| ObjectiveTo observe the mechanism of Shenmayizhi Formula in improving synaptic plasticity by regulating brian microvascular endothelium in rats with vascular dementia.ContentIn this study,the vascular dementia model rats were established by permanent ligation of bilateral common carotid arteries.After three weeks of intervention with Shenmayizhi Formula,Morris water maze test was conducted to observe the pathological morphology and micro vessel density of nerve cells in hippocampal CA1 area,and to detect synapse-related proteins and VEGF/Flk-1/p38 MAPK signal pathway-related proteins,so as to explore the effect and mechanism of Shenmayizhi Formula on improving synaptic plasticity by regulating cerebral microvascular endothelium in vascular dementia rats.MethodsIn this study,the rat model of vascular dementia was established by permanent ligation of bilateral common carotid arteries.100 10-week-old male Wistar rats were randomly divided into 7 groups after successful modeling:sham operation group,model group,blocker group,high-dose Shenmayizhi Formula group,low-dose Shenmayizhi Formula group,blocker of high-dose Shenmayizhi Formula group and blocker of low-dose Shenmayizhi Formula group,with at least 10 rats in each group.The blocking agent SU5416 was injected intraperitoneally at 25mg/kg,the high dose of Shenmayizhi Formula was given by gavage at 16.5g/kg,and the low dose of Shenmayizhi Formula was given by gavage at 3.3g/kg.Morris water maze test was used to evaluate the learning and memory ability of each group of rats.Pathological morphology of hippocampal CA1 region was observed by hematoxylin-eosin staining.Glutamate vesicle transporter and brain microvascular endothelial cells were labeled by immunohistochemical staining,and microvessels were counted.The expression of NMDAR1,PSD-95,VEGF,Flk-1 and p38 MAPK protein was detected by Western-Blot.The mRNA levels of NMDAR1,PSD-95,VEGF,Flk-1 and p38 MAPK mRNA detected by real-time fluorescence quantitative PCR.Results1 Effect of Shenma Yizhi Formula on Learning and Memory Ability of Vascular Dementia RatsIn the navigation test,the latency of each group is significantly different(P<0.05)and there is no significant difference in the interaction between time and grouping(P<0.05).On the 3rd,4th and 5th day,the latency of model group was significantly longer than that of sham operation group(P<0.05).On the 3rd day,compared with the model group,the latency of the high-dose Shenmayizhi Formula group and the low-dose Shenmayizhi Formula group was significantly shortened(P<0.05).On the 4th day,compared with the model group,the latency of the high-dose Shenmayizhi Formula group and the low-dose Shenmayizhi Formula group was significantly shortened(P<0.05).On the 5th day,compared with the model group,the latency of the high-dose Shenmayizhi Formula group was significantly shortened(P<0.05).In the space exploration experiment,compared with the sham operation group,the number of times of crossing the platform in the model group was significantly reduced(P<0.05).Compared with the model group,the times of crossing the platform in the high-dose Shenmayizhi Formula group and the low-dose Shenmayizhi Formula group increased significantly(P<0.05).Compared with the high-dose Shenmayizhi Formula group,the times of crossing in blocker of high-dose Shenmayizhi Formula group were significantly reduced(P<0.05).Compared with the low-dose Shenmayizhi Formula group,the number of crossing the platform in the blocker of low-dose Shenmayizhi Formula group was significantly reduced(P<0.05).In the space exploration experiment,compared with the sham operation group,the distance of the model group in the quadrant where the platform is located was significantly reduced(P<0.05).Compared with the model group,the distance in the quadrant where the platform is located in the high-dose Shenmayizhi Formula group nd the low-dose Shenmayizhi Formula group increased significantly(P<0.05).Compared with the high-dose Shenmayizhi Formula group,the times of crossing in blocker of high-dose Shenmayizhi Formula group were significantly reduced(P<0.05).Compared with the low-dose Shenmayizhi Formula group,the number of crossing the platform in the blocker of low-dose Shenmayizhi Formula group was significantly reduced(P<0.05).2 Effect of Shenmayizhi Formula on pathological morphology of hippocampal CA1 region in rats with vascular dementiaMicroscopically,the pathological morphology of pyramidal cells in CA1 area of hippocampal slices of rats in each group was observed,showing that the nucleus of neurons was light blue and the cytoplasm was pink.In sham operation group,pyramidal cells were arranged tightly and firmly,and the nuclei were full,clear,orderly and clearly demarcated.Compared with the sham operation group,the pyramidal cells in the model group are loosely arranged,and the pyramidal cell layer is detached and thinned,the nucleus of the remaining pyramidal cells is reduced,the axons are scattered and broken,and the boundary is unclear.Compared with the model group,pyramidal cells were loosely arranged and axons were scattered and broken in the blocker group.Compared with the model group,the pyramidal cell layers in the high-dose Shenmayizhi Formula group and the low-dose Shenmayizhi Formula group are arranged regularly and have less axon breakage.3 Effect of Shenmayizhi Formula on brain microvascular endothelial cellsCD31 count:Immunohistochemical staining of CD31 showed that brain microvascular endothelial cells were brown,and in sham operation group,brain microvascular endothelial cells were complete in structure,regular in shape and flat in size.In the model group,the brain microvascular endothelial cells showed deep staining,open vascular malformation,irregular edge contour,different diameters and dense distribution.Compared with the model group,the number of brain microvascular endothelial cells decreased in the blocker group.Compared with the model group,the outline of brain microvascular endothelial cells in the high-dose Shenmayizhi Formula group and the low-dose Shenmayizhi Formula group is regular,the blood vessels are closed,the edge outline is clear,and the diameter is close.Microvessel density count:Compared with sham operation group,the microvessel density count of model group increased significantly(P<0.05)..Compared with the model group,the count of microvessel density in the high-dose Shenmayizhi Formula group and the low-dose Shenmayizhi Formula group decreased significantly(P<0.05)..Compared with the high-dose Shenmayizhi Formula group,the micro vessel density count of blocker of high-dose Shenmayizhi Formula group decreased significantly(P<0.05)..Compared with the low-dose Shenmayizhi Formula group,the microvessel density count in the blocker of low-dose Shenmayizhi Formula grou decreased significantly(P<0.05)..4 Effect of Shenmayizhi Formula on Synaptic Related ProteinsImmunohistochemical staining of glutamate vesicle transporter:Compared with sham operation group,the positive expression of glutamate vesicle transporter in model group decreased significantly.Compared with the model group,the positive expression of VGLUT in blocker group,blocker of high-dose Shenmayizhi Formula group and blocker of low-dose Shenmayizhi Formula group decreased significantly,while the positive expression of VGLUT in high-dose Shenmayizhi Formula and low-dose Shenmayizhi Formula groups increased significantly.N-methyl-D-aspartate receptor protein 1:Compared with the sham operation group,the expression of N-methyl-D-aspartate receptor protein 1 protein and mRNA in the model group decreased significantly(P<0.05).Compared with the model group,the expressions of N-methyl-D-aspartate receptor protein 1 protein and mRNA in the high-dose Shenmayizhi Formula and low-dose Shenmayizhi Formula groups were significantly higher(P<0.05).Compared with the high-dose Shenmayizhi Formula group,the expression of N-methyl-D-aspartate receptor protein 1 protein and mRNA in blocker of high-dose Shenmayizhi Formula group was significantly lower(P<0.05).Post-synaptic dense protein-95:Compared with sham operation group,the expression of post-synaptic dense protein-95 protein and mRNA in model group decreased significantly(P<0.05).Compared with the model group,the protein expression of post-synaptic dense protein-95 in the high-dose Shenmayizhi Formula and low-dose Shenmayizhi Formula groups were significantly higher(P<0.01).Compared with the model group,mRNA content of post-synaptic dense protein-95 in the high-dose Shenmayizhi Formula and low-dose Shenmayizhi Formula groups were significantly higher(P<0.05).Compared with the high-dose Shenmayizhi Formula group,the protein content and mRNA content of post-synaptic dense protein-95 in blocker of high-dose Shenmayizhi Formula group decreased significantly(P<0.05).Compared with the low-dose Shenmayizhi Formula group,the protein content and mRNA content of post-synaptic dense protein-95 in blocker of low-dose Shenmayizhi Formula group decreased significantly(P<0.05).5 Effect of Shenmayizhi Formula on Expression of VEGF,FLK-1 and p38 MAPK Protein in Hippocampus of Vascular Dementia RatsVEGF:Compared with sham operation group,VEGF protein and mRNA content in model group decreased significantly(P<0.05).Compared with the model group,the expression of VEGF protein and mRNA in the high-dose group of Shenmayizhi Formula increased significantly(P<0.05),while the expression of VEGF protein in the low-dose group of Shenmayizhi Formula increased significantly(P<0.01)and VEGF mRNA in the low-dose group of Shenmayizhi Formula increased significantly(P<0.05).Compared with the high-dose Shenmayizhi Formula group,the content of VEGF protein and mRNA in blocker of high-dose Shenmayizhi Formula group decreased significantly(P<0.05).Compared with the low-dose Shenmayizhi Formula group,the expression of VEGF protein in the blocker of low-dose Shenmayizhi Formula group decreased significantly(P<0.01).Compared with the low-dose Shenmayizhi Formula group,VEGF mRNA in the blocker of low-dose Shenmayizhi Formula group decreased significantly(P<0.05).FLK-1:Compared with the sham operation group,the content of FLK-1 protein and mRNA in the model group decreased significantly(P<0.05).Compared with the model group,the content of FLK-1 protein and mRNA in the high-dose Shenmayizhi Formula group increased significantly(P<0.05).Compared with the model group,the content of FLK-1 protein in the low-dose Shenmayizhi Formula group increased significantly(P<0.01),FLK-1 mRNA in the low-dose Shenmayizhi Formula group increased significantly(P<0.05).Compared with the high-dose Shenmayizhi Formula group,the content of FLK-1 protein and mRNA in blocker of high-dose Shenmayizhi Formula group decreased significantly(P<0.05).Compared with the low-dose group of Shenmayizhi Formula,the expression of FLK-1 protein in the blocker of low-dose Shenmayizhi Formula group decreased significantly(P<0.01),FLK-1 mRNA in the blocker of low-dose Shenmayizhi Formula group decreased significantly(P<0.05).P38 MAPK:Compared with sham operation group,the content of P38 MAPK protein and mRNA in model group decreased significantly(P<0.05).Compared with the model group,the contents of p38 MAPK protein and mRNA in the high-dose and low-dose groups of Shenmayizhi Formula were significantly higher(P<0.05).Compared with high-dose Shenmayizhi Formula group,the content of p38 MAPK protein and mRNA in high-dose Shenmayizhi Formula group was significantly lower(P<0.05),while that in low-dose Shenmayizhi Formula group was significantly lower(P<0.05).ConclusionShenmayizhi Formula can improve the spatial learning and memory ability of vascular dementia rats and protect pyramidal cells in hippocampal CA1 region.At the same time,it can improve the morphology and function of brain microvascular endothelial cells,promote the expression of synaptic protein vesicular glutamate transporter,N-methyl-D-aspartate receptor protein 1 and mRNA,promote the expression of postsynaptic dense substance 95 protein and mRNA,and promote the expression of VEGF/FLK-1/P38 MAPK signal pathway related protein and mRNA.This study shows that Shenmayizhi Formula can protect the learning and memory function of VaD model rats by improving the function of brain microvascular endothelial cells,activating VEGF/FLK-1/P38 MAPK signal pathway and improving synaptic plasticity of nerve cells.The regulatory effect of Shenmayizhi Formula reveals a new treatment strategy for vascular dementia,and lays a mechanism foundation for further studying the interaction between neuronal synapses and brain microvascular endothelial cells.At the same time,it confirms the theoretical connotation of TCM that "blood is harmonious with benefits,and spirit is dwelling",and opens up a new idea of regulating five gods from collaterals. |
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