3- n-butylphthalide exerts neuroprotective effects by enhancing anti-oxidation and attenuating mitochondrial dysfunction in an in vitro model of ischemic stroke

Abstract

Purpose: This study examined whether the neuroprotective drug, 3-n-butylphthalide (NBP), which is used to treat ischemic stroke, prevents mitochondrial dysfunction.

Materials and methods: PC12 neuronal cells were pretreated for 24 hours with NBP (10 μmol/L), then exposed to oxygen and glucose deprivation (OGD) for 8 hours as an in vitro model of ischemic stroke. Indices of anti-oxidative response, mitochondrial function and mitochondrial dynamics were evaluated.

Results: OGD suppressed cell viability, induced apoptosis and increased caspase-3 activity. NBP significantly reversed these effects. NBP prevented oxidative damage by increasing the activity of superoxide dismutase and lowering levels of malondialdehyde (MDA) and reactive oxygen species (ROS). At the same time, it increased expression of Nrf2, HO-1 and AMPK. NBP attenuated mitochondrial dysfunction by enhancing mitochondrial membrane potential and increasing the activity of mitochondrial respiratory chain complexes I-IV and ATPase. NBP altered the balance of proteins regulating mitochondrial fusion and division.

Conclusion: NBP exerts neuroprotective actions by enhancing anti-oxidation and attenuating mitochondrial dysfunction. Our findings provide insight into how NBP may exert neuroprotective effects in ischemic stroke and raise the possibility that it may function similarly against other neurodegenerative diseases involving mitochondrial dysfunction.

Keywords: ischemic stroke; mitochondrial dynamics; mitochondrial dysfunction; neuroprotective.

Figure 1

Effect of NBP on cell viability and apoptosis following OGD. Notes: PC12 cells were pretreated with different concentrations of NBP, then exposed to OGD. (A) Viability was measured in the MTT assay. (B) Apoptosis was detected using TUNEL staining. (C) Caspase-3 activity was measured using a commercial kit. Bars show mean±SD of three independent experiments. *P<0.05 vs control, ^#P<0.05 vs OGD-treated. **P<0.01 vs control, ^##P<0.01 vs OGD-treated. Abbreviations: NBP, 3-n-butylphthalide; OGD, oxygen and glucose deprivation.

Figure 2

The drug NBP protects PC12 cells and mitochondria from damage due to OGD. Notes: (A) Ultrastructure of PC12 cells and mitochondria by transmission electron microscopy. (B) Morphology of mitochondria by fluorescence microscopy after staining with Mito Tracker Green FM. Abbreviations: NBP, 3-n-butylphthalide; OGD, oxygen and glucose deprivation.

Figure 3

The drug NBP protects PC12 cells against oxidative stress induced by OGD. Notes: (A) Effect of NBP on SOD activity and MDA levels. (B) Effect of NBP on production of ROS. (C) Effect of NBP on expression of anti-oxidant proteins. **P<0.01 vs control, ^##P<0.01 vs OGD-treated. Abbreviations: MDA, malondialdehyde; NBP, 3-n-butylphthalide; OGD, oxygen and glucose deprivation; ROS, reactive oxygen species; SOD, superoxide dismutase.

Figure 4

Effect of NBP on mitochondrial membrane potential in PC12 cells after OGD. Abbreviations: NBP, 3-n-butylphthalide; OGD, oxygen and glucose deprivation.

Figure 5

Effect of NBP on the activity of MRCC I–IV and ATPase in PC12 cells after OGD. Note: **P<0.01 vs control, ^##P<0.01 vs OGD-treated. Abbreviations: MRCC, mitochondrial respiratory chain complexes; NBP, 3-n-butylphthalide; OGD, oxygen and glucose deprivation.

Figure 6

Effect of NBP on mitochondrial dynamics in PC12 cells after OGD. Notes: (A) Western blotting was used to determine relative expression levels of Mfn1, Mfn2, OPA1, Drp1 and Fis1. (B) The bars represent Mfn1, Mfn2, OPA1, Drp1 and Fis1 protein/β-ACTIN levels in NBP, OGD, NBP+OGD group compared to control. **P<0.01 vs control, ^##P<0.01 vs OGD-treated. Abbreviations: NBP, 3-n-butylphthalide; OGD, oxygen and glucose deprivation.