Harpagide Alleviates Cerebral Ischemic Injury via AMPK-MCU Phosphorylation-Dependent Inhibition of Mitochondrial Calcium Overload

Mitochondrial calcium homeostasis may offer therapeutic benefits for ischemic stroke. Harpagide has been shown to inhibit mitochondrial calcium uptake, but the mechanism remains unclear. In this study, a male ICR mouse model of transient middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia and PC12 and SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) were used. Apoptosis, lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), and the ATP levels were measured to assess the neuroprotective effects of harpagide. DCFH-DA, JC-1, and Rhod-2A probes were used to analyze the mitochondrial function. RT-qPCR and western blot were used to determine the mitochondrial calcium uniporter (MCU), the apoptosis-inducing factor (AIF), Endo G, Cyt C, Caspase-3, and AMPK-MCU phosphorylation expression levels. Additionally, Longa scoring and triphenyl tetrazolium chloride (TTC) staining were used. Transmission electron microscopy (TEM) was used to examine the mitochondrial ultrastructural changes. An immunohistochemical analysis was conducted to detect the MCU expression in the temporal cortex. We found that harpagide exhibits robust neuroprotective effects both in vivo and in vitro , decreased LDH release and MDA levels while increasing SOD activity and ATP content. Harpagide also inhibited mitochondrial calcium overload, mitigated mitochondrial damage, and regulated the MCU, AIF, Endo G, Cyt C, and Caspase-3 expression to reduce mitochondria-mediated apoptosis. Mechanistically, harpagide exerted its neuroprotective effects by inhibiting excessive AMPK activation and subsequent MCU phosphorylation at serine 57. The present study suggests that harpagide regulates mitochondrial calcium homeostasis via the AMPK-MCU phosphorylation cascade, providing a reliable strategy for therapies that target ischemic stroke.