Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: The highly vascularized nature of glioblastoma multiforme (GBM) underlies its high lethality. Xihuang pill (XHP), a renowned proprietary Chinese medicine, has arisen as a potential candidate for clinical GBM management. Nonetheless, its effects and mechanisms on glioma-associated endothelial cells (GECs), which are key components of angiogenesis, remain unexplored. AIM OF THE STUDY: This study addresses the effect and potential mechanisms of XHP on GECs. MATERIALS AND METHODS: GEC viability and proliferation were assessed using CCK-8 and EdU assays, respectively. Drug interaction between XHP and temozolomide (TMZ) was assessed via CompuSyn package and Combenefit software. Cell cycle distribution was detected by flow cytometry. Pyroptosis was evaluated using optical microscopy, scanning electron microscopy, Hoechst 33342/PI double staining, and LDH release assay. Mitochondrial function was examined by measuring membrane potential (MMP) and mitochondrial superoxide (MitoSOX) production. The impact of the AMPK pathway was investigated using an AMPK agonist. In vivo, GBM growth was evaluated in an orthotopic GBM model in C57BL/6 mice. Histopathological analysis was performed via hematoxylin and eosin (H&E) staining. GEC proliferation and pyroptosis within neoplastic tissues were detected using immunofluorescent staining for PCNA, CD31, NLRP3, and GSDME-N terminal. Western blot was utilized to evaluate cell cycle proteins, pyroptosis markers, and key signaling pathway proteins. RESULTS: XHP and TMZ demonstrated a synergistic effect in GECs. XHP dose-dependently inhibited GEC proliferation by downregulating Cyclin E1, CDK2, and CDK1, and upregulating p21, leading to S-phase arrest. XHP also triggered GEC pyroptosis, characterized by membrane bubbling, NLRP3 inflammasome activation, and cleavage of caspase-1/GSDMD and caspase-3/GSDME. Moreover, XHP promoted mitochondrial reactive oxygen species (mtROS) release, causing mitochondrial impairment and exacerbating pyroptosis in GECs. XHP also decreased AMPK signaling, activating dual pyroptosis-associated pathways, which could be reversed by an AMPK agonist. In vivo, XHP suppressed proliferation and triggered pyroptosis in GECs, prevented angiogenesis, and, when combined with TMZ, significantly inhibited GBM progression and prolonged survival. CONCLUSION: Our study offers the first proof that XHP causes AMPK inhibition-mediated mitochondrial dysfunction, activates both the NLRP3/caspase-1/GSDMD and caspase-3/GSDME signaling, and promotes pyroptosis in GECs, thereby effectively suppressing tumor angiogenesis and prolonging survival of tumor-bearing mice when combined with TMZ.