| Objective:Vascular dementia(VD)is a brain-destroying disease caused by various cerebrovascular diseases,causing dementia syndrome characterized by cognitive dysfunction.It is the second most common type of Alzheimer's disease.The pathogenesis is still unclear and there is no effective treatment.Traditional Chinese medicine has certain advantages and broad development prospects in the treatment of vascular dementia.Therefore,it is of great significance to further clarify the therapeutic effects and mechanisms of traditional Chinese medicine on VD.The pathogenesis of VD is still unclear.In recent years,researchers have recognized that synaptic damage is a common neuronal pathological manifestation of VD,Alzheimer's disease,and Lewy body dementia,and it has been shown that synaptic damage begins at the axis.The disorder of the terminal vesicle signaling pathway eventually progresses to apoptosis in related cerebral cortical neurons.The latest study of VD synaptic damage found that clathrin is the most prominent neurosynaptic-associated protein in VD brain,and clathrin-mediated endocytosis(CME)is a receptor-mediated protein.One of the main ways of endocytosis plays an important role in the regulation of plasma membrane protein endocytosis and intracellular and extracellular signal transduction processes.The N-methyl-D-aspartic acid(NMDA)receptor is a kind of ionotropic glutamate receptor,which is involved in both the learning and memory physiology in the central nervous system.The process,in turn,is closely related to the glutamate excitotoxicity produced by VD,and endocytosis of NMDA receptors is dependent on clathrin-mediated endocytosis.Based on the above studies,we found that changes in early NMDA receptors in VD are likely to be associated with clathrin-mediated abnormalities in endocytic processes,and decreased expression of clathrin in the brain of VD patients,which mediated a large reduction in endocytosis of NMDA receptors,neuronal surface Increased NMDA receptors,increased excitatory toxicity of glutamate causes neuronal death.Therefore,clathrin-mediated endocytosis of NMDA receptors can be used as a new therapeutic target.Shenzhi jiannao recipe is based on the etiology and pathogenesis theory of academician wang yongyan,"deficiency of qi and toxin damage collaterals obstruction",combined with many years of clinical experience,the effective ingredients of traditional Chinese medicine are combined under the guidance of TCM compound theory,which has the effect of strengthening qi,detoxifying and clearing collaterals.Previous research has confirmed that Shenzhi jiannao recipe can reduce the neurotransmitter Glu in the cortex and the content of GABA,improve VD rats learning and memory function,can improve the bilateral common carotid arteries ligation dementia induced by chronic hypoperfusion rats model of brain ischemia state,protect the damaged neurons,inhibit the reduction in the number of neurons to prevent neuronal degeneration necrosis.On this basis,in order to further clarify the pharmacodynamic effect and mechanism of shenzhi jiannao recipe,we propose the hypothesis that shenzhi jiannao recipe can protect the damaged neurons,whether it is related to the increase of endocytosis of clathrin-mediated NMDA receptor on the surface of neurons,so as to reduce the excitatory toxicity of glutamate.This experiment will be divided into two parts,the first part:to establish animal model of vascular dementia rats by behavioral testing and histomorphology observation to evaluate VD model,through the VD rats serum BDNF in the cortex and the detection of the content of MMP-9 to observe and know the curative effect of VD rats on the brain party,further validation and knowledge of brain cerebral protection;Part ii:based on clathrin-mediated endocytosis of NMDA receptor on neuron surface,the changes of clathrin expression in VD state were discussed,and the characteristics of clathrin-mediated endocytosis of NMDA receptor and the effect of shenzhen-jiannao recipe on this change were discussed.Methods:1.Observation of brain protection effect of shenzhi jiannao recipe on rats with vascular dementia.SD rats were randomly divided into normal group,sham-operated group and model group.The VD rat model was prepared by repeated occlusion and reperfusion of bilateral common carotid artery and intraperitoneal injection of sodium nitroprusside.In the sham-operated group,only bilateral common carotid artery was separated without sodium nitroprusside injection and occlusion of vessel.The model rats were evenly divided into VD model group,shenzhi jiannao recipe treatment group and memantine group.The drugs were administered by gavage on the second day after the operation,and the corresponding drugs were administered by gavage in the administration group respectively.The normal group,the sham-operated group and the VD model group were given the same amount of distilled water once a day for 14 consecutive days.The learning and memory abilities of rats were tested by step down test,Novel Object Recognition and Morris water maze.The hippocampal pathological changes of VD rats were observed by HE staining and Nissl staining,and the contents of BDNF and MMP-9 in the cortex and serum of rats in each group were determined by ELISA method,and observe the effect of shenzhi jiannao recipe on its changes and further clarify the pharmacodynamic mechanism of shenzhi jiannao recipe on brain protection.2.Effects of shenzhi jiannao recipe on clathrin-mediated endocytosis of NMD A receptor.SD rats were randomly divided into normal group,sham-operated group and model group.The VD rat model was prepared by repeated occlusion and reperfusion of bilateral common carotid artery and intraperitoneal injection of sodium nitroprusside.In the sham-operated group,only bilateral common carotid artery was separated without sodium nitroprusside injection and occlusion of vessel.The model rats were evenly divided into VD model group,shenzhi jiannao recipe treatment group and memantine group.The drugs were administered by gavage on the second day after the operation,and the corresponding drugs were administered by gavage in the administration group respectively.The normal group,the sham-operated group and the VD model group were given the same amount of distilled water once a day for 14 consecutive days.The mRNA expression level of clathrin,NMDA-NR1 in the hippocampus was detected by RT-PCR.Western blot was used to detect the expression level of clathrin and its endocytosis marker RAB5B in the hippocampal tissue and the expression level of NMDA-NR1 on the surface of hippocampal neurons.Immunohistochemical staining was performed to observe the distribution and expression changes of clatbrin,RAB5B and NMDA-NR1,and to explore the effect of shenzhijiannao recipe on clathrin-mediated NMDA endocytosis in VD.Results:1.Observation of brain protection effect of shenzhi jiannao recipe on rats with vascular dementia.1.1 Behavioral Test Results1.1.1 Step down test.Compared with the normal group,there was no statistical difference in the incubation period and the number of errors between the sham-operated group and the normal group(P>0.05),The incubation period was significantly shortened and the number of errors significantly increased in the VD model group(P<0.01),indicating that the learning and memory function of the VD model rats was decreased and the model was successful.1.1.2 Novel Object Recognition.Compared with the sham-operated group,the discrimination index of the VD model group was significantly reduced(P<0.01).Compared with the VD model group,the discrimination index of the rats in the shenzhijiannao recipe treatment group and the memantine group was significantly increased(P<0.01).1.1.3 Positioning navigation experiment of Morris water maze.Compared with the sham-operated group,the escape latency of the VD model group was significantly prolonged(P<0.01).Compared with the VD model group,the escape latency was significantly shortened in the shenzhijiannao recipe treatment group and the memantine group(P<0.01,P<0.05).1.1.4 Space exploration experiment of Morris water maze.Compared with the sham-operated group,the duration of stay in the target quadrant and the times of crossing the platform in the VD model group were significantly reduced(P<0.01).Compared with the VD model group,the duration of stay in the target quadrant and the number of times of crossing the platform in the shenzhijiannao recipe treatment group and the memantine group were significantly increased(P<0.01,P<0.05).1.2Morphology test result.1.2.1 HE staining.The neurons in the hippocampal CA1 area of the rats in the sham-operated group had normal morphological structure,and the neurons were closely arranged,with abundant layers,clear nucleoli and abundant cytoplasm.In the VD model group,the number of hippocampal CA1 decreased,the Neuronal cell body Shrink,and the neurons were scattered,with neuron loss and nuclear shrinkage.The neurons in the CA1 area of the hippocampus in the shenzhijiannao recipe treatment group and the memantine group were sparsely arranged,and the cell morphology and number were improved compared with the model group.1.2.2 Nissl staining.In the sham-operated group,hippocampal neurons were arranged in a neat and compact arrangement,and staining was blocky or granular with dense and regular staining.In the VD model group,the hippocampal neurons were disordered in fine arrangement,the cell body was reduced,the number of cells was significantly reduced,and a large number of nerve cells appeared apoptosis and Nissl body fragmentation.Compared with VD model group,the hippocampal neurons in the shenzhijiannao recipe treatment group had clear layers and thin cell bands,and the morphology and arrangement of hippocampal cells in memantine group were improved,but there were still a large number of neuronal apoptosis.1.3 The changes of BDNF and MMP-9 in VD rat cortex and serum.ELISA results showed that,compared with the sham-operated group,the BDNF content in serum and cortex of the VD model group decreased(P<0.01),and the MMP-9 content increased significantly(P<0.01).Compared with the VD model group,the BDNF content in serum and cortex of the shenzhijiannao recipe treatment group and the memantine group increased(P<0.01),and MMP-9 content decreased significantly(P<0.01).2.Effects of Shenzhi jiannao Recipe on clathrin-mediated endocytosis of NMDA receptor.2.1 RT-PCR results.Compared with the sham-operated group,the level of clathrin mRNA was significantly decreased(P<0.01)and the level of NMDA-NR1 mRNA increased in the hippocampus of rats in VD model group(P<0.01).Compared with the VD model group,the level of clathrin mRNA increased in shenzhijiannao recipe treatment group,and the expression of NMDA-NR1 mRNA in shenzhijiannao recipe treatment group and memantine group was decreased(P<0.01,P<0.05).2.2 Western blot results.Compared with the sham-operated group,the expression of clathrin and RAB5B in the hippocampal tissues of the VD model group decreased significantly(P<0.01),and the expression of NMDA-NR1 increased(P<0.01).Compared with the VD model group,the level of clathrin,RAB5B in the hippocampus of shenzhijiannao recipe treatment group increased significantly(P<0.01),and the level of NMDA-NR1 decreased significantly(P<0.01).There was no statistical difference in the expression of clathrin,RAB5B and NMDA-NR1 in the hippocampus of memantine group(P>0.05).2.3Immunohistochemical Results.Clathrin protein is mainly expressed in the cytoplasm and membrane of nerve cells,NMDA-NR1 receptor is mainly expressed in the membrane and protuberances of nerve cells,and RAB5B protein is mainly expressed in the cytoplasm and nucleus of nerve cells.Compared with the sham-operated group,the expression of clathrin and RAB5B in the hippocampal tissues of the model group decreased ssignificantly(P<0.01),and the expression of NMDA-NR1 increased significantly(P<0.01).Compared with the VD model group,the level of clathrin,RAB5B in the hippocampus of shenzhijiannao recipe treatment group increased significantly(P<0,01),and the protein levels of NMDA-NR1 were significantly decreased(P<0.01).There was no statistical difference in the expression levels of clathrin,RAB5B and NMDA-NR1 in the hippocampus of memantine group(P>0.05).Conclusion:1.Shenzhi jiannao recipe can reduce the damage of hippocampal neurons in VD rats,reduce MMP-9 content in cortex and serum,up-regulate the BDNF content in serum and cortex,improve the learning and memory ability of VD rats,and have brain protective effect.2.Shenzhi jiannao recipe may promote the endocytosis of NMD A-NR1 on the surface of neurons by up-regulating the expression of clathrin and RAB5B,and play a brain protective role by alleviating the damage of excitatory toxicity on neurons. |
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