| ObjectiveConnexin（Cx）on the surface of astrocyte membrane has a regulatory effect on glutamic acid（Glu）in vascular dementia.Meanwhile,myelin sheath formed by oligodendrocyte（OL）plays an important role in learning and memory.On this basis,this study speculated that the cognitive impairment of VaD might be related to the accumulation of Glu caused by excessive opening of Cx,and thus induced myelin sheath injury.Modified Shu Yu Wan（SYW）has been used in the treatment of various types of dementia for many years.Combined with this prescription,this topic summarized the etiology and pathogenesis of traditional Chinese medicine of VaD and the pharmacological mechanism of SYW in the treatment of VaD through theoretical research,so as to clarify its rationality.In animal experiments,the effects of SYW on the repair of myelin damage in CA1 region of hippocampus in VaD rats were investigated by behavioral de-tection,immunofluorescence,electron microscopy and other methods from the aspects of hippocampal nerve inflammation,oxidative stress,the role of glial cells in the excitatory toxicity of Glu,Cx expression,and myelin damage repair.The objective is to clarify the mechanism of SYW improving the learning and memory of VaD rats,so as to better understand the pathogenesis of the disease and provide new research ideas and drug selection for the treatment of the dis-ease.Methods1 Theoretical research partThis section discusses the understanding of VaD in TCM and Western med-icine from the perspectives of risk factors,pathogenesis,etiology and pathogen-esis of TCM.In addition,the theoretical basis of the treatment of VaD by SYW was clarified from two parts:formulation analysis of SYW and modern phar-macology study of prescriptions.2 Experimental research partAfter adaptive feeding of healthy SPF grade 3-month-old Sprague Dawley male rats for 1 week,rats with no significant difference in escape latency were selected through the morris water maze（MWM）.VaD rat model was prepared using an improved method of bilateral carotid artery ligation.After surgery,48successfully modeled rats were selected through MWM and randomly divided into model group（Model group）,high-dose group（SYW-H group）,medium dose group（SYW-M group）,and low-dose group（SYW-L group）.The rest were sham operated group（Sham group）with 12 rats in each group.The high,me-dium,and low dose groups of Dioscorea pill were treated with different con-centrations of SYW decoction,while the sham group and model group were treated with equal amounts of physiological saline and continuously gavaged for 28 days.At the end of the drug treatment,we tested the learning and memory abilities of the rats by two behavioral measures,MWM and NOR.After behavioral testing,brains were harvested by perfusion and sectioned for HE staining and LFB staining of the hippocampus.Hippocampal neuronal apoptosis and myelin sheath injury were observed by staining,as well as subse-quent IF observation.Fresh hippocampal tissue was used for Elisa,WB,and TEM observation after glutaraldehyde fixation:The levels of tumor necrosis factor-α（TNF-α）,interleukin 6（IL-6）,reactive oxygen species（ROS）,superoxide dismutase（SOD）and Glu in hippocampus were detected by Elisa.The protein levels of excitatory amino acid transporter（EAAT）1,N-methyl-D-aspartate receptor（NMDAR）andα-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor（AMPAR）,Cx43 and Cx30 were observed by WB.TEM was used to observe the microstructure of myelin sheath in hippocampal CA1 region.Myelin basic protein（MBP）,oligodendrocyte lin-eage transcription factor 2（Olig2）,glial fibrillary acidic protein（GFAP）and connexin 43（Cx43）,GFAP and Cx30 were observed by IF.Result1 The effect of SYW on learning,memory,and recognition function in VaD ratsIn the localization cruise test,compared with the rats in sham group,the escape latency of the rats in model group was significantly prolonged on the 3rd and 5th day（*P<0.05）.Compared with the rats in model group,the escape latency of the SYW high,medium and low dose groups was significantly short-ened on the 3rd and 5th day（#P<0.05）.In the space exploration test,compared with the rats in sham group,the number of crossing the platform and the time of staying in the target quadrant in the rats in model group were significantly reduced(**P<0.01).Compared with the rats in model group,the number of crossing the platform in the SYW-H and SYW-M groups was significantly increased,and the residence time of the target quadrant was significantly prolonged(##P<0.01).The number of crossing the platform and the time of staying in the target quadrant in the SYW-L group were significantly increased（#P<0.05）.In the novel object recognition test,the recognition index（RI）of the model group was significantly lower than that of the sham group(**P<0.01).Com-pared with the rats in model group,the RI of SYW-H and SYW-M groups was significantly increased（#P<0.05）.The RI of SYW-L group was higher than that of model group,but the difference was not statistically significant.2 The effect of SYW on the morphology of hippocampal neurons in VaD ratsHE staining showed that the cells in CA1 region of hippocampal tissue in the sham group had a complete structure,orderly and compact arrangement,regular morphology,distinct hierarchy,full cytoplasm and clear outline of the cell body.The nuclei were mostly round and large,and the nuclei were distrib-uted in the center of the cytoplasm.The proportion of cell damage in unit visual field was the lowest,and there was no obvious loss of neurons.Compared with the rats in sham group,the cell structure in CA1 region of hippocampal tissue in model group was loose,disordered and incomplete,with obvious intercellular space,necrosis or degeneration of a large number of neu-rons,shrinkage of cytoplasm,turbidness and deepened staining.The cell size was uneven and the arrangement was disordered,so the cell count deviation in unit visual field was large,and the proportion of damaged cells increased sig-nificantly(**P<0.01).After SYW intervention,hippocampal tissue damage degree was signifi-cantly reduced in SYW high-dose,medium-dose and low-dose groups,cell structure was relatively complete,cell morphology was more regular than model group,cell arrangement was more orderly,the number of normal cells was significantly increased,cytoplasmic staining was reduced,and nuclear swelling was reduced.Cell counts in unit visual field tended to be consistent,indicating that cells in hippocampal CA1 region of rats treated with SYW were more regular in size and more closely arranged than those in model group.The proportion of damaged cells in SYW high,medium and low dose groups was significantly improved compared with the rats in model group(##P<0.01),and SYW-H group was better than SYW-M and SYW-L group.3 The effect of SYW on hippocampal inflammation,oxidative stress,and Glu levels in VaD RatsElisa showed that compared with the rats in sham group,the levels of TNF-αand IL-6 in hippocampus of model group were significantly increased（*P<0.05）,and Glu level was significantly increased(**P<0.01).Compared with the rats in model group,the levels of TNF-αand IL-6 in SYW-H group were significantly decreased（#P<0.05）,and Glu was significantly decreased(##P<0.01).The levels of TNF-αand Glu were significantly decreased in SYW-M group,and the levels of IL-6 were decreased,but the difference was not statis-tically significant.Glu level in SYW-L group was significantly decreased（#P<0.05）,and inflammatory factors were decreased compared with the rats in model group,but the difference was not statistically significant.SYW-H group showed the most obvious inhibitory effect on inflammatory factors and increased Glu content in hippocampus.Compared with the rats in sham group,ROS level in hippocampus of model group was significantly increased(**P<0.01),and SOD level was significantly decreased（*P<0.05）.Compared with the rats in model group,ROS levels in SYW high,medium and low dose groups were significantly decreased(##P<0.01).SOD level in SYW-H group was significantly increased（#P<0.05）,SOD level in SYW-M and SYW-L groups was higher than that in model group,but the difference was not statistically significant.4 The effect of SYW on glutamate transporters and glutamate receptors in the hippocampus of VaD ratsWB showed that compared with the rats in sham group,EAAT1 and p-AM-PAR Glu R1 levels in model group were significantly increased(**P<0.01),while AMPAR and NMDAR1 levels were significantly decreased(**P<0.01).Compared with the rats in model group,EAAT1 and p-AMPAR Glu R1 levels in SYW-H group and SYW-M group were significantly decreased(##P<0.01),while AMPAR Glu R1 and NMDAR1 levels were significantly increased(##P<0.01).EAAT1 in SYW-L group was significantly decreased（#P<0.05）,AM-PAR and NMDAR1 levels were significantly increased(##P<0.01),and p-AM-PAR was decreased compared with the rats in model group,but the difference was not statistically significant.Compared with the rats in sham group,Cx43 and Cx30 levels in hippocam-pus of model group were significantly increased(**P<0.01).Compared with the rats in model group,Cx43 levels in SYW-H and SYW-M groups were sig-nificantly decreased(##P<0.01),and Cx43 levels in SYW-L group were slightly lower than those in model group,but the difference was not statistically significant.The down-regulation effect on Cx43 was most obvious in SYW-H and SYW-M groups.Compared with the rats in model group,Cx30 expression in SYW-H group was significantly decreased（#P<0.05）,and Cx30 expression in SYW-M and SYW-L groups was decreased compared with the rats in model group,but the difference was not statistically significant.The down-regulation effect on Cx30 was most obvious in SYW-H group.5 The effect of SYW on the ultrastructure of hippocampal myelin sheath in VaD ratsElectron microscopy showed that the structure of myelin sheath in hippo-campal CA1 region of rats in the sham group was complete,the lamella was dense,the arrangement was regular,the edge was clear and smooth,the bound-ary was obvious,the vacuoles were less,the cytoplasm was clear,and the cel-lular contents were evenly distributed.Compared with the rats in sham group,the structure of hippocampal myelin in model group was loose,the lamellar and marginal structures were blurred,the dense layer was separated,and more vacuole-like changes occurred,and the cytoplasm was cloudy and the cellular contents were blurred.Compared with the rats in model group,after SYW intervention,the myelin lamellar structure in SYW-H and SYW-M groups was significantly improved,with clear edges,reduced vacuole-like structure,clear cytoplasm and visible organelles.Compared with the rats in model group,the structure of myelin la-mella in SYW-L group was improved,but it was still loose,the edge of myelin sheath was blurred,the cytoplasm was cloudy,and there were vacuole-like changes.The improvement of myelin structure in SYW-H group was the most obvious.6 The effect of SYW on hippocampal myelin blue staining in VaD ratsLFB staining showed that the hippocampal myelin in the sham group was uniformly blue-stained,and the cells were closely arranged with fewer vacuoles.In model group,myelin sheath staining was decreased,cells were arranged loosely,and more vacuole-like changes appeared.After SYW intervention,the staining of myelin sheath in hippocampal CA1 region of rats in each group was more uniform and deeper than that in model group.7 The effect of SYW on hippocampal myelin sheath,glial cell markers,and gap junction proteins in VaD ratsImmunofluorescence staining showed that compared with the rats in sham group,the cell structure of hippocampus in model group was disordered,and the fluorescence intensity of MBP was significantly decreased(**P<0.01).Compared with the rats in model group,after SYW intervention,MBP fluores-cence intensity of SYW-H and SYW-M was significantly increased(##P<0.01),and MBP of SYW-L group was significantly increased（#P<0.05）.The im-provement of MBP in SYW-H and SYW-M groups was the most obvious.Compared with the rats in sham group,the fluorescence intensity of OL marker Olig2 in hippocampus of model group was significantly decreased（*P<0.05）,and the Olig2 was distributed in both vertebral somatic layer and py-ramidal cell.Compared with the rats in model group,Olig2 fluorescence inten-sity in SYW-H group was significantly increased（#P<0.05）,and that in SYW-M and SYW-L groups was significantly increased(##P<0.01).Compared with the rats in sham group,the fluorescence intensity of Cx43and Cx30 in hippocampus of model group was significantly increased（*P<0.05）,and the fluorescence intensity of GFAP was significantly increased(**P<0.01).Gfap-cx43 and GFAp-CX30 co-labeled cells were increased in unit visual field,and distributed in the pyramidal layer and outside the pyramidal layer,suggested that Astrocyte（AC）activation increased in the hippocampus at the fourth week after modeling,while Cx43 and Cx30 expression increased.Compared with the rats in model group,Cx43 fluorescence intensity in SYW-H and SYW-M groups was significantly decreased（#P<0.05）,and GFAP fluorescence intensity was significantly decreased(##P<0.01).The fluores-cence intensity of Cx43 in SYW-L group was lower than that in model group,but the difference was not statistically significant.The fluorescence intensity of GFAP was significantly decreased（#P<0.05）.GFAP-Cx43 co-labeled cells de-creased in unit visual field.Compared with the rats in model group,Cx30 fluo-rescence intensity in SYW high,middle and low dose groups was significantly decreased(##P<0.01).The fluorescence intensity of GFAP in SYW-H group was significantly decreased(##P<0.01),and that in SYW-M group was signif-icantly decreased（#P<0.05）.The fluorescence intensity of GFAP in SYW-L group was decreased compared with the rats in model group,but the difference was not statistically significant.The number of GFAP-Cx30 co-labeled cells decreased in the unit field of vision.Conclusion1 In this experiment,the modified 2-VO method was used to induce the VaD rat model.After observing the pathological changes of hippocampus by MWM and NOR behavioral tests and HE staining,the model was considered to be sta-ble and reliable,which was an ideal animal model for studying the pathophysi-ological mechanism of VaD,and laid an experimental foundation for exploring clinical drug action targets.In addition,SYW improves the spatial learning memory and object recognition of VaD rats,and reduce the damage degree of hippocampal neurons.2 The hippocampus of VaD model rats showed increased inflammation and oxidative stress damage,excessive accumulation of Glu,and dysfunction of EAAT1,which increased activation of downstream NMDAR and AMPAR,in-ducing cell damage and apoptosis,which may be the cause of the impairment of learning and memory and cognitive function of VaD rats.SYW improves the spatial learning memory and object recognition ability of VaD rats,which may be accomplished by reducing the inflammatory response and oxidative stress level of hippocampus,reducing the excitatory toxic injury caused by Glu accu-mulation,promoting the normal function of EAAT1,and reducing the activation of NMDAR and AMPAR.3 The inflammatory response and oxidative stress injury in hippocampus of VaD model rats are aggravated,Glu is over-accumulated,EAAT1 dysfunction is dysregulated,which increases the activation of downstream NMDAR and AMPAR and induces cell damage and apoptosis,which may be the reasons for the impairment of learning and memory and cognitive function in VaD rats.SYW improves the spatial learning and memory and object recognition of VaD rats,which may be achieved by reducing the inflammatory response and oxida-tive stress level in the hippocampus,reducing excitotoxic damage caused by Glu accumulation,promoting the recovery of EAAT1 function,and reducing the activation of NMDAR and AMPAR.These results suggest that SYW has a neuroprotective effect.4 AC activation was increased in the hippocampus of VaD model rats,and Cx expression of AC was increased.Meanwhile,OL marker expression was decreased,MBP expression in hippocampus was decreased,myelin was lost,myelin ultrastructure was destroyed,and neurons were damaged,which might be one of the mechanisms of neuron damage and cognitive impairment in VaD rats.SYW reduces the activation of AC and reduce the expression of Cx,while the expression of OL is significantly increased,and the expression of MBP pro-tein is up-regulated in the hippocampus of VaD rats to protect the myelin and myelin sheath ultrastructure,thus reducing the damage of neurons and improv-ing cognitive function.Therefore,it can be used as an effective treatment for VaD. |
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