This study aimed to assess the neuroprotective effects of salidroside (SAL) on cerebral ischemia-reperfusion injury (CIRI) in a rat model and to elucidate the underlying mechanisms, with a focus on the role of estrogen receptor beta (ERβ) and BCL2 interacting protein 3 (BNIP3)-mediated mitochondrial autophagy as potential therapeutic targets in ischemic stroke. A total of 165 female Sprague-Dawley rats were randomly assigned into 11 groups (n = 15 per group). One group served as the control. The remaining animals underwent bilateral ovariectomy and were subsequently allocated into the following groups: ovariectomy-only, middle cerebral artery occlusion/reperfusion (MCAO/R), estradiol control, ERβ inhibitor, two inhibitor arms (inhibitor-only and inhibitor-plus-SAL), three SAL treatment groups (low, mediummitochondrial division, high dose), and a positive control (edaravone). All groups, except the control and ovariectomy-only groups, were subjected to MCAO for one hour followed by 24 h of reperfusion. Neurological function, cerebral infarct volume, blood-brain barrier (BBB) permeability, and brain water content were evaluated. Histopathological alterations were assessed, and transmission electron microscopy was employed to detect autophagosomes. Western blot analysis was performed to quantify protein expression levels of ERβ, BNIP3, NIP3-like protein X, and microtubule-associated protein 1 A/1B-light chain 3. Administration of SAL and edaravone significantly reduced neurological impairment, infarct volume, BBB disruption, and cerebral edema in the MCAO/R model. SAL treatment upregulated ERβ and BNIP3 expression and enhanced mitochondrial autophagy-associated protein levels. These effects were attenuated by the use of ERβ and mitochondrial division inhibitors, indicating a mechanistic link between SAL-mediated neuroprotection and activation of the ERβ/BNIP3 signaling axis. SAL exerts a neuroprotective effect against CIRI in rats, primarily through activation of ERβ and enhancement of BNIP3-mediated mitochondrial autophagy. These findings suggest that modulation of the ERβ/BNIP3 pathway may represent a promising therapeutic approach for ischemic stroke.
