| ObjectiveThe cerebrovascular disease, which nowadays has become the third cause of death and the first cause of disability, severely harms the human health. While the aging society is coming, its morbidity is obviously on the rise. Therefore, it is significant to study the mechanism of the ischemic neuronal injury to prevent and cure the disease.The ischemia can sharply interfere with the neuronal Ca2+ homeostasis, but the abnormity of the neuronal Ca2+ message plays the important role in the ischemic neuronal injury. Therefore, to study the dynamic alternation of the Ca2+ concentration in the neuron after the ischemia at different time points is of the importance to the experimental research and the clinical treatment of the ischemic neuronal injury.Simultaneously, to study the opening and closing of the Ca2+ L-channel will be helpful to reveal the mechanism of the ischemic neuronal injury and provide the favorable evidence for the prevention and treatment.The cerebral ischemia pertains to the stroke in Traditional Chinese Medicine(TCM). Qi deficiency and blood stasis are the important pathogenic factors, 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 is approved and taken seriously, but there are some questions remaining to be answered about the experimental research on the mechanism of action: the research on prevention, the study on time-window treatment, the deepening of the existing observational index, etc. To intensively study the pathophysiology of the ischemic stroke and the mechanism of action of supplementing qi and activating blood circulation from the cell-and-molecule level gives play to superiority to prevent and treat the ischemic stroke with TCM, and is to make this method more practical and operational.Buyang Huanwu Tang(补阳还五汤), having the function of invigorating qi and promoting blood circulation to remove blood stasis, acquired the outstanding effects while it was applied to cure the ischemic cerebrovascular disease and its sequel. For this reason, increased attention had been paid to the research on the mechanism of protecting the ischemic lesion. As for theexperimental research on the mechanism of action, the possible effects of Buyang Huanwu Tang on Ca2+ were discussed. However, the report about the research on the effects of Buyang Huanwu Tang on Ca2+ in terms of monoplast was not yet seen.The complex prescription of the Chinese meteria medica is the important part of TCM, and is the main style and means of the clinical medication of TCM. At present, the complex prescription of the Chinese meteria medica has been a common area of research from the general level, and the single Chinese drug from the cell-and-molecule level. The complex prescription is prominent in the application of the Chinese medicine. Many Chinese drugs always have the synergistic effects and the compound has the complicated internal metabolism. The study on the single drug is not enough to reveal the mechanism of action of the complex prescription. Therefore, it is more significant to intensively study the mechanism of action of the complex prescription of the Chinese medicine from the cell-and-molecule level.To sum up, the research plans to apply the patch clamp and the ion-image analytical technique to simultaneously study the effects of Buyang Huanwu Tang on the Ca2+ L-channels and the intracellular Ca2+ concentrations of the ischemic rats' cerebral cortical neurons, intensively discuss the mechanism that the abnormity of the neuronal Ca2+ message joins in the ischemic neuronal injury and the molecular mechanism of cerebral anti-ischemia with Buyang Huanwu Tang, provide the theoretical basis for the prevention and treatment of the ischemic encephalopathy with this prescription, and explore new ideas and methods for the research on the complex prescription.MethodsThe experiment was divided into two sections.1) Changes of the free-Ca2+ concentrations of the cerebral cortical neurons of rats with ischemia and reperfusion and the effects of Buyang Huanwu Tang on the free- Ca2+ concentrations at different time points:Laboratory animals: health adult SD rats(male and female) weighting 200-250g Laboratory groups: the experiment was divided into 11 groups: (1)the sham-operated group; (2) five groups with ischemia and reperfusion at time points(2h, 12h, 24h, 48h, 72h); (3)five groups with ischemia and reperfusion preintervened by the Buyang Huanwu Tang at time points(2h, 12h, 24h, 48h, 72h);Consulted the modified Pulsinelli's tetra-vessel occlusion to prepare the global cerebral ischemic rats.The rat's cerebral cortical cell was acquired in the acute separation method. And the Ca2+ concentration of a single neuron was tested by the Ca2+ image-forming system. 2) Changes of Ca2+ L-channels of the cerebral cortical neurons of rats with ischemia and reperfusion and the effects of Buyang Huanwu Tang on the Ca2+ L-channels at different time points:The laboratory animals and groups, the preparation of rats with the global ischemia, and the acute separation of the neuron were the same as section one.Records and analyses of the single-channel electricity: the recording single-channel current was amplified by the EPC-9 patch-clamping amplifier(the feedback resistancewas over 50GΩ; the high-pass filtering was 10KHz,and the low was 2.9KHz), and collected into the computer by Pulsefit+Pulse(the sampling frequency was 50KHz and the sampling time was 10s). It was tested by the analytical softwareTAC that the resolution of the channel's opening and closing was 0.2ms. Fifty percent of the electrical amplitude at some recording voltage was considered as the transition to judge the channel's opening and closing. The acquired data was statistically processed by the softwareTACFIT. The channel opened at the single stage or the multi stage while being recorded, but the single-stage opening was chosen to analyse the temporal data. There were analytical indexes at each clamping voltage-the average opening time and the opening probability.Statistical process of the data: all the data were indicated by Mean Value(MV)± Standard Deviation(SD). They were processed in the single-variable analysis of variance and the paired T test(the significant level was 0.05).Results1) Changes of the Ca2+ concentrations in the cerebral cortical neurons of rats with global cerebral ischemia and reperfusion at different time points:At 2h after reperfusion, the Ca2+ concentration was increasing. This group was in significant difference with the sham-operated group.At 12h, it was dropping. Compared with the sham-operated group, there was no significant difference.At 24h, it was increasing again. A significant difference was found between this group and the sham-operated group.At 48h, compared with the sham-operated group, there was a significant difference.At 72h, it was dropping. Compared with the sham-operated group, there was a significant difference.2) Effects of Buyang Huanwu Tang on the Ca2+ concentrations of global cerebral cortical neurons of rats with ischemia and reperfusion at different time points:In the sham-operated group, the Ca2+ concentration in the rat preintervened by Buyang Huanwu Tang had a significant drop compared with the rat without intervention.At 2h after reperfusion, the Ca2+ concentration in this group was obviously increasing compared with the sham-operated group; but compared with the model group, it was significantly dropping.At 12h, the Ca2+ concentration in this group was obviously increasing compared with the sham-operated group; but compared with the model group, there was no significant difference.At 24h, 48h, and 72h, the Ca2+ concentrations in these groups were obviously increasing compared with the sham-operated group; but compared with the model group, they were significantly dropping.It showed that the preintervention by Buyang Huanwu Tang had the definite effect on dropping the Ca2+ concentration of the global cerebral cortical neuron of the rat with ischemia and reperfusion. Especially, the effect was more significant when the Ca2+concentration reached the peaks(at 2h and 24h after reperfusion).3) Changes of the average opening time of Ca2+ L-channels after ischemia and reperfusion at different time points:At different time points, the average opening times of Ca2+ L-channels after ischemia and reperfusion were higher than the normal group.At 2h, 12h, and 24h, the average opening times of Ca2+ L-channels with the high plateaus had significant differences compared with the sham-operated group.At 48h and 72h, both the average opening times were sharply increasing and there were significant differences compared with the sham-operated group, which showed that the times respectively reached the second peaks.4) Effects of Buyang Huanwu Tang on the Ca2+ L-channels of the cerebral cortical neurons of rats with ischemia and reperfusion at different time points:At different time points, the average opening times of Ca2+ L-channels in the global cerebral ischemic rats preintervened by Buyang Huanwu Tang were higher than the sham-operated group.At 72h, the time after reperfusion with Buyang Huanwu Tang was obviously lower than the niodel group, which showed that the preintervention by Buyang Huanwu Tang had the definite effect on dropping the average opening time of the Ca2+ L-channel in the global cerebral cortical neuron of the rat with ischemia and reperfusion. 5) Changes of the opening probability of the Ca2+ L-channels of the rats' cerebral cortical neurons after reperfusion at different time points:At 2h after reperfusion, the opening probability was obviously higher than the sham-operated group.At 12h, it was dropping and in no significant difference with the sham-operated group.At 24h, it was sharply increasing to the peak and in significant difference with the sham-operated group.At 48h, it was gradually dropping and in significant difference with the sham-operated group.At 72h, it was dropping to the level that there was no significant difference with the sham-operated group.The results suggested that the opening probability of the Ca2+ L-channel in the cerebral cortical neuron of the rat with global ischemia and reperfusion reached two peaks and the second peak was higher than the first one.6) Effects of Buyang Huanwu Tang on the opening probability of the Ca2+ L-channels of the rats' cerebral cortical neurons after reperfusion at different time points:At 2h, the opening probability was in no significant difference with the sham-operated group; but compared with the model group, it was significantly dropping.At 12h, the opening probability was in significant difference with the sham-operated group; but compared with the model group, there was no significant difference.At 24h, the opening probability was in significant difference with the sham-operated group; but compared with the model group, there was no significant difference.At 72h, the opening probability was significantly dropping compared with the sham-operated group; it was in significant difference with the model group.It showed that the preintervention by Buyang Huanwu Tang had the definite effect on dropping the opening probability of the Ca2+ L-channels of the rats' cerebral cortical neurons after reperfusion. Especially, the effect on drop was more significant when the probability reached the peak(at 2h after reperfusion).Conclusions1) The changes of the Ca2+ concentrations in the cerebral cortical neurons of rats with global cerebral ischemia and reperfusion at different time points had two transitions: the Ca2+ concentration reached the first peak at 2h after ischemia and reperfusion, dropped to the normal level at 12h, increased into the second peak at 24h which was much higher than the first one, and dropped again at 48h and 72h but was still higher than the normal level.2) The preintervention by Buyang Huanwu Tang could drop the Ca2+ concentration of the global cerebral cortical neurons of rats with ischemia and reperfusion at all time points except 12h, and obviously inhibit the two Ca2+ accumulations after ischemia and reperfusion. It was one of the main mechanisms of the cerebral anti-ischemia with Buyang Huanwu Tang to drop the Ca2+ concentration of the global cerebral cortical neurons of the rats with ischemia and reperfusion at different time points.3 ) The average opening time of Ca2+ L-channels of the global cerebral cortical neurons of the rats with ischemia and reperfusion at different time points(2h, 12h, 24h, 48h, 72h) had two peaks: the first one appeared at 2h, 12h and 24h after reperfusion and the second one which was higher than the first one appeared at 48h and 72h. The two climaxes of the opening probability of the Ca2+ L-channels in the rats' cerebral cortical neurons also appeared: the first one which was higher than the normal group was at 2h after reperfusion, but at 12h it dropped approximately to the normal level; the second one appeared at 24h.4) The changes of the average opening time and probability of Ca2+ L-channels in rats withischemia and reperfusion at different time points suggested that the mechanisms of the effects of the ischemic injury on Ca2+ L-channels were different after ischemia and reperfusion at different time points, that was to say that the effects on the availability and the opening of Ca2+ L-channels were different. The effects were probably controlled by the oxido-reduction of the oxidant and reductant, the phosphorylation and dephosphorylation of the phosphate, and the protein kinase.5) Buyang Huanwu Tang dropped the opening time and probability of Ca2+ L-channel in theneuron with ischemia and reperfusion. The effects of significantly dropping the opening probability of Ca2+ L-channel after ischemia and reperfusion at 2h had received obvious attention. And the first calcium overload also appeared in the cerebral cortical neuron at 2h.6) The inhibition of Buyang Huanwu Tang to the second Ca2+ accumulation might be relativeto the effects of Buyang Huanwu Tang on the Ca2+ storeroom, and might be the follow-up result of the inhibition of Buyang Huanwu Tang to the first Ca2+ accumulation. However, the specific mechanisms about them remained to be intensively discussed.
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