Matrine attenuates focal cerebral ischemic injury by improving antioxidant activity and inhibiting apoptosis in mice

Abstract

Matrine, an active constituent of the Chinese herb, Sophora flavescens Ait., and it is known for its antioxidant, anti-inflammatory and antitumor activities. It has been demonstrated that matrine exerts protective effects against heart failure by decreasing the expression of caspase-3 and Bax, and increasing Bcl‑2 levels. In this study, we aimed to determine whether these protective effects of matrine can be applied to cerebral ischemia. Following 7 successive days of treatment with matrine (7.5, 15 and 30 mg/kg) and nimodipine (1 mg/kg) by intraperitoneal injection, male Institute of Cancer Research (ICR) mice were subjected to middle cerebral artery occlusion (MCAO). Following reperfusion, the neurobehavioral score and brain infarct volume were estimated, and morphological changes were analyzed by hematoxylin and eosin (H&E) staining and electron microscopy. The percentage of apoptotic neurons was determined by flow cytometry. The levels of oxidative stress were assessed by measuring the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the total antioxidant capacity (T-AOC). Western blot analysis and immunofluorescence staining were used to examine the expression of the apoptosis-related proteins, caspase-3, Bax and Bcl-2. Our results revealed that pre-treatment with matrine significantly decreased the infarct volume and improved the neurological scores. Matrine also reduced the percentage of apoptotic neurons and relieved neuronal morphological damage. Furthermore, matrine markedly decreased the MDA levels, and increased SOD, GSH-Px and CAT activity, and T-AOC. Western blot analysis and immunofluorescence staining revealed a marked decrease in caspase-3 expression and an increase in the Bcl-2/Bax ratio in the group pre-treated with matrine (30 mg/kg) as compared with the vehicle-treated group. The findings of the present study demonstrate that matrine exerts neuroprotective effects against cerebral ischemic injury and that these effects are associated with its antioxidant and anti-apoptotic properties.

Figure 1

Structure of matrine (Mat) and oxymatrine.

Figure 2

Protective effects of matrine (Mat) against cerebral ischemic injury in mice. (A) 2,3,5-Triphenyltetrazolium chloride (TTC) staining of representative coronal sections at 24 h following reperfusion. (B) Quantitative analysis of the infarct volume at 24 h following reperfusion. (C) Quantification of neurological deficit scores at 24 h following reperfusion. Data are expressed as the means ± SEM (n=6). ^##p<0.01 vs. sham-operated group; ^*p<0.05 and ^**p<0.01 vs. vehicle-treated group.

Figure 3

Effects of matrine (Mat) pre-treatment on histological alterations in the ischemic cerebral cortex at 24 h following reperfusion (hematoxylin and eosin staining, ×400 magnification). (A) Sham-operated group. (B) Vehicle-treated group. (C) Mat 7.5 mg/kg-treated group. (D) Mat 15 mg/kg-treated group. (E) Mat 30 mg/kg-treated group. (F) Nimodipine 1 mg/kg-treated group. Arrows indicate necrotic changes with neurons having a scalloped shrunken form in the ischemic lesions.

Figure 4

Ultrastructural changes induced by cerebral ischemia and inhibition by matrine (Mat) (×3,000 magnification). (A) Sham-operated group. In the hippocampus, the nerve cell (NC) shows a normal ultrastructure. The nucleus (N), granular endoplasmic reticulum (ER), mitochondrion (M), and Golgi apparatus (G) are indicated. (B) Vehicle-treated group. In the hippocampus, the nerve cell exhibits nuclear chromatine clumping, enlargement of granular ER cisternae, increase in lysosomes (L) and cytoplasmic blebbing. The Golgi apparatus (G) and mitochondrion (M) are indicated. (C–F) Mat (7.5, 15 and 30 mg/kg)- and nimodipine-treated group, respectively. In the hippocampus, the nerve cell (NC) shows nuclear (N) chromatine clumping and slight dilatation of the granular ER cisternae and mitochondrion (M). Lysosomes (L) are indicated. In the Mat 30 mg/kg- and nimodipine-treated groups, the nerve cells (NC) had a relatively normal ultrastructure.

Figure 5

Matrine (Mat) attenuates oxidative stress following focal cerebral ischemia. (A) Effect of Mat on the content of malondialdehyde (MDA) at 24 h following reperfusion. (B) Effect of Mat on the activity of superoxide dismutase (SOD) at 24 h following reperfusion. (C) Effect of Mat on the glutathione peroxidase (GSH-Px) level at 24 h following reperfusion. (D) Effect of Mat on the catalase (CAT) level at 24 h following reperfusion. (E) Effect of Mat on the total antioxidant capacity (T-AOC) at 24 h following reperfusion. Data are expressed as the means ± SEM (n=6). ^##p<0.01 vs. sham-operated group; ^*p<0.05 and ^**p<0.01 vs. vehicle-treated group.

Figure 6

Apoptosis in the ischemic brain cortex neurons was measured by flow cytometry (quadrants 1, 2, 3 and 4 represent dead neurons, late apoptotic neurons, normal neurons and early apoptotic neurons, respectively). (A–D) Apoptotic neurons in the sham-operated group, vehicle-treated group, matrine (Mat) (30 mg/kg)-treated group and nimodipine-treated group, respectively. (E) The percentage of apoptotic neurons in the different groups at 24 h following reperfusion. Data are expressed as the means ± SEM (n=6). ^##p<0.01 vs. sham-operated group; ^*p<0.05 vs. vehicle-treated group.

Figure 7

Effects of matrine (Mat) on the expression of caspase-3. (A) Representative photomicrographs of caspase-3 immunofluorescence staining in the ischemic cortex (×400 magnification) and hippocampus CA1 region (×200 magnification). (B and C) Quantification of caspase-3 fluorescence intensity in the ischemic cortex and hippocampus CA1 region in the different groups. (D) Representative western blot of caspase-3 activation in the ischemic cortex at 24 h following reperfusion. (E) Effect of Mat (30 mg/kg) on caspase-3 activation in the cortext in mice subjected to middle cerebral artery occlusion (MCAO) at 24 h following reperfusion. Data are expressed as the means ± SEM (n=6). ^##p<0.01 vs. sham-operated group; ^*p<0.05 and ^**p<0.01 vs. vehicle-treated group.

Figure 8

Effects of matrine (Mat) on the expression of Bax. (A) Representative photomicrographs of Bax immunofluorescence staining in the ischemic cortex (×400 magnification) and hippocampus CA1 region (×200 magnification). (B and C) Quantification of Bax fluorescence intensity in the ischemic cortex and hippocampus CA1 region in the different groups. (D) Representative western blot of Bax activation in the ischemic cortex at 24 h following reperfusion. (E) Effect of Mat (30 mg/kg) on Bax activation in the cortext of mice subjected to middle cerebral artery occlusion (MCAO) at 24 h following reperfusion. Data are expressed as the means ± SEM (n=6). ^##p<0.01 vs. sham-operated group; ^*p<0.05 and ^**p<0.01 vs. vehicle-treated group.

Figure 9

Effects of matrine (Mat) on the expression of Bcl-2. (A) Representative photomicrographs of Bcl-2 immunofluorescence staining in the ischemic cortex (×400 magnification) and hippocampus CA1 region (×200 magnification). (B and C) Quantification of Bcl-2 fluorescence intensity in the ischemic cortex and hippocampus CA1 region in the different groups. (D) Representative western blot of Bcl-2 activation in the ischemic cortex at 24 h following reperfusion. (E) Effect of Mat (30 mg/kg) on Bcl-2 activation in the cortex of mice subjected to middle cerebral artery occlusion (MCAO) at 24 h following reperfusion. (F) Effect of Mat (30 mg/kg) on the Bcl-2/Bax ratio in the cortext of mice subjected to MCAO at 24 h following reperfusion. Data are expressed as the means ± SEM (n=6). ^##p<0.01 vs. sham-operated group; ^*p<0.05, and ^**p<0.01 vs. vehicle-treated group.