| ObjectiveIschemic stroke is a clinically common and frequently-occurred disease.Its prevalence, mortality and morbidity are very high. In China every year about1.2～1.5million people suffer from stroke for the first time, about0.8～1million people died by stroke. Ischemic stroke on the accounted for75%～90%.Stroke which is seriously affecting people’s normal life, has become the first or the second reason for death. With the advent of an aging society, its incidence is showing a rising trend. Besides treatment with for this disease needs a lot of money, and it is occurring on younger people. Therefore, it is significant to study the mechanism so as to help to prevent and treat it.According to Traditional Chinese Medicine (TCM) theory, Qi deficency and blood stasis are the important pathogenic factor, so the therapeutic principle is to supplement qi and activate blood circulation. This rule is the main method of prevention and treatment of the ischemic stroke, which has been approved and taken popularly, but there are some questions remaining to be answered about the experimental research on the mechanism:the research on prevention, the study on time-window treatment, the deepening of the existing observational index, etc. If the pathophysiology of the ischemic stroke and the mechanism of supplementing qi and activating blood circulation are studyied from the cell-and-molcule level, it would be helpful for TCM to prevent and treat ischemic stroke, which would make TCM more practical and operational.Buyang Huanwu Decoction has been used to treat ischemic cerebrovascular disease and its sequel because of its ability to invigorate qi and promote blood circulation to remove blood stasis. For this reason, increased attention had been paid to the research on the mechani sm of protecting the ischemic lesion. Our preliminary studies showed that the following two points:①The pre-intervention with Buyang Huanwu Decoction can reduce the Ca" concentration in cerebral cortical neurons of global cerebral ischemia and reperfusion model of rat,②it also can reduce the average opening time and probability of Ca2+-channels after ischemia and reperfusion.AMPA receptor is very important for mediating fast excitability nerve transmission, and more and more attention has been paid to its role in glutamate excitotoxicity. GluR2subunit lead to low calcium permeability due to its mRNAQ/R sites of editing, so the relative amount of AMPA receptors determines the functional properties of AMPA reptors. GluR2subunit highly expressed in various neurons can inhibit calcium inflow through AMPA receptors. In avariety of neurological injury or degenerative diseases, the GluR2expression decreases, and the calcium inflow increases, which causing neurons death, that is "the GluR2hypothesis". However, this hypothesis is still in dispute. For example:down-regulation of GluR2does not necessarily indicate cell death, also GluR2downward exists in survival neurouns in some excitotoxicity stimulation, ischemia or ischemic preconditioning. Therefore, the role of GluR2in glutamate excitotoxicity injury remains to be further investigated.During cerebral ischemia/reperfusion injury, each subunit of NMDAR occur in reversible process or shift through tyrosine, serine/threonine phosphorylation/dephosphorylation, regulating NMDAR excitotoxic injury to neurons. The latest study found that ischemia-reperfusion-induced neuron injury is not only related with the amount of Ca^" influx, but also more closely related with the ways of Ca" influx. Inflow of Ca2+y the NMDAR channel is closely related to ischemic neuron injury, but it has not been studied extensively until now.Therefore, we plans to apply the immunohistochemical and RT-PCR to study The effects of the pre-intervention with Buyang Huanwu Decotion on GluR2, NR1, NR2B in cerabral cortical neurons of ischemical/reperfusion rats’. Inve-stigating the mechanism of Buyang Huanwu Decoction regulating calcium homostasis in the protection of cerebral ischemia and reperfusion injury of neurons from the cellular, molecular and genie level would be helpful to provide theoretical basis for the prevention and treatment of the ischemic encephalopathy with this prescription, to give full play to unique advantages of its multi-target, multi-channel role, significant effect and inexpensiv-e, and to explore new ideas and methods for the research on the complex prescription.MethodsThe experiments were divided into three sections.1. The effects of Buyang Huanwu Decoction pre-intervention on AMPA receptor subunit GluR2expression in cerebral cortical neurons were observed in rats with global cerebral ischemia and reperfusion.Experimental animals:healthy adult SD rats(male and female), weighting180～220g,72rats.Experimental groups:the experiment was divided into12groups:(1the normal group;(2)the sham-operated group;(3)the model group, five groups with ischemia and reperfusion at different time points(2h,12h,24h,48h72h);(4) the herb group, five groups with ischemia and reperfusion by the Buyang Huanwu Decoction pre-intervention at time points(2h,12h,24h,48h,72h).Consulted the modified Pulsinelli’s tetra-vessel occlusion to prepare the global cerebral ischemic rats.Apply immunohistochemistry techniques2. The effects of Buyang Huanwu Decoction pre-intervention on NMDA receptor subunit NR1.NR2B expression of the cerebral cortical neurons were studied in rats with global cerebral ischemia and reperfusion.Experimental animals:healthy adult SD rats(male and female) weighting180-220g,36rats.Experimental groups:the experiment was divided into6groups:(1)the normal group;(2)the sham-operated group;(3) the model group, two groups with ischemia and reperfusion at time points(2h,24h);(4) the herb group, two groups with ischemia and reperfusion with Buyang Huanwu Decoction pre-intervention at different time points(2h,24h)Consulted the modified Pulsinelli’s tetra-vessel occlusion to prepare the global cerebral ischemic rats.Apply immunohistochemistry techniques3. The effects of Buyang Huanwu Decoction pre-intervention on NMDA receptor subunit NR1, NR2B mRNA expression in cerebral cortical neurons were observed in rats with global cerebral ischemia and reperfusion.Experimental animals, experimental groups, and the preparation of rats with the global ischemia were the same as section one.Apply RT-PCR techniquesResultsThe first experiment1. The normal group compared with the sham group, the GluR2expression was not statistically significant.2. The GluR2expression of the model group was significantly lower than the normal group, but the GluR2expression of five different time points was not statistically significant.3. The GluR2expression of the herb group was significantly lower than the normal group, however, compared with the model group at each time point, the herb group GluR2expression was significantly higher, which indicated that the Buyang Huanwu Decoction pre-intervention could inhibit the downregulation of GluR2expression induced by global cerebral ischemia and reperfusion. Especially the upregulation in12h,24h,48h,72h are higher, which are significantly different from2h, it indicated that Buyang Huanwu Decoction pre-intervention have different effects on the GluR2expression of the cerebral cortical neurons of rats during different stages.The second experiment1. The normal group compared with the sham group, the NR1, NR2B expression was not statistically significant.2.The NR1, NR2B expression of the model group was significantly higher than the normal group, which has significant difference.3. The NR1, NR2B expression of the herb group was significantly lower than the model group, which indicate that Buyang Huanwu Decoction pre-intervention can inhibit the increasement of NR1, NR2B expression of the cerebral cortical neurons of rats with global cerebral ischemia and reperfusion.The third experimentThe results of this experiment was consistent with the second experiment. Cone IusionsThe results of this thesis combined with our prelimiary research results, we hypothesize the following points:①he GluR2, NR1, NR2B play an important role of in neuron damage in global cerebral ischemia and reperfusion.②The GluR2, NR1, NR2B participate in global cerebral ischemia and reperf-usion-induced neuron-calcium overload.The reduced expression of GluR2, the increased expression of NR1/NR2B and the opening of the L-type calcium channel all mediated extracellular calcium influx, causeing intracellular calcium overload and neuron damage; Buyang Huanwu Decoction pre-intervention could inhibit the reduced expression of GluR2, the increased expression of NR1in global cerebral ischemia and reperfusion.③Buyanghuanwu decoction has positive role in preventing cerebral ischemia and reperfusion injury. |
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