Fig. 4. Autophagy promoted silibinin-induced increase of H₂O₂ via causing GSH depletion.

a Western blotting proved that silibinin induced downregulation of HIF-1α, but upregulation of GPX4 in a time-dependent manner. B H₂O₂ assay showed that intracellular H₂O₂ was improved time-dependently in silibinin-treated glioma cells. c GSH assay proved that silibinin treatment resulted in time-dependent depletion of GSH. d H₂O₂ assay demonstrated that supplement of exogenous GSH prevented silibinin-induced increase of H₂O₂. e LDH release assay showed that silibinin-induced glioma cell death was prevented when GSH was supplemented. f Western blotting revealed that pretreatment with GSH prevented silibinin-induced BNIP3 overexpression and accumulation on mitochondria. g Western blotting showed that H₂O₂ not only induced time-dependent upregulation of BNIP3, but also improved BNIP3 levels in mitochondrial fractions in a time-dependent manner. h GSH assay showed that silibinin-induced GSH depletion was inhibited in the presence of 3MA or bafilomycin A1. i H₂O₂ assay showed silibinin-induced improvement of H₂O₂ was reversed in the cells pretreated with 3MA or bafilomycin A1. ^#p < 0.05 versus control group; *p < 0.01 versus control group. The values are expressed as mean ± SEM (n = 5 per group).