DL-3-n-Butylphthalide Protects Mitochondria Against Ischemia/Hypoxia Damage via Suppressing GCN5L1-Mediated Drp1 Acetylation in Neurons and Mouse Brains.

BACKGROUND: Mitochondrial dysfunction is an initial event of the cascade reactions triggered by ischemic stroke, contributing to the pathogenesis of ischemic brain injury. DL-3-n-butylphthalide (NBP), a compound originally isolated from the seeds of Apium graveolens Linn, exerts neuroprotective effects by improving mitochondrial function in ischemic brain tissues; however, the exact molecular mechanisms underlying its action remain poorly understood. METHODS: The OGD-exposed neuronal cells and dMCAO mice were used to investigate the effects of ischemia/hypoxia on mitochondrial function and the protective action of NBP on mitochondrial damage. Co-immunoprecipitation and immunofluorescence staining were performed to identify the interaction between Drp1 and GCN5L1. Western blotting, immunofluorescence and immunohistochemical staining were conducted to detect the expression of GCN5L1, Drp1, ERK1/2, Bax, Bcl2, and caspase-3. The mitochondrial function was analyzed by measuring mitochondrial ROS, ATP production, mitochondrial membrane potential (MMP) and mPTP opening. RESULTS: We observed that mitochondrial dysfunction occurs in OGD-treated neuronal cells and brain tissues of dMCAO mice, as evidenced by the alteration in the mPTP, MMP, ATP content, and ROS levels, which are accompanied by a significant increase in mitochondrial fission and neuronal apoptosis, as shown by TUNEL staining and the changes in Bcl-2, Bax and caspase-3 expression. Importantly, NBP intervention significantly attenuates ischemia/hypoxia-induced mitochondrial dysfunction and cellular apoptosis in the neuron and mouse brains. Mechanistically, NBP not only reverses the upregulation of Drp1 and GCN5L1 expression by ischemia/hypoxia, but also inhibits the ischemia/hypoxia-induced phosphorylation of Drp1 by blocking the ERK1/2 signaling, which in turn suppresses the interaction between Drp1 and GCN5L1, thereby decreasing Drp1 acetylation by GCN5L1 and excessive mitochondrial fission. CONCLUSION: Our findings provide a novel insight into the molecular mechanism whereby NBP protects mitochondria against ischemia/hypoxia damage, offering a promising drug for mitochondria-targeting therapeutics for ischemic stroke.