| Objective:To evaluate sagacious confucius’ pillow elixir(SCPE)effect on mild cognitive impairment mainly based on metabolomics and systems pharmacology,analyze metabolites and pathways of different sagacious confucius’ pillow elixir and combining with bioinformation mining provides the reference for the basic and clinical research of mild cognitive impairment.Methods:Seven month of age male SAMP8 mice as the research object,with seven months male SAMR1 mice for the control,using Morris water maze,Neuropathological staining,ELISA,TUNEL and Western bloting technology for sagacious confucius’ pillow elixir intervention on model of mild cognitive impairment as the pharmacological effects evaluation,from the perspective of chronic inflammation induced neuronal pyroptosis to research the pharmacological mechanism of sagacious confucius’ pillow elixir improve cognitive function.Based on The ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry technology to reveal the effects of sagacious confucius’ pillow elixir on mice with mild cognitive impairment.Metabolomic biomarkers and pathways of brain tissue were screened and identified to elucidate the change mechanism of biomarkers and pathways of sagacious confucius’ pillow elixir in different doses.Based on the ADME/T model,systems pharmacology methods were used to screen the main components of sagacious confucius’ pillow elixir that play a role in improving cognitive function,so as to explore more possible mechanisms of action.Results:1.Morris water maze results: compared with the control group before administration(0d),the escape latency was significantly prolonged in the model group,but the number of crossing the platform was not statistically different(P>0.05).After 7 days of administration,compared with the control group,the learning and memory ability of mice in the model group and the low,medium and high dose groups(LD/MD/HD)decreased significantly(P<0.01),but the improvement of learning and memory ability among SCPE was not obvious.After 14 days of administration,SCPE showed significant improvement in learning and memory(P<0.01).SCPE improved the learning and memory ability of mice at 21 d and 28 d after administration,and was positively correlated with the dose(P<0.01).Neuropathological results:compared with the control group,the number of neurons in the hippocampal CA1~CA3 area of the model group was significantly reduced,and abnormal pathological changes such as degeneration,necrosis,large deposition of amyloid substance,decreased nidosomes and granulation were observed in the model group.The above pathological changes were improved after intervention with different doses of SCPE,especially in the high dose group.TUNEL staining results: compared with the control group,the number and ratio of TUNEL positive cells in the hippocampal CA1~CA3 region were significantly increased in the model group(**P < 0.01).Compared with the model group,the number of TUNEL positive cells in the hippocampus of the low,medium and high dose SCPE groups was significantly reduced(##P < 0.01)and the ratio was also significantly reduced(##P < 0.01),which has dose dependent to a certain extent.ELISA results: serum levels of inflammatory mediators IL-1β,IL-6,IL-18 and TNF-a were significantly higher in the model group than in the control group(P < 0.01).Compared with the model group,the levels of IL-1β,IL-6,IL-18 and TNF-a in serum decreased after different doses of SCPE administrated 28 days,and the high dose group(HD)showed the most obvious effect.The changes of IL-6 and IL-18 levels were strongly correlated with the dose of SCPE.Western blot results:compared with the control group,the expression levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,IL-18 and Aβ in the hippocampus of the modelgroup were significantly higher(P < 0.01).After continuous administration of SCPE for 28 days,the expressions of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,IL-18 and Aβ proteins in the hippocampal of each dose group was down-regulated to varying degrees,especially in the high dose group.2.After metabolomics analysis,we found 84 MCI biological metabolic markers,including angiotensin,leukotriene D4,hexadecanoic acid 2,(3 z,6 z)3,6-nonyl diene aldehyde,isopropyl adrenaline,D-ornithine,methoxyl amine,citrulline,coniferyl aldehyde,isopropyl malic acid,N-acetyl-L-histidine,etc al.8 main metabolic pathways related to MCI were obtained,which were D-arginine and D-ornithine metabolic pathways;Alanine,aspartate and glutamate metabolic pathways;Phenylalanine metabolic pathway;Arginine and proline metabolic pathways;Biosynthesis of valine leucine and isoleucine;Cysteine and methionine metabolic pathways;Aminoacyl t RNA biosynthesis;D-glutamine and D-glutamic acid metabolic pathways.Among them,the low dose SCPE can callback 40 metabolic markers,including: Hexadecanedioic acid,Eicosadienoic acid,L-Isoleucine,Leukotriene D4,11Z-Eicosenoic acid,D-Phenylalanine,Isopropylmaleate,trans-Cinnamic acidacetyldiacid,etc.Four metabolic pathways were obtained by metabolite analysis: Phenylalanine metabolism;Alanine,aspartate and glutamate metabolism;Valine,leucine and isoleucine biosynthesis;D-Glutamine and D-glutamate metabolism.Medium dose of SCPE can callback 48 biological metabolic markers including:Hexadecanedioic acid,Eicosadienoic acid,L-Isoleucine,Leukotriene D4,11Z-Eicosenoic acid,D-Phenylalanine,Isopropylmaleate,trans-Cinnamic acidacetyldiacid,etc.Four main metabolic pathways were obtained by metabolite analysis:Phenylalanine metabolic pathway;Alanine,aspartate and glutamate metabolic pathways;Cysteine and methionine metabolic pathways;Dglutamine and D-glutamic acid metabolic pathways.High dose SCPE can reverse 54 biomarkers,including: Hexadecanedioic acid,Eicosadienoic acid,L-Isoleucine,Leukotriene D4,11Z-Eicosenoic acid,trans-Cinnamic acid,N1-Acetylspermine,L-Phenylalanine,Gibberellin A15 open lactone,etc.Four main metabolic pathways were obtained by metabolite analysis:Phenylalanine metabolic pathway;Alanine,aspartate and glutamate metabolic pathways;Cysteine and methionine metabolic pathways;Dglutamine and D-glutamic acid metabolic pathways.3.By means of system pharmacology,28 active components in sagacious confucius’ pillow elixir(OB≥30%、DL≥0.1、BBB≥-0.3)were screened according to the ADME/T(absorption/distribution/metabolism/excretion/toxicity)model,and 84 targets were predicted.72 target proteins directly and indirectly associated with MCI were collected and set up target protein libraries.Cytoscape 3.7.1 software was used to construct MCTD network of "drug-component-target-disease".Three most significant active compounds were 2’-O-Methyliso liquiritigenin and alloaromadendrene from Acorus tatarinowii,6-Dihydroxy-3,7-dimethoxyxanthone from Polygala tenuifolia Willd.PTGS2,GABRA1 and CHRM1 were the top three important targets.72 target proteins were imported into STRING database to construct a protein interaction network.Cytoscape 3.7.1 software was used for visual processing and topological analysis,and 15 important targets whose degrees and betweenness centrality were greater than their average were screened out.According to PPI network graph,TP53,NOS3 and Caspase-3 were the top three targets.By DAVID,Metascape,JTre View platform respectively to extract the 15 key targets of PPI network conduct Heatmap,GO,Kegg analysis,found the main GO items:regulate the blood circulation,participate in synaptic membrane composition and synaptic transmission,organic polyol of transhipment,balance in the body of a multicellular organism,response to toxic substances,etc.The Kegg items included neuroactivity of receptor ligand,c AMP signaling pathway,c GMP-PKG signaling pathway, prostate cancer,morphine addiction,thyroid hormone signaling pathway,p53 signaling pathway,and IL-17 signaling pathway.Conclusion:1.Sagacious confucius’ pillow elixir play pharmacological role in improving cognitive function by resisting the classic pyroptosis NLRP3/Caspase-1 signaling pathway mediating Aβ protein deposition.Moreover,the high dose of Sagacious confucius’ pillow elixir has a more obvious effect on improving cognitive function.2.The pathogenesis of mild cognitive impairment may be caused by the changes in the content of metabolic markers such as angiotensin,leukotriene D4 and cetane diacid in brain tissue,which affect the changes in related metabolic pathways.3.The mechanism of Sagacious confucius’ pillow elixir in the treatment of mild cognitive impairment is to regulate the metabolism of phenylalanine,alanine,aspartate and glutamate,D-glutamine and D-glutamate by regulating the levels of metabolic markers such as cetyl diacid,eicosauric acid and isoleucine.4.Sagacious confucius’ pillow elixir could through2’-O-Methylisoliquiritigenin,-Alloaromadendrene,6-Dihydroxy-3,7-dim ethoxyxanthone active ingredients act on PTGS2,GABRA1,CHRM1,TP53,NOS3,Caspase-3 receptor play the "multicomponent-multitargets-multipathways" effect. |
