Effects And Mechanisms Of Acorus Tatarinowii On Cognitive Impairment In AD Model Mice Induced By D-Galactose

Alzheimer’s disease（AD）is a progressive neurodegenerative disease that is the most common of dementia with a high prevalence in the elderly population.With the acceleration of population aging,the incidence of AD is also increasing.Despite the current stage of progress in AD research,its underlying pathogenic factors and effective treatment methods are still unclear.At present,the most important hypothesis about AD is the Aβcascade hypothesis,which is accepted by most scholars and believes that Aβmonomers are obtained by the hydrolysis of amyloid precursor APP byβ-secretase andγ-secretase.When the production and clearance rates of Aβare out of balance,Aβwill be excessively deposited in the brain,forming toxic aggregates,and then causing a series of pathological changes.Aβdeposition near cerebral microvessels can lead to the loss of pericyte cells near cerebral microvascular,causing oxidative stress response and cerebral microangioscular contraction.Acorus tatarinowii is an essential medicine for awakening the mind,with sedative,antiarrhythmic,anticonvulsant,antidepressant,antioxidant and other pharmacological effects,which is commonly used in clinical treatment of AD,however,there is no report on the study of it on AD brain microvascular pericytes.In this study,the objectives of this study were to explore the improvement effect and mechanism of the Acorus tatarinowii water extract on cognitive impairment in mice with AD,and to further explore the protective effect ofβ-Asarone,the main active ingredient of Acorus tatarinowii,on pericyte cells,in order to provide more experimental basis for the clinical treatment of AD.Objective:1.To investigate the effect and mechanism of Acorus tatarinowii water extract on cognitive impairment in mice with D-galactose-induced AD model.2.To investigate the protective effect ofβ-Asarone,the main active ingredient of Acorus tatarinowii,on Aβ_1-40 damage to MBVP cells.Methods:1.The mechanism and target of action of Acorus tatarinowii component in the treatment of AD was predicted by network pharmacological methods.2.Healthy C57BL/cnc male mice weighing about 25 g at 6 weeks of age were random Ly divided into 6 groups,normal control group（control）,model group（model）,positive drug donepezil hydrochloride group（donepezil）,Acorus tatarinowii water extract low-dose group（1g/kg）,medium-dose group（2 g/kg）,and high-dose group（4 g/kg）.Mice in the control group were injected with 0.4 m L of normal saline subcutaneously per item per day.According to the dose of 150 mg/kg,the mice in the model group and each administration group were subcutaneously injected with 0.4 m L of 9.35 mg/m L D-galactose solution every day.The mice in the control group and the model group were each given 0.2 m L of normal saline daily,at a dose of 0.8 g/kg,mice in the positive drug group were each given 0.2m L of 0.1mg/m L donepezil solution every day,and as prescribed dose,each mouse in the Acorus tatarinowii water extract group was given 0.2m L of the prescribed concentration of drug solution（0.125g/m L,0.25g/m L,0.5g/m L）per day,continuous modeling administration for 13 weeks.（1）Detected the weight of each group of mice every week after 4 weeks of molding administration;The object location recognition experiments were performed on mice at 4,6,and 8 weeks of model administration,and novel object recognition experiments on mice at weeks 5,7,and 9;the passive avoidance experiment was carried out on mice in the 10th week of mold administration;the active avoidance experiment was conducted on mice in the 11th week of mold administration;the cerebral blood flow of each mouse was detected by Doppler flowmeter at the 12th week of model administration.,and took materials.（2）HE staining was performed to observe the pathological changes in mouse brain tissue.Immunohistochemical staining was used to detect Aβdeposition in mice brain tissue.The expression of Aβmetabolism-related proteins LRP-1,BACE1,pericyclic marker PDGFR-β,and PI3K and AKT were detected by Western-blot.3.The Aβ_1-40 oligomers were identified by transmission electron microscope and silver staining method.Aβ_1-40 incubated for 4 days was selected to induce mouse brain microvascular perivascular cells（MBVP）to establish an in vitro AD injury model,and treated withβ-Asarone,the main active ingredient of Acorus tatarinowii.x CELLigence RTCA DPlus Instrument was used to screen out the appropriate administration concentration ofβ-Asarone;the expression of ROS was detected by reactive oxygen species detection kit.The expression of Aβmetabolism-related proteins LRP-1,BACE1 in MVBP cells were detected by Western-blot.Results:1.Network pharmacological results mainly screened out 40 common core targets of Acorus tatarinowii and Alzheimer’s disease,mainly including AKT1,HSP90AA1,CASP3,EGFR,ESR1,VEGFA,etc.,enrichment analysis of 191 intersection targets,76 KEGG signal pathways and 448 GO enrichment entries were obtained,mainly involving cancer-related signaling pathways,PI3K/AKT signaling pathways,HIF-1 signal pathways,calcium signal pathways,etc.2.Compared with the control group,the mice in the model group had abnormal memory-related behaviors（P<0.05）,decreased cerebral blood flow（P<0.001）,damaged brain tissue cell structure,and had significant Aβdeposition in the cortex and hippocampus（P<0.05）,and BACE1 protein expression level increased（P<0.01）,while the protein expression levels of LRP-1,PDGFR-β,p-PI3K and p-AKT decreased（P<0.05）;Various concentrations of Acorus tatarinowii water extract can improve the recognition ability of new objects（P<0.05）and the cell structure of brain tissue in AD mice,reduce the deposition of Aβin the cortex（P<0.05）,and increase the expression level of AKT phosphorylated protein（P<0.01）.In addition,medium-dose and high-dose Acorus tatarinowii water extract can significantly reduce the deposition of Aβin the hippocampus（P<0.05）,reduce the expression of BACE1 protein（P<0.001）,and increase the expression of LRP-1 and p-PI3K protein（P<0.05）.And medium-dose Acorus tatarinowii water extract can increase the expression of PDGFR-β（P<0.001）,low-dose group Acorus tatarinowii water extract can significantly increase cerebral blood flow in AD model mice（P<0.05）.3.Aβ_1-40 incubated at 37℃for 4 days,a large number of short filamentous oligomers could be seen under the electron microscope,and the silver staining method showed that the molecular weight of the oligomers is 4-30 KDa.20μM and 40μM Aβ_1-40 oligomers could significantly impair MBVP cell morphology,significantly reduce cell viability（P<0.01）,increase the leakage rate of cell LDH（P<0.01）,increase the release of ROS from MBVP cells（P<0.01）,increase BACE1 protein expression level（P<0.01）.1μMβ-Asarone significantly increased cell viability（P<0.01）,reduced the release of cellular ROS（P<0.01）,and reduced BACE1protein expression level（P<0.01）.Conclusions:1.Acorus tatarinowii water extract can improve the cognitive function of AD model mice induced by D-galactose,increase cerebral blood flow,regulate the expression of Aβmetabolism-related proteins,and reduce Aβdeposition in the brain.The mechanism of its efficacy may be related to PI3K/AKT signaling pathway.2.The main active ingredient of Acorus tatarinowii,β-Asarone,exerts protective effects on MBVP cells damaged by Aβ_1-40 oligomers.