Neuroprotective effect of a formula, moschus combined with borneolum synthcticum, from traditional chinese medicine on ischemia stroke in rats

Abstract

Moschus compatible with borneolum synthcticum is a well-known herb pair in Traditional Chinese Medicine and the present study aims to assess the neuroprotective effect of a formula composed of this herb pair on ischemia stroke in rats. The middle cerebral artery occlusion model of focal cerebral ischemia in rat was performed by using intraluminal suture method. The behavioral scores, infarct volume, and neuron ultrastructure of model and formula-treated rats were investigated after the 2 h of ischemia and 24 h of reperfusion. Meanwhile the expression levels of caspase-3, caspase-9, Bcl-2, and Bax were measured by western blot analysis. The formula treatment showed obvious neuroprotective effect according to significant decrease of the neurological scores (P < 0.01) and the infarct volumes (P < 0.05) when compared to the MCAO group. We also observed that this formula had antiapoptosis activity on neuron cell under electron microscope. Furthermore, our result supported the idea that pro- and postadministration of this formula had an antiapoptosis effect by decreasing remarkably the expression of caspase-3 and caspase-9 (P < 0.05) as well as increasing significantly the ratio of Bcl-2 to Bax (P < 0.01). All evidences demonstrated the neuroprotective effect of this formula on ischemia stroke due to decrease of brain infract volume and modulation of the expression of apoptosis-related proteins.

Figure 1

The major components of the formula were determined by GC-FID method as follows. Helium at a flow rate of 1.1 mL/min was used as the carrier gas. The initial oven temperature was 80°C, followed by a ramp to 180°C at 20°C/min, and then ramped to 280°C at 30°C/min (held at 280°C for 5 min). The injection port and detector temperatures were at 280°C, respectively. The hydrogen and air flow rates in FID 40 and 430 mL/min, respectively. (a) Was the GC-FID chromatogram of the standard references mixed with muscone and borneol. (b) Was the GC-FID chromatogram of the tested formula composed of moschus compatible with borneolum synthcticum.

Figure 2

The comparison on the protective effects of treatments on the neurological deficits and the infarct volume after ischemia stroke. The rats were administrated intraperitoneally by the tested formula drug at 30 min before ischemia and at 2 h, 12 h, and 24 h after ischemia. After 2 h of ischemia and 24 h of reperfusion, the neurological scores were evaluated according to a graded scoring system described in the method in Section 2.3, and the infarct volume was calculated by the method described in Section 2.4. (a) Represented the different neurological deficits scores of sham group, MCAO group, and formula-treated group; (b) was the representative images of TTC-staining brains from MCAO group and the formula-treated group; the normal brain tissue displayed the color of rose red; meanwhile the infarct area displayed a color of pale white; (c) showed the different infarct volumes from rats of sham group, MCAO group, and formula-treated group. Bars represent means ± SEM of six rats; *significant difference at P≦0.05. **Remarkably significant difference at P≦0.01.

Figure 3

The results of dose-dependent and window time experiments: (a) the MCAO rats were administrated intraperitoneally with the prepared formula drug at high dose of 15 mL/kg body weight, moderate dose of 10 mL/kg, and low dose of 5 mL/kg. (b) The MCAO rats were administrated intraperitoneally 10 mL/kg at 2 h, 4 h, 6 h, and 9 h of ischemia. *Significant difference at P≦0.05 and **remarkably significant difference at P≦0.01.

Figure 4

The representative images of neurons in MCAO rats' brains from different groups under electron microscope (×12 k). (a) Was a representative image of neuron cell from sham group, (b) was an representative image of neuron cell from MCAO group, and (c) was a representative image of neuron cell from formula-treat group.

Figure 5

The comparison on the expression levels of caspase-3 and caspase-9 in the brain tissue after ischemia. (a) Was the representative graphs of caspase-3 and caspase-9, and β-actin was used as loading control. (b) Showed quantitative comparison on ratio of caspase-3 to β-actin among different groups; (c) showed quantitative comparison on ratio of caspase-9 to β-actin among different groups. Bars represent means ± SEM of eight rats. *Significant difference at P≦0.05. **Remarkably significant difference at P≦0.01.

Figure 6

The comparison on the expression levels of Bcl-2 and Bax in the brain tissue after ischemia. (a) Was the representative graphs of Bcl-2 and Bax, and β-actin was used as loading control. (b) Showed quantitative comparison on ratio of Bcl-2 to β-actin among different groups, (c) showed quantitative comparison on ratio of Bax to β-actin among different groups, and (d) was quantitative comparison on ratio of Bcl-2 to Bax. Bars represent means ± SEM of eight rats. *Significant difference at P≦0.05. **Remarkably significant difference at P≦0.01.