| Objective: Through the animal experiments, it is to compare the effects of Buyanghuanwu Tang and its separate components on the protection of cerebral ischemia in hippocampus neurons, anti-oxidative stress and the regulation of apoptosis-related genes Bax, bcl-2 protein, to seek the effective component of the BYHWT against cerebral ischemia, and to discuss its structure and significance, which will provide more laboratory evidence for the research and development of new Chinese medicine for prevention and treatment of cerebral ischemic.Methods: 120 clean Kunming mice were randomized equally into 6 groups: sham group, model group, BYHWT group, BQHX group, BQTL group and HXTL group. Mice of every Chinese medicine group were fed with its corresponding Chinese medicine. Each mouse was fed to 15ml/kg dose by its weight(BYHWT group, BQHX group, BQTL group, HXTL group, equivalent to 21.45,20.10,19.35,3.45g/Kg dosage), Mice of sham group and model group were fed with the same dose of physiological saline, once a day until the experience was end. Three days later, two hours after gavage, all mice were intraperitoneal-anesthetized by 10% hydration chlorine Alde Hyde with 0.33ml/100g dose, fixed on the operating table supine, incited in the middle of the neck, dissected muscles and blood vessels bluntly, and what's more, their bilateral common carotid artery should be exposed fully. In the operation, attention should be paid to protect the vague nerve. Mice of sham group were only exposed bilateral common carotid artery, while others were clipped bilateral common carotid artery with micro-artery clip for 30min, perfusion restored. Keep warm during and after operation. Then put them back to the cage for being raised with a healthy diet in the same feeding way. 24h removed. Then the blades were used to make the brain hemisphere separated along the cerebral longitudinal fissure, the left hemisphere in cryopreservation. SOD activity and MDA content of brain tissue were observed by xanthine oxidase assay, thiobarbituric acid assay; the right hemisphere was fixed with 10% Formaldehyde solution. By means of paraffin section, immunohisto chemistry and analyse system of Motic Med 6.0 image, the expression of Bax and bcl-2 protein were detected and analysed. The remaining mice were killed on the 7th day after operation with brain tissue removed, whole brain stripped rapidly, coronary 1～4mm brain slice cut behind optic chiasma, fixed in 10% Formaldehyde solution. Paraffin section and HE staining were used to observe the histological grade of hippocampal CA1 area in the light microscope and count the neuronal density.Results: 1 Histopathological Changes in Hippocampal CA1 area on 7d after IschemiaThe damage of hippocampal CA1 area in mice of model group (most of the histological rank was 2 and 3 grade), compared with sham group (most was 0 and 1 grade), was more obvious (p<0.01), which prompted that delayed neuron death was very serious. While the hippocampal CA1 area in mice of every Chinese medicine treatment group had no significant damage; most of the histological rank was 0, 1 and 2 grade, obviously lower than the model group(p<0.05 or p<0.01), and BYHWT, BQHX and BQTL group were significantly lower than HXTL group(p<0.05 or p<0.01), but there were no significant difference among the former three groups (P>0.05). By counting the neuronal density of hippocampal CA1 we found: The neuronal density of model group (72±22n/mm) was significantly lower than sham group (211±16n/mm)(P < 0.01); BYHWT(188±19n/mm) and the separate component groups were significantly higher than model group(p<0.05 or p<0.01); Among the Chinese medicine treatment groups, BYHWT, BQHX (175±20n/mm) and BQTL(178±16n/mm) group were more higher than HXTL group (119±17n/mm)(p<0.01), but there were no significant difference among the former three groups (P>0.05).2 Changes of SOD Activity and MDA Content in Brain Tissue 24h after IschemiaThe SOD activity in brain tissue of model group (SOD: 23.39±2.60 U/mgport, MDA: 1.204±0.157 nmol/mgprot. The following as the same), compared with sham group (8.01±1.66), decreased obviously (P<0.01), and the MDA content in brain tissue increased significantly. Compared with model group, the SOD activity of BYHWT group(SOD: 22.55±2.37, MDA: 1.297±0.199) and in all its separate groups increased significantly, and the MDA content decreased obviously(p<0.05 or p<0.01). BYHWT group, BQHX group(SOD: 21.28±1.92, MDA: 1.352±0.146) and BQTL group(SOD: 21.37±2.32, MDA: 1.321±0.152) compared with HXTL group increased obviously and histological grade decreased significantly (P<0.01), but there were no significant difference among the former three groups (P>0.05).3 Expression of Bax and Bcl-2 Protein in Hippocampal CA1 Area 24h after IschemiaThe expression of Bax (0.1438±0.0145. AOD, the following as the same) protein and Bcl-2 (0.2008±0.0209) protein in hippocampal CA1 area in mice of sham group was feeble. Compared with sham group, the expression of Bax protein(0.3883±0.0310) in model group enhanced significantly(P<0.01), but the expression of Bcl-2 protein (0.2177±0.0316) enhanced so slightly that there were no significant difference between them(P>0.05). Compared with model group, the expression of BYHWT group (Bax: 0.2035±0.0212, Bcl-2: 0.3807±0.032) and all its separate groups Bax all weakened obviously, the expression of Bcl-2 enhanced obviously(P<0.01). Compared with HXTL group(Bax: 0.3499±0.0294, Bcl-2: 0.2779±0.0331), the expression of Bax in BYHWT, BQHX(Bax: 0.2187±0.0250, Bcl-2: 0.3616±0.0258) and BQTL group(Bax: 0.2188±0.0211, Bcl-2: 0.3629±0.0290) weakened obviously, the expression of Bcl-2 enhanced obviously(P < 0.01), but there were no significant difference among the former three groups (P>0.05).Conclusions: 1 7 days reperfusion after cerebral ischemia for 30min resulted in the damage of hippocampus, which was characterized by histological grade rising in the damage hippocampus and density of neurons reducing. BYHWT and the separate components could reduce the damage of hippocampus caused by cerebral ischemia to some extent and the function of BYHWT was the most obvious; the effect of BQHX and BQTL was superior to HXTL, which indicated that Qi-reinforce drug may be the most effective components of BYHWT at anti-cerebral ischemia injury.2 BYHWT and the separate components could increase the SOD activity of ischemic brain tissue, reduce the MDA content to some extent, and the function of BYHWT was the most obvious; the effect of BQHX and BQTL was superior to HXTL, which indicated that BYHWT could significantly enhance the ability of ischemic brain tissue at anti-oxidative stress injury, reduce the oxidation of noxious stimulation of neurons, and play a protective role in neurons, during which Qi-reinforce drug might play the most important role.3 BYHWT and the separate components could, to some extent, decrease the expression of brain protein of Bax and increase the expression of Bcl-2 in ischemic hippocampus to inhibit apoptosis and reduce the DND, which may be one of the important mechanisms that BYHWT and the separate components resisted cerebral ischemic injury. The effect of BQHX and BQTL were better than HXTL, which indicated that Qi-reinforce drug might be the most effective components at anti-cerebral ischemia jury.
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