Harpagide from radix scrophulariae attenuated ischemic stroke injury through calcium homeostasis regulation between mitochondria-associated membranes via the IP3R1/GRP75/VDAC1 complex.

ETHNOPHARMACOLOGICAL RELEVANCE: Numerous studies have provided evidence supporting the significant roles of Radix Scrophulariae in the treatment of fevers, bacterial infection and encephalopathy. In particular, harpagide, a prominent iridoid glycosides derived from Radix Scrophulariae, the representative component of Radix Scrophulariae, has demonstrated noteworthy neuroprotective against cerebral ischemic injury. However, the precise mechanisms that underlie its anti-ischemic effects remain unclear.

PURPOSE: The aim of this study is to elucidate the in vitro and in vivo neuroprotective effects of harpagide and the mechanisms involved in mitochondria-associated membranes (MAMs).

METHODS AND MATERIALS: The neuroprotective effects of harpagide were evaluated using 2,3,5-Triphenyltetrazolium chloride (TTC) staining and the Longa Neurological Function Score in vivo. JC-1, fluorescein diacetate (FDA)/propidium iodide (PI) staining, a western blot assay, and RT-PCR were used to assess the mitochondria-associated neuroprotective impacts of harpagide on primary cortical neurons. The harpagide effects on the endoplasmic reticulum (ER), mitochondrial calcium ion concentrations, and double immunofluorescence was used to assess the harpagide effects on the expression and co-localization of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1)/glucose-regulated protein 75 (GRP75)/voltage-dependent anion channel 1 (VDAC1) complex in the MAMs of neurons. Additionally, RT-PCR and a western blot assay were used to quantify these protein expression levels on primary cortical neurons. Furthermore, MAMs were evaluated using specific calcium ion fluorescent probes and immunofluorescence both in vivo and in vitro.

RESULTS

Harpagide mitigated cerebral injury in the middle cerebral artery occlusion (MCAO) model. In addition, neuronal mitochondrial damage and mitochondria-mediated apoptosis factors in the oxygen and glucose deprivation (OGD) model significantly decreased by harpagide. Harpagide also significantly ameliorated the neuronal calcium homeostasis imbalance and mitigated MAM alterations in both the MCAO and OGD models. Harpagide also reduced the co-localization and expressions of inositol IP3R1, GRP75, and the VDAC1 in the OGD model.

CONCLUSION

This study provided evidence that harpagide regulated calcium homeostasis within MAMs via the IP3R1/GRP75/VDAC1 complex, thereby reducing mitochondria-mediated neuronal apoptosis and alleviating IS injury both in vivo and in vitro. These findings offer a novel research direction for the therapeutic application of harpagide in ischemic stroke (IS).