Astragaloside IV attenuates cerebral ischemia-reperfusion injury by suppressing PERK/eIF2α/ATF4-mediated endoplasmic reticulum stress through activation of the SHH signaling pathway.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) triggers endoplasmic reticulum stress (ERS), a key pathological driver of neuronal apoptosis and neurological impairment. While the neuroprotective effects of astragaloside IV (AS-IV) are well-documented, the precise regulatory bridge between the Sonic Hedgehog (SHH) pathway and ERS remains to be elucidated. METHODS: Ischemic conditions were simulated in vitro using HT22 hippocampal neurons subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability, apoptosis, and ERS/SHH-related markers were evaluated via Cell Counting Kit-8 (CCK-8), Western blotting, and immunofluorescence assays. Bidirectional rescue experiments were conducted using dithiothreitol (DTT, an ERS inducer), Cyclopamine (Cyc, an SHH inhibitor), and GLI family zinc finger 1 (Gli1) Short hairpin RNA (shRNA) (sh-Gli1). In vivo validations were performed in Middle cerebral artery occlusion/reperfusion (MCAO/R) rats pretreated with AS-IV (28 mg/kg/day, i.p. for 3 days). Neurological deficits, infarct volume, and penumbral neuronal survival were assessed alongside brain tissue immunoblotting. RESULTS: AS-IV pretreatment rescued HT22 cells from OGD/R-induced death in a dose-dependent manner and significantly suppressed the PKR-like ER kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4)-mediated ERS/apoptosis axis. Expanded analysis revealed that AS-IV also partially inhibited the Inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response (UPR) without affecting activating transcription factor 6 (ATF6). Mechanistically, AS-IV significantly elevated the expression of SHH, Patched 1 (PTCH1), and Gli1. These protective benefits were largely reversed by pharmacological SHH inhibition, ERS reactivation, or genetic silencing of Gli1, supporting a canonical Gli1-dependent transcriptional mechanism. In MCAO/R rats, AS-IV pretreatment significantly reduced infarct volumes and improved neuronal survival and functional recovery. Consistent with cellular findings, AS-IV attenuated cerebral ERS markers through SHH activation, a mechanism reversed by Cyc. CONCLUSION: AS-IV pretreatment protects neurons from CIRI-induced impairment by selectively suppressing the PERK and IRE1α UPR branches of the UPR through activation of the canonical SHH/Gli1 signaling pathway. These findings highlight the SHH/Gli1/ERS regulatory axis as a potential therapeutic target for ischemic stroke.