Protective multi‑target effects of DL‑3‑n‑butylphthalide combined with 3‑methyl‑1‑phenyl‑2‑pyrazolin‑5‑one in mice with ischemic stroke

Abstract

DL‑3‑n‑butylphthalide (NBP) and 3‑methyl‑1- phenyl‑2‑pyrazolin‑5‑one (edaravone) are acknowledged neuroprotective agents that protect against ischemic stroke. However, the underlying mechanisms of a combination therapy with NBP and edaravone have not yet been fully clarified. The aim of the present study was to explore whether the co‑administration of NBP and edaravone had multi‑target protective effects on the neurovascular unit (NVU) of mice affected by ischemic stroke. Male C57BL/6 mice were randomly divided into the following three groups: i) Sham operation control, ii) middle cerebral artery occlusion (MCAO) and reperfusion, iii) and MCAO/reperfusion with the co‑administration of NBP (40 mg/kg) and edaravone (6 mg/kg) delivered via intraperitoneal injection at 0 and 4 h after reperfusion (NBP + edaravone). After ischemia and reperfusion, infarct volumes and neurological deficits were evaluated. The immunoreactivity of the NVU, comprising neurons, endothelial cells and astrocytes, was determined using immunofluorescence staining of neuronal nuclei (NeuN), platelet and endothelial cell adhesion molecule 1 (CD31) and glial fibrillary acidic protein (GFAP). Western blotting was used to detect the expression levels of apoptosis‑related proteins. The infarct volume, neurological function scores and cell damage were increased in the MCAO group compared with the sham operation group. Furthermore, the MCAO mice had reduced NeuN and CD31 expression and increased GFAP expression compared with the sham group. By contrast, the NBP + edaravone group exhibited reduced cell damage and consequently lower infarct volume and neurological deficit scores compared with the MCAO group. The NBP + edaravone group exhibited increased NeuN and CD31 expression and decreased GFAP expression compared with the MCAO group. Furthermore, the expression levels of Bax and cleaved caspase‑3 in the NBP + edaravone group were decreased significantly compared with the MCAO group, while the expression levels of Bcl‑2 and mitochondrial cytochrome c were increased. In conclusion, the results of the present study demonstrated that NBP and edaravone effectively prevented ischemic stroke damage with multi‑target protective effects. In addition, NBP + edaravone may be a promising combination therapy for ischemic stroke.

Keywords: 3‑methyl‑1‑phenyl‑2-pyrazolin‑5‑one; NBP; NVU; combination therapy; ischemic stroke.

Figure 1.

NBP combined with edaravone reduces infarct volumes and neurological deficit scores. Effects of NBP combined with edaravone on infarct volumes and neurological deficit scores in mice following 1 h of MCAO and 24 h of reperfusion. (A) Representative 2,3,5-triphenyltetrazolium chloride staining images of the brain slices. (B) Percentage of infarct volumes in the whole brain (n=4 for each group). (C) Neurological deficit scores in the groups (n=4 for each group). **P<0.01 vs. Sham; ^#P<0.05, ^##P<0.01 vs. MCAO. NBP, DL-3-n-butylphthalide; edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one; MCAO, middle cerebral artery occlusion.

Figure 2.

Morphological cell changes in the three groups. Representative images of (A) hematoxylin-eosin staining and (B) Nissl staining in the sham operation, MCAO and NBP + edaravone groups (n=3 in each group). Magnification, ×200. MCAO, middle cerebral artery occlusion; NBP, DL-3-n-butylphthalide; edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one.

Figure 3.

GFAP immunofluorescence staining to evaluate astrocyte immunoreactivity. The merged image indicates that the structure of GFAP was closely connected with the nucleus stained by DAPI to demonstrate that this was an astrocyte. Immunofluorescence staining for GFAP (arrows suggest the localization of GFAP) in the ischemic cortex of mice following 1 h of MCAO and 24 h of reperfusion. (A) Representative GFAP immunofluorescence staining images of brain slices. Magnification, ×100. (B) Quantification of GFAP expression. The GFAP expression in the MCAO group was stronger compared with that in the sham operation group, while it was significantly decreased in the NBP combined with edaravone group compared with that in the MCAO group (n=3 in each group). ***P<0.001. GFAP, glial fibrillary acidic proteins; MCAO, middle cerebral artery occlusion; NBP, DL-3-n-butylphthalide; edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one.

Figure 4.

NeuN immunofluorescence staining to appraise neuron immunoreactivity. Immunofluorescence staining for NeuN in the ischemic cortex of mice following 1 h of MCAO and 24 h of reperfusion. (A) Representative NeuN immunofluorescence staining images of brain slices. Magnification, ×100. (B) Quantification of NeuN expression. NeuN expression in the MCAO group was reduced compared with that in the sham operation group, while NBP combined with edaravone significantly increased NeuN expression compared with that in the MCAO group (n=3 in each group). *P<0.05, ***P<0.001. NeuN, neuronal nuclei; MCAO, middle cerebral artery occlusion; NBP, DL-3-n-butylphthalide; edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one.

Figure 5.

Relationship between CD31 and vascular endothelial cell immunoreactivity. Immunofluorescence staining for CD31 (arrows suggest the localization of CD31) in the ischemic cortex of mice following 1 h of MCAO and 24 h of reperfusion. (A) Representative CD31 immunofluorescence staining images of brain slices. Magnification, ×100. (B) Quantification of CD31 expression. CD31 expression was significantly decreased in the MCAO group compared with the sham operation group, while NBP combined with edaravone significantly increased CD31 expression compared with both the sham and the MCAO groups (n=3 in each group). *P<0.05, ***P<0.001. CD31, platelet and endothelial cell adhesion molecule 1; MCAO, middle cerebral artery occlusion; NBP, DL-3-n-butylphthalide; edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one.

Figure 6.

NBP combined with edaravone increases the expression levels of PSD95, SYP and ZO-1. Expression levels of (A) PSD95, (B) SYP and (C) ZO-1 in the different groups (n=3 in each group). **P<0.01, ***P<0.001 and ****P<0.0001. MCAO, middle cerebral artery occlusion; NBP, DL-3-n-butylphthalide; edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one; PSD95, post synaptic density protein 95; SYP, synaptophysin; ZO-1, zonula occludens-1.

Figure 7.

Apoptosis-related protein expression. NBP combined with edaravone inhibited apoptosis. Expression levels of (A) Bax, (B) Bcl-2, (C) cleaved caspase-3 and (D) Cyt-c in the different groups (n=3 in each group). Western blot analysis of Cyt-c was performed only for the mitochondrial fraction. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MCAO, middle cerebral artery occlusion; NBP, DL-3-n-butylphthalide; edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one; Cyt-c, cytochrome c.