Cerebrovascular Protective Effect of Boldine Against Neural Apoptosis via Inhibition of Mitochondrial Bax Translocation and Cytochrome C Release

Abstract

BACKGROUND In the present study, we explored the protective effect and mechanism of action of boldine (BOL) against neural apoptosis, which is a mediator of TBI. MATERIAL AND METHODS The effect of BOL on mitochondrial and cytosol proteins of extracted from cerebral cortical tissue of mice was evaluated. The grip test was used to assess the neurological deficit and brain water content of the subjects after administration of BOL to assess its effect on SOD, GSH, and MDA activity in brain ischemic tissues. To further confirm the effect of the BOL, the histopathological analysis and morphology of neurons were studied by Nissl staining. The effect of BOL against TBI-induced neural apoptosis by immuno-histochemistry and Western blotting assay were also studied. RESULTS BOL showed significant improvement against TBI in a dose-dependent manner. In the BOL-treated group, the apoptotic index was significantly reduced, but the level of caspase-3 was greatly diminished. Additionally, the level of the Bax in mitochondria (mit) and cytosol was elevated in the TBI-treated group as compared to the sham group. Further BOL at the test dose causes significant reduction in the level of mitochondrial MDA together with increase in SOD activity as compared to the TBI alone group. CONCLUSIONS BOL showed a cerebroprotective effect against TBI by attenuating the oxidative stress and the mitochondrial apoptotic pathway. It also inhibited mitochondrial Bax translocation and cytochrome c release.

Figure 1

Effect of BOL on the neurological function of treated and un-treated group. Data are presented as mean ±SEM. * p<0.05 vs. sham group; ^# p<0.05; ^## p<0.01 vs. TBI alone group.

Figure 2

Effect of BOL (30 mg/kg) on the neuronal apoptosis followed by TBI insult. (A) Nissl staining of the cortical tissue under different treatments; (B) Quantitative scoring of neuron survival. Data are presented as mean ±SEM. ** p<0.01 vs. sham group; ^# p<0.05 vs. TBI alone group.

Figure 3

Effect of BOL (30 mg/kg) on apoptotic index as determined by TUNEL assay. (A) Immunohistochemistry of the apoptotic cells; (B) Apoptosis index in the cortex (%). Data are presented as mean ±SEM; ** p < 0.01, vs. sham; ^## p<0.01 vs. TBI alone group.

Figure 4

Effect of BOL (30 mg/kg) as determined by Western blot analysis of the (A) expression of caspase-3 following TBI. (B) and (C) cleaved caspase-3 expression in cortical neural cells. Data represent the mean ±SEM. ** p<0.01 vs. sham; ^# p<0.05 vs. TBI alone group.

Figure 5

Effect of BOL (30 mg/kg) on proapoptotic protein expression as determined by Western blot analysis. (A, B) the expression of Bax and cytochrome c (Cyt c) in the ipsilateral cortex, respectively; (C–F) relative expression of Bax and cytochrome in mitochondria (mit) and cytosol. Data represent the mean ±SEM. ** p<0.01; *** p<0.001 vs. sham; ^# p<0.05 vs. TBI alone group.

Figure 6

Effect of BOL (30 mg/kg) on oxidative stress. (A) MDA activity; (B) SOD level. Data represent the mean ±SEM. ** p<0.01; *** p<0.001 vs. sham; ^# p<0.05 vs. TBI alone group.