[Xihuang Pills induce mitochondria-associated ferroptosis to enhance therapeutic efficacy of temozolomide against glioblastoma via Nrf2/HO-1/GPX4 signaling axis].

This study aimed to investigate whether Xihuang Pills(XHP) enhance the therapeutic efficacy of temozolomide(TMZ) against glioblastoma(GBM) U251 cells by inducing mitochondria-associated ferroptosis via regulation of Nrf2/HO-1/GPX4 signaling axis. The XHP-containing serum was prepared, and the effects of various concentrations of TMZ(0-200 μmol·L~(-1)) and XHP(0-20%) on U251 cell viability were assessed by CCK-8 assay. The optimal synergistic dose(200 μmol·L~(-1) TMZ + 10% XHP) was identified via the SynergyFinder platform for establishing a combined intervention model. Control, XHP, TMZ, and XHP + TMZ groups were designed. Cell proliferation was evaluated by EdU staining and colony formation assay, and cell migration and invasion were assessed by Transwell and wound healing assays. FerroOrange and DCFH-DA fluorescent probes were used to measure the intracellular levels of Fe~(2+) and ROS, respectively. The JC-1 fluorescent probe was used to assess mitochondrial membrane potential, while Mito-PeDPP fluorescent probe to examine mitochondrial lipid peroxidation. Mito-Tracker Green and MitoSOX~(TM) Red were used to examine mitochondrial morphology and oxidative stress. Transmission electron microscopy was adopted to observe the mitochondrial ultrastructure. GSH and MDA levels were measured by colorimetric assays. Western blot was employed to assess the protein levels of Nrf2, HO-1, xCT, and GPX4. The results showed that, XHP + TMZ suppressed cell viability, proliferation, migration, and invasion(P