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

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.