Study On The Mechanism Of Mongolian Medicine Eerdun-Wurile In Improving Learning And Memory Ability In Aβ_1-40 Induced Alzheimer’s Disease Model Rats Based On Metabolomics

Objective:The increasing number of Alzheimer’s disease（AD）patients is a threat to human health,but there is still a lack of effective treatment.The Mongolian medicine Eerdun-Wurile has a remarkable clinical effect on the treatment of“Bai Mai”disease.In this study,AD model rats were used to study the effect,material basis and target of Mongolian medicine Eerdun-Wurile in improving learning and memory ability,in order to clarify the mechanism of action.Methods:The rat model of AD was established by injecting Aβ_1-40 into CA1 region of hippocampus.They were divided into normal group,sham operation group,Aβ_1-40 model group,Eerdun-Wurile group（0.62mg/kg/d）,Eerdun-Wurile extract group（0.124 mg/kg/d）and positive control group（Donepezil,1 mg/kg/d）,and treated by gavage for 22 days.From the15th day,Morris water maze method was used to conduct space exploration and positioning cruise ability test,and the effect of Mongolian medicine Eerdun-Wurile on improving learning and memory was analyzed.After completion,Eerdun-Wurile was administered at 0.54g/kg/d and extract group at 0.108g/kg/d.60 min later,blood and brain tissues were collected.TUNEL fluorescence staining was used to analyze the apoptosis of hippocampal cells,and ELISA was used to detect the content of Aβ_1-40 in serum and CA1 region of hippocampus.Secondly,the untargeted metabolomics method was used to detect the small molecule compounds in Eerdun-Wurile and its serum.Differential metabolites,potential biomarkers of AD induction model and Eerdun-Wurile components in blood were analyzed and verified by HPLC.At the same time,the Discovery Studio molecular simulation method was used to predict and screen the active constituents of Eerdun-Wurile.Rusults:The water maze results showed that the incubation period,that is,the time to find the platform,was significantly longer in the model group than in the sham operated group（P<0.001）,indicating that the learning and memory were significantly decreased,and the model was successfully established.After Eerdun-Wurile administration,the latency period was significantly shortened（P<0.001）,and the number of crossing the original platform was increased（P<0.05）,which significantly improved the learning and memory.ELISA results showed that the serum and hippocampal Aβ_1-40 contents in model group were significantly higher than those in sham operated group（P<0.05）,which again proved the establishment of the model.Eerdun-Wurile significantly decreased the serum and hippocampus Aβ_1-40 content（serum P<0.05,hippocampus P<0.001）.Eerdun-Wurile extract showed similar effects.At the same time,apoptosis of hippocampal cells in AD model rats was obvious,and Eerdun-Wurile effectively inhibited apoptosis.Non-targeted metabolomics data showed 115 differential metabolites（up-regulated 76,down-regulated 39）involved in 19 metabolic pathways in the model group compared with the normal group.In addition,11 potential serum biomarkers of AD model rats were screened to participate in three metabolic pathways.There were 147 differential metabolites（up-regulated 101,down-regulated 46）involved in 22 metabolic pathways after Eerdun-Wurile treatment.There were115 differential metabolites（up regulated 68,down regulated 47）in Eerdun-Wurile extract after treatment,and they were involved in 13 metabolic pathways.179 components of Eerdun-Wurile were identified,of which 65 were prototypic components and 114 were transitional or metabolic components.Among the 179,15 components with neuroprotective effects reported in the literature and chemical structure can be searched in the Chemspider database were selected for target prediction.Molecular simulation analysis showed that 188 target proteins with Fit value>0.5 were GSK-3β,HS90A,CHK1,CDK2,MAPK14 and DHI1.Among them,geniposide binds to Alzheimer’s disease-related targets GSK-3β,MAPK and BACE1 proteins.Geniposide was detected by HPLC,which was consistent with the results of non-targeted detection.Conclusions:This study proves that Mongolian medicine Eerdun-Wurile and its extract can improve learning and memory ability.The main mechanism of action is Eerdun-Wurile blood components（179 kinds in this study）,including geniposide.Through participating in 22 metabolic pathways,147 metabolites can be regulated to reduce the content of Aβ_1-40 in serum and hippocampus,and effectively inhibit the apoptosis of hippocampal cells.MAPK,GSK-3β,BACE1 and other AD-related target proteins may play a role.