A Novel Mechanism by which harpagide protects against cerebral ischemia:Focus on Grp75 and MAMs Calcium Homeostasis

Maintenance of Ca²⁺ homeostasis at endoplasmic reticulum-mitochondria contact sites (MAMs) is a critical determinant of amelioration of post-ischemic brain injury and repair. Harpagide is a bioactive compound isolated from traditional Chinese medicine Radix Scrophulariae that shows potent anti-ischemic injury by regulating calcium homeostasis between MAMs. Nevertheless, the exact molecular pathways by which harpagide exerts these actions and identification of its direct cellular targets remain unresolved.

This study aims to elucidate the time-dependent therapeutic potential and molecular mechanism of harpagide in ischemic injury, with particular emphasis on Grp75 and calcium homeostasis at MAMs.

Mouse middle cerebral artery occlusion (MCAO6h,8h)and PC12 cell oxygen-glucose deprivation (OGD4h,6h,8h)models were established. Relative cerebral blood flow, 2,3,5-triphenyltetrazolium chloride (TTC) staining, and hematoxylin-eosin (H&E) staining were used to analyze the neuroprotective effects of harpagide in MCAO mice. In vitro assays included apoptosis, Ca²⁺ transfer from the ER to the mitochondria, ER-mitochondria contact site analysis, molecular docking, Western blotting, fluorescent immunocytochemistry, co-immunoprecipitation, RNA interference of Grp75, and LDH release measurement. These assays were used to evaluate the therapeutic effects and mechanisms of harpagide in cerebral ischemic injury.

Harpagide reduced infarct volume and neurological deficits in MCAO6h and 8h mice and attenuated mitochondria-mediated apoptosis at 4 h, 6 h, and 8 h post- OGD in vitro. Harpagide regulated calcium homeostasis between MAMs following ischemic stroke via the IP₃R1-Grp75-VDAC1 axis. Harpagide exhibited a time-dependent modulation of Grp75: a trend toward increased expression after 4 h of OGD observed along with enhancement of ER-to-mitochondria Ca²⁺ transfer and ER-mitochondria contact, but significantly downregulating Grp75 at 6 h and 8 h of OGD resulted in decreased Ca²⁺ overload and reduced contact. Co-immunoprecipitation revealed that harpagide transiently enhanced IP₃R1-Grp75 binding, peaking at 4 h and declining by 6 h. Harpagide also attenuated OGD-induced Grp75-VDAC1 co-localization at 6 h. Further assays showed that the protective effect was associated with targeting of Grp75. Notably, the anti-OGD-mediated neuronal damage effect of harpagide was markedly inhibited by Grp75 knockdown, but reinforced by the overexpression of Grp75 at 4 h OGD.

Harpagide reduced injury due to cerebral ischemia in vivo and in vitro. The mechanism may be through the modulation of Grp75 to regulate calcium homeostasis at MAMs and the phase-dependent, dichotomous function of Grp75 in cerebral ischemia.