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.