg/mL. Prion replication was significantly inhibited in SMB-S15 cells treated with ethyl acetate extracts at this concentration of 0.1×2⁻⁸ mg/mL for 3 days (P<0.01). While there were no inhibitory effects on prion replication in SMB-S15 cells treated with dichloromethane extracts and n-butanol extracts. In addition, ethyl acetate extracts decreased the levels of reactive oxygen species and hydrogen peroxide, increased the expression level of HO-1, GCLC, total glutathione and enhanced the activity of SOD, with statistical difference. These data suggested that the ethyl acetate extracts from Inonotus obliquus could inhibit prion replication in vitro which might be related to the activation of Nrf2 signaling pathway.