| Objective: In the process of ischemic brain injury, MMP–9 brings about acute blood brain barrier damage by degrading the composition of ECM, which is one of the important mechanisms, while TIMP–1 can regulate the activity of MMP–9 by combining with MMP–9 specificaly with 1:1 proportion at the protein level. In this experiment, histopathology and immunohistochemistry were used to observe the protective effect on hippocampal neurons by benefiting qi, activating blood circulation, eliminating phlegm and dredging channels therapy and the effect on the expressions of MMP–9 protein and TIMP–1 protein in cerebral ischemia mice so as to study its action of anti-cerebral ischemic injury and a part of mechanism for providing certain experiment basis for clinical application of the method in prevention and treatment of cerebral ischemia disease.Methods: 80 healthy Kunming mice weight 30-40g were equally divided at random into 4 groups: sham group, model group, Chinese medicine treatment group and control group. Treatment group were fed with compound decoction for benefiting qi, activating blood circulation, eliminating phlegm and dredging channels (each 1ml crude containing 1.56 g) while control group were fed with Naoxuekang Solvent water pills (each 1ml contains 7mg Naoxuekang pills), sham group and model group with equivalent physiological saline, all with the dosage of 15ml/kg , once a day. On the 3rd day, 2 hours after administration, all mice were intraperitoneal-anesthetized with 10% hydration chlorine aldehyde of 350mg/kg dosage, fixed on the operating table supine, incited in the middle of the neck, and dissected muscles and blood vessels bluntly. Meanwhile, attention should be paid to protect the vagus nerve and have bilateral common carotid artery exposed. Mice of sham group only exposed bilateral common carotid artery, others were clipped bilateral common carotid artery with non-invasive micro-artery clip for 30min, and then perfusion was restored. Keep warm during and after the operation, and ensure animal spontaneous breathing, then reencaging for being raised and repeating the former feeding method after operation. 48h later, 10 mice of each group were randomly selected, given deep anesthesia with 10%chloral hydrate, and perfusion-fixed with 4% paraformaldehyde PBS, following with brain tissue removed, 1-4mm brain slice cut behind optic chiasma and fastened for 48h in 4% paraformaldehyde PBS for the purpose to observe histological grade in hippocampal CA1 area in the light microscope and counted neuronal density through conducting paraffin section and HE staining, and to detect the expression of MMP–9 and TIMP–1 protein by means of immunohistochemistry. For the remaining mice, the same approach was adopted to collect specimen on 7th day after operation and the same targets observed.Results:1 Histopathological changes in hippocampus CA1 area: (1) Postoperative 48h:The damage of hippocampus CA1 area in mice of model group (ND:184±16n/mm) compared with sham group (ND:211±12n/mm) was more obvious (P<0.05) and most of the histological grades were 1 and 2, while the hippocampus CA1 area in mice of Chinese medicine treatment group (ND:206±12n/mm) and control group (ND:204±13n/mm) had no significant damage (P>0.05).(2) Postoperative 7d: The neuronal density of hippocampus CA1 area in mice of model group (ND:87±23n/mm) compared with sham group (ND: 215±19n/mm) decreased obviously (P<0.01) and histological grade increased significantly (P<0.01),which prompted delayed neuron death serious. The damage of hippocampus in mice of Chinese medicine treatment group (ND:187±20n/mm) and control group (ND:182±21n/mm) compared with model group was lighter, and the histological grade and neuron density in CA1 area had obvious difference (P<0.01 or P<0.05). There was no significant difference between Chinese medicine treatment group and control group (P>0.05). 2 The expression of MMP–9 protein and TIMP–1 protein in hippocampus CA1 area: (1) Postoperative 48h: The expression of MMP–9 protein and TIMP–1 protein in hippocampus in mice of sham group was feeble (MMP–9: 0.1392±0.0146; TIMP–1: 0.1652±0.0384). Compared with sham group, the expression of MMP–9 protein and TIMP–1 protein in mice of model group (MMP–9: 0.2365±0.0130; TIMP–1: 0.2198±0.0196), Chinese medicine treatment group (MMP–9: 0.1971±0.0134; TIMP–1: 0.2779±0.0385) and control group (MMP–9: 0.2022±0.0164; TIMP–1: 0.2566±0.0264) all significantly enhanced (P<0.01 or P<0.05). Compared with model group, the expression of MMP–9 protein in mice of Chinese medicine treatment group and control group decreased significantly while the expression of TIMP–1 protein increased obviously (P<0.01 or P<0.05). The expression of MMP–9 protein and TIMP–1 protein between the two groups had no significant difference (P>0.05). (2) Postoperative 7d: Compared with sham group (MMP–9: 0.1482±0.0123; TIMP–1: 0.1667±0.0338), the expression of MMP–9 protein in mice of model group increased significantly (P<0.01),and the expression of TIMP–1 protein also increased without statistical significance (P>0.05). Compared with model group, the expression of MMP–9 protein in mice of Chinese medicine treatment group (MMP–9: 0.1659±0.0053; TIMP–1: 0.2618±0.0295) and control group (MMP–9: 0.1712±0.0102; TIMP–1: 0.2370±0.0259) all reduced obviously (P<0.01) while the expression of TIMP–1 protein increased distinctly (P<0.01),but there was no significant difference between the two groups (P>0.05).Conclusion: 1 Cerebral ischemia caused by clipping bilateral common carotid artery for 30min, damage of hippocampus will appear more obviously with postoperative 7d, which is characterized by histological grade rising and density of pyramidal neurons reducing. The prescription of benefiting qi, activating blood circulation, eliminating phlegm and dredging channels can significantly reduce the damage of hippocampus caused by cerebral ischemia.2 The prescription of benefiting qi, activating blood circulation, eliminating phlegm and dredging channels can obviously decrease the expression of MMP–9 protein in hippocampus CA1 area while enhance the expression of its natural inhibitor—TIMP–1 protein at 48h and 7d after cerebral ischemia reperfusion, thus reduce the degradation of ECM , decrease the damage of BBB, and inhibite apoptosis, which may be one of the important mechanisms for resisting cerebral ischemic injury though benefiting qi, activating blood circulation, eliminating phlegm and dredging channels therapies. |
PDF Full Text Request