| BackgroundsVascular dementia (VD) is the second most common form of dementia after Alzheimer’s disease (AD).VD is a syndrome includeing Severe cognitive impairment caused by a series of cerebral vascular factors(ischemic stroke, hemorrhagic stroke, etc). In China, the prevalence rate of VD was1.1%～3%, the annual incidence rate in5-9/1000, and the incidence rate increased year by year. VD not only seriously affects the life quality of the patients, but also caused heavy economic burden on society and family. The prevention and treatment of vascular dementia has become a serious medical and social problem.At present,VD is one of the dementia type which has the hope to get prevention and treatment,but the pathogenesis of vascular dementia is not clear,and the research in this area has become a research focus.Hippocampus is an important functional area which has close relationship with the human learning and memory, and it has susceptibility to ischemia hypoperfusion injury.The apoptosis of hippocampal neurons plays an important role in the pathogenesis of vascular dementia. The study found that decreased Bcl-2, increased the expression of Bax could activate Caspase-3and p53, consequently induce neuronal apoptosis in rat hippocampus.Bcl-2and Caspase-3are two important regulation molecular in apoptosis, overexpression of Bcl-2can effectively inhibit caspase-3activation and apoptosis.Activation of the p38MAPK (mitogen-activated protein kinase) signaling pathway by hypoxia may initiate neuronal apoptosis, leading to the functional deficits of VaD, P38MAPK signaling pathway can affect apoptosis by regulating the expression of Bcl-2, Bax and Caspase-3. At present, SB202190, the specific inhibitor of p38MAPK alleviate cerebral ischemia reperfusion injury were reported. No previous study has examined the effect of SB202190for sustaining hippocampus-dependent spatial memory or the effects of this inhibitor on the expression of apoptotic regulators under chronic ischemia, especially the expression of Bcl-2, Caspase-3.To these ends, we examined the effects of SB202190on hippocampal neuron apoptosis, Bcl-2and caspase-3expression, and p38MAPK phospho-activation in a rat model of VaD by Western blotting, immunofluorescence and confocal laser scanning microscopy, and tested whether p38MAPK inhibition can rescue deficits in hippocampus-dependent Morris water maze spatial learning and memory. Our results indicate that blockade of the p38MAPK signaling pathway can indeed protect hippocampal neurons against chronic ischemic injury and rescue spatial learning and memory deficits, at least in part by suppressing caspase-3-dependent apoptosis. Part1The making of vascular dementia rat model and the effect of P38MAPK inhibitor on the behavior test of vascular dementia ratsVascular dementia (VD) is a common cause of senile dementia, learning and memory function as the advanced neural physiological activity in the brain, has become important in experimental vascular dementia animal model.The hippocampus involved in learning and memory function, the advanced nerve activity. The hippocampus is very sensitive to ischemia and hypoxia, especially the chronic cerebral hypoperfusion. Previous experiments have confirmed that the ischemia-reperfusion of brain could damage the hippocampus,and lead to learning and memory impairment.Chronic cerebral hypoperfusion is the main cause of vascular dementia, and the making of vascular dementia experiment animal model by two vessels method is recognized the classical method at home and abroad.In our experiment the rat in sham operation group only been isolated bilateral common carotid artery without ligation. Morris water maze was used in the experimental animal behavior experiments, the hidden platform in escape latency denote the animal’s spatial memory, learning ability; exploring experimental in Morris water maze is maintained to the test animal memory. We use two vessel method to establish vascular dementia rat model, the effects of P38MAPK inhibitors on the ability of learning and memory of vascular dementia rats.Objective and methodsThis experiment used the bilateral common carotid artery ligation caused by chronic ischemia injury of rat brain tissue, and through behavior tests were carried out to determine the existence of learning and memory disorders, make the model of vascular dementia rats, sham operation group only isolated bilateral common carotid artery ligation and normal control is not, as the two blood tube method VD model. The intracerebroventricular injection way to give P38MAPK inhibitor SB202190, affect learning and memory from the7day after the model was tested by Morris water maze in rats of each group.ResultOne week after ICV injection, hippocampus-dependent spatial learning was examined by the MWM hidden platform trail. On each of three testing days, the deficit in spatial learning as measured by escape latency was significantly longer in the model group than the sham-operated group (all P<0.01), suggesting successful induction of hippocampal deficits. Latencies were significantly lower in rats injected with SB202190compared to the model group on each day of testing (all P<0.01). indicating spatial learning, whilst latency in the VaD model group was not significantly reduced by training (from82.71±8.27s to77.74±6.33s, P>0.05). Escape latencies in the SB202190group decreased from48.72±7.01s to40.34±2.46s (P<0.05), indicating spatial learning improvementOn day4, the hidden platform was removed from the MWM in order to conduct a probe test of spatial memory. sham operation group rats remain platform quadrant time was35.21±3.78s, SB202190group was26.45±4.66s,18.67±5.39s in model group, there were significant differences between the SB202190group and sham operation group and model the residence time of the original platform quadrant group (P<0.01); rats crossing the original platform times had significant difference (P<0.01);there was no significant difference between the speed of the rats in each group (P>0.05).ConclusionIn this experiment, the platform escape latency of each rats was recorded and compared, in the first day, the escape latency of VDmodel group was significantly longer than that in sham operation group, indicate learning ability of fake operation group rats was better than the model group, the learning ability of VDmodel group by ligation of bilateral common carotid artery was significantly decreased, which confirmed the success of VD model. Furthermore after SB202190treatment, escape latent period of SB202190group rat is shortened obviously, the original platform quadrant time was significantly prolonged, indicate the learning ability of rats after SB202190treatment improved. SB202190, one of the P38MAPK inhibitors, which has definite effect on improve the learning memory ability in vascular dementia rats model. Part2The effect of P38MAPK inhibitor on P38MAPK phosphorylation、cell apoptosis、and the expression of Bcl-2、Caspase-3in hippocampus of vascular dementia ratApoptosis as a basic biological phenomena in cell, is a multi gene controlled process.Bcl-2and Caspase-3are two important member in of apoptosis molecules family, especially the function in the regulatiing on brain cell apoptosis has been widely confirmed. Studies have shown that the expression of Bcl-2is closely related to cell survival, and increase the expression of Bcl-2in brain may reduce cerebral infarct size and has protective effects on neural cells. Caspase-3is a downstream regulated protein of Bcl-2, also is the initial factor of apoptosis. The overexpression of Bcl-2can effectively inhibit the activation of Caspase-3and the apoptosis.P38MAPK signal transduction pathway plays an important role in apoptosis. Phosphorylated p38MAPK,the activated form can regulate gene transcription, induce the expression of specific genes, cause cell proliferation、differentiation、apoptosis and cytokine synthesis, and induce p38-Caspase-3apoptosis pathway. The inhibition of neuronal apoptosis after ischemia by SB202190may be the multi-channel and multi targets.lt can inhibite mitochondrial cytochrome C release and reduce apoptosis by Caspase3/Caspase2dependent proteolytic cascade which inhibited by Bcl-2. Studies showed that learning memory ability and the expression of P38MAPK was closely related to the VD rats. Our study want to confirme that the P38MAPK pathway can regulate the expression of apoptosis factor Bcl-2and Caspase-3, by which P38MAPK pathway can affect the learning and memory ability of VD rats model.Objective and methodsvascular dementia (VD) model was established by two vessels method (bilateral common carotid artery occlusion,2VO),60male Wistar rats3month old were randomly divided into sham operation group, vascular dementia model group and SB202190group. Rats were injected medication into the lateral ventricle, the rats of SB202190group injected5μL SB202190(10μmol/L)solution by a micro syringe into lateral ventricle. The model group and sham operation group rats were injected5μL1%DMSO solution The hippocampus cell apoptosis was detected by TUNEL method after the injection of drugs, western blot method was used to detect p-P38MAPK in the rat hippocampus, and mmunofluorescence and confocal laser scanning microscopy were used into observe Bcl-2and Caspase-3expression in hippocampus of rats.ResultThe results of the fluorescence intensity display, TUNEL positive cells expressed green fluorescence. The sham operation group slices express few TUNEL positive cells (green fluorescence), in the model group and the SB202190group positive cells increased obviously, there was significant difference; compared TUNEL positive cells decreased in SB202190group than in the model group.Observe the changes of p38MAPK phosphorylation in rats hippocampus by Western blot method, the expression of p-p38MAPK in different groups of rat hippocampus have obvious differences, the expression of p-p38MAPK increased significantly in the model group and a small amount of p-p38MAPK expression in sham operation group.the expression of p-p38MAPK in SB202190group decreased obviously compared with the model group, the difference was significant.Bcl-2protein in this experiment was marked as green fluorescence. The expressionof Bcl-2protein in the sham operation group was only few immunofluorescence, Bcl-2fluorescence intensity in the model group increased, and fluorescence intensity in SB202190group significantly.Bcl-2immune fluorescence intensity increased significantly in SB202190group compared with model group. The Bcl-2positive cells expressed green fluorescent, positive cells could be occasionally found in sham operation group, and increased in model group, significantly increased in SB202190group. the number of Bcl-2positive cells increased obviously in SB202190group compared with the model group, there was statistical significance.Caspase-3protein in this experiment was also marked as green fluorescence. only few immunofluorescence expressed in sham operation group, fluorescence intensity of Caspase-3in SB202190group increased, and significantly increased in the model group. The fluorescence intensity of Caspase-3decreased significantly in SB202190group compared with model group, and there was statistical significance. The Caspase-3positive cells expressed as green fluorescent, positive cells in sham operation group were occasionally observed, the number of Caspase-3positive cells in the model group increased significantly, the number in SB202190group increased. SB202190group compared with the model group, the number of Caspase-3positive cells decreased significantly, there was statistical significance.ConclusionThis study demonstrated that the P38MAPK pathway can regulate the expression of apoptosis factor Bcl-2and Caspase-3, which may be the way that P38MAPK pathway affect the learning and memory ability of VD rats. Application of P38MAPK inhibitor SB202190can inhibit the apoptosis of cells in hippocampus in vascular dementia rats model, may be a proteolytic cascade by the inhibition of Bcl-2on Caspase3, improving the ability of learning and memory in rats by the reduction of apoptosis.All of these changes confirmed the unique protective effect of SB202190on hippocampal neurons in VD rats model. |
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