| Background:Alzheimer’s disease（AD）is an irreversible neurodegenerative disease characterized by learning decline,memory impairment,and cognitive dysfunction in the clinic.It seriously affects the daily life of patients and brings a heavy burden to society and families.With the increase in the population and the aggravation of the aging of the population,the incidence rate of AD will increase.The existing drugs have unsatisfactory efficacy and serious side effects.Effective drugs or treatment methods are in urgent demand.Arctium lappa L.leaves,named“burdock leaves”,are non-medicinal parts of the Composite plant Burdock.China is the largest exporter of burdock in the world,which lead bring abundant burdock resources,especially the production of the leaves.Burdock leaf has a long history as a medicinal and edible plant.“Nong Sang Ji Yao”recorded that burdock leaves can be used as food to brighten the eyes and resist aging.“Jiu Huang Ben Cao”also recorded that burdock leaves are very beneficial to our health and resistance to aging.Both the records indicated that burdock leaves can improve aging-related diseases.Moreover,burdock leaves can treat stroke as food in“Tai Ping Sheng Hui Fang”,which manifested that burdock leaves can treat brain diseases.In recent years,scholars proposed that“toxic damage to the brain meridians”is an important pathogenesis of AD from the perspective of traditional Chinese medicine theory.The theory holds that the elimination of harmful substances from the body,such as Aβand inflammation,can improve AD.“Heat clearing and detoxification”therapy guided by the theory of“toxic damage to brain meridians”has been used in clinical practice,such as Huanglian Jiedu Decoction.Meantime,“Chinese Materia Medica”and“Dictionary of Traditional Chinese Medicine”recorded that burdock leaves have the effect of clearing heat and detoxifying,indicating that burdock leaves could improve AD.In addition,modern research has shown that burdock leaves are rich in polyphenols,flavonoids,lignans,and other components,most of which have the effect of improving learning,memory,and cognitive functions.Based on the above analysis,it is suggested that burdock leaf may have the effect of improving AD.However,there are few studies on the anti-AD effects of burdock leaves.Therefore,it is significant to focus on the pharmacological effects,material basis,and mechanism of the burdock leaves improving AD for the utilization of resources and the exploration of new pharmacological activities of burdock leaves.Objective:1.To evaluate the improvement effect of burdock leaves on memory,cognitive function,and pathological injury based on a rat model of AD induced by D-galactose（D-gal）combined with aluminum chloride（AlCl3）;2.To explore the material basis and mechanism of burdock leaves in improving memory and cognitive function based on Ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS/MS）,network pharmacology,and molecular docking;3.To investigate the regulatory effect of burdock leaves on the differential metabolites related to AD,and to explore the regulation mechanism from the perspective of metabolic pathways based on the hippocampal metabonomics operated by LC-MS;4.From the perspective of signaling pathways related to metabolic pathways and core targets,to explore the anti-AD mechanism of burdock leaves in improving AD.Methods:1.A AD rat model was constructed by intraperitoneal injection of D-gal（60 mg/kg）and intragastric administration of AlCl3（20 mg/kg）for 12 weeks,and the pharmacological effect was evaluated based on this model.72 rats were randomly divided into 6 groups,namely Control group,Model group,low-dosage,medium-dosage,and high-dosage groups of burdock leaves（0.75 g/kg,1.5 g/kg,and 3.0 g/kg,respectively）,and donepezil positive control group（1 mg/kg）.To evaluate the improvement effect of burdock leaves on memory decline by behavioral indicators,including new object recognition experiment,passive avoidance experiment,and Morris water maze experiment;The protective effect of burdock leaves on the brain tissue of AD model rats was further investigated through pathological indicators of the hippocampus,including HE staining,Nissl staining,and immunohistochemical staining.2.First,the chemical components of Burdock plants were summarized by exploring the databases,such as PubMed,CNKI,and TCMSP databases.Relevant information,including chemical formulas,molecular weights,and fragment ions,was collected to construct a local database of Burdock plants based on literature and chemical databases,such as Pub Chem and Chemi Spider.Secondly,UPLC-Q-TOF-MS/MS was used to analyze the burdock leaves ingredients in both positive and negative ion modes.The analysis results were sequentially matched with the secondary database TCMMS/MS Library provided by AB SCIEX Corporation,the local database constructed by ourselves,and the GNPS database.The chemical compositions of burdock leaf were determined based on retention time,molecular ion peaks,secondary fragment ions,and mass spectrometry fragmentation rules.On this basis,using network pharmacology technology,corresponding targets of the 61 components identified above were collected through databases such as TCMSP and Swisstarget Prediction,and AD-related genes were obtained using disease databases（TTD,OMIM,Drugbank,Genecard,and Pharma GKB）to obtain anti-AD target genes of burdock leaves;By constructing a“component-target”network,key active ingredients are selected based on the“degree”standard;Constructing a protein interaction network to screen potential targets using the Cyto NCA;Analyzing the pathological correlation between potential targets and aging,Aβprotein,Tau protein;The target genes associated with the three indexes were identified as key targets for subsequent analysis.The human gene expression database was used to analyze the gene expression of key targets in the hippocampus and cortex,and molecular docking was conducted on key components and key targets to further clarify the material basis and mechanism of burdock leaves in improving AD.3.The metabolic profile of hippocampus tissue of rats in the control group,model group,and burdock leaves dose group was characterized based on LC-MS non-targeted metabonomics technology.Through metabolite identification,multivariate statistical analysis,and metabolic pathway enrichment analysis,the metabolic pathways regulated in the process of improving AD by burdock leaves were found.The metabolic pathway network was constructed to clarify the mechanism of improving AD by burdock leaves at the metabonomics level.Secondly,further focus on key metabolic pathways based on the analysis of metabolic pathway networks and the number of differential metabolites involved in metabolic pathways.4.Integrate the screening results of key targets in network pharmacology and metabolomics analysis results,and combine with the literature to identify signal pathways closely related to burdock leaves improving AD.Western blot technology,a molecular biological method,was used to analyze the expression of proteins related to this signal pathway,and the effect of burdock leaves on the expression of key proteins in the hippocampus of the AD rat model was deeply studied.Results:1.The results of behavioral experiments showed that rats in the model group exhibited memory decline and cognitive dysfunction compared with the control group,specifically manifested by a decrease in the cognitive index of non-spatial learning and memory（new object recognition experiment）,a shorter latency of conditional fear memory（passive avoidance experiment）,a longer latency of spatial learning and memory,a decrease in the number of crossing platforms,and a shorter dwell time in the target quadrant（Morris water maze experiment）.After burdock leaves were administered,memory abilities and cognitive impairment improved,and the effects of medium-dose groups and high-dose groups were better than those of low-dose groups.Pathological indicators showed that compared with the control group,the model group exhibited significant AD features in the hippocampal tissue,such as pathological injury,neuronal loss,and Aβabnormal deposition,excessive phosphorylation of Tau protein;burdock leaves can significantly reduce pathological damage caused by AD.2.Based on LC-MS/MS technology,a total of 61 chemical components were identified from the burdock leaves,including 12 flavonoids,12 phenolic acids,8 amino acids,5 lignans,5 fatty acids,4 organic acids,3 coumarins,and other components.The results of network pharmacology research showed that the 61 components correspond to792 targets,and 1661 AD-related target genes were obtained from TTD,OMIM,Drugbank,Genecard,and Pharma GKB.After crossing,345 anti-AD genes were obtained.Protein interaction network analysis found 36 potential targets,including 30 genes related to“aging”,9 targets related to“Aβpathology”,and 9 targets related to“Tau protein pathology”.However,there are only 4 targets related to aging,Aβpathology and Tau protein pathology,namely STAT3 and RELA（NF-κB p65）,MAPK8,and AR.STAT3,RELA and MAPK8 have significant differences in the hippocampus and cortex of AD patients and normal people at gene level.“Component-target”network analysis found that10 active components in the network have higher“degree”values,namely quercetin,kaempferol,isorhamnetin,naringin,isoferulic acid,sage phenol,benzoyllinoleide,caffeic acid ethyl ester,6,7-dimethylcoumarin and caffeic acid.Molecular docking results showed that 10 key active ingredients had strong physical binding capabilities with 4 key targets.3.Metabolomic analysis revealed that a total of 23 differential metabolites were identified,which indicated that metabolic disorders occur in the hippocampus of AD rats.Among 23 metabolites,burdock leaves could significantly reverse 15 metabolites（9metabolites in the low-dose group,12 metabolites in the medium-dose group,and 10metabolites in the high-dose group）,involving 6 metabolic pathways,namely phenylalanine,tyrosine,and tryptophan biosynthesis;taurine and taurine metabolism;phenylalanine metabolism,histidine metabolism,starch and sucrose metabolism,and TCA cycle.Among them,the TCA cycle regulates the most metabolites,namely citric acid,α-ketoglutaric acid and fumaric acid.In addition,integrated metabolic network analysis found that certain metabolism（taurine and taurine metabolism;starch and glucose metabolism;and phenylalanine,arginine,and serine biosynthesis）are mainly involved in regulating the TCA cycle by affecting the production of acetyl-Co A;another metabolism（phenylalanine,aspartate,and serine biosynthesis;and phenylalanine metabolism）regulate the TCA cycle by affecting the levels of fumaric acid and succinic acid;while the histidine metabolism can affect the level ofα-ketoglutaric acid and subsequently interfere the TCA cycle.Therefore,TCA cycle may be a key metabolic pathway for burdock leaves to ameliorate AD.4.Numerous studies have shown that TCA cycle also participates in the regulation of inflammatory responses.Three of the four key targets screened in network pharmacology（STAT3,RELA and MAPK8）are associated with inflammation.Therefore,STAT3/NF-κB pathway and inflammatory factor-related proteins were analyzed by Western blot.The results showed that the protein expression of IL-1β,IL-6,and p-NF-κB in the hippocampus was significantly increased compared with control group,while the protein expression of STAT3 protein did not change significantly.After administration of burdock leaves,the protein expression of IL-1β,IL-6,and p-NF-κB was significantly inhibited.Conclusion:1.The AD animal model induced by D-gal/AlCl3 could cause learning and memory decline,cognitive dysfunction,and typical pathological injury of AD.Burdock leaves can improve memory decline and alleviate pathological damage caused by AD.2.The main active ingredients of burdock leaves improved AD may be quercetin,kaempferol,isorhamnetin,naringin,isoferulic acid,sage phenol,benzoyllinoleide,caffeic acid ethyl ester,6,7-dimethylcoumarin,and caffeic acid.3.Comprehensive studies of network pharmacology,metabolomics,and molecular biology have shown that burdock leaves improve AD by regulating metabolic disorders dominated by TCA cycle and inhibiting NF-κB-mediated inflammatory response. |
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