BACKGROUND: Ischemic stroke (IS) is linked to dysregulated mitophagy. Timosaponin B-II (TBII), a complex furostan steroid saponin extracted from Anemarrhena asphodeloides Bunge, has been demonstrated to play a crucial role in regulating autophagy. However, whether TBII exerts therapeutic effects in cerebral ischemia through the regulation of autophagy, particularly Parkin-dependent pathways remained unexplored.
PURPOSE: The present study investigated whether TBII can alleviate cerebral ischemic injury by enhancing Parkin-dependent mitophagy.
METHODS: We evaluated TBII's effects on cerebral ischemic injury using permanent middle cerebral artery occlusion (pMCAO) in mice and oxygen-glucose deprivation (OGD)-treated neurons. Mitophagy and mitochondrial function were assessed by Western blot, transmission electron microscopy (TEM), and immunofluorescence techniques. Parkin knockdown (shPrkn lentivirus) and the mitophagy inhibitor Mdivi-1 were employed to validate TBII' mechanisms.
RESULTS: Intragastrical (i.g.) administration of TBII (10, 20, 40 mg/kg) for 7 days significantly reduced cerebral infarction volume, brain water content, and neurological deficits in pMCAO mice, while attenuating neuronal death in vivo and in vitro. Molecular docking, cellular thermal shift assays (CETSA), drug affinity responsive target stability (DARTS), and molecular dynamics simulations confirmed that TBII specifically binds and stabilizes to Parkin, suggesting its potential to enhance mitophagy. TBII mitigated the impairment of mitophagy by upregulating Parkin and p-Parkin (Ser65), promoting the ubiquitination of mitochondria, the degradation of autophagy substrate SQSTM1 and damaged mitochondria after IS. TBII also preserved mitochondrial membrane potential (MMP), suppressed oxidative stress, and restored mitochondrial function and ultrastructure. These benefits were reversed by the mitophagy inhibitor Mdivi-1 and Parkin knockdown.
CONCLUSION: The present study demonstrates that TBII reduces oxidative stress, preserves mitochondrial function, and ultimately attenuates ischemic brain injure by enhancing Parkin-dependent mitophagy. Our study provides the first evidence supporting TBII as a promising therapeutic agent for IS.
