| Objective:In this study,based on PI3K/AKT signaling pathway,we explored the effect of Velvet antler peptide(VAP)on neuron repair and protection,and explained the mechanism of VAP in improving Mild Cognitive Impairment(MCI),which provided evidence for clinical prevention and treatment of MCI.Method: In this study,we used in vivo and in vitro experiments to explore the mechanism of VAP improving MCI by regulating PI3K/AKT signaling pathway.1 VAP identification and detectionIdentification of physical and chemical properties of VAP by biuret method;The molecular weight of VAP was determined by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).The content of different components in VAP was determined by pre-column derivatization-high performance liquid chromatography(PD-HPLC).2 Study on the mechanism of antler improving MCI based on network pharmacologyScreening the active components and targets of pilose antler by TCMID database and Pubchem database;Screening MCI action targets by OMIM database,Disgenet database and Genecards database;Screening the target of antler-MCI by Venny 2.1 online mapping tool platform;The Protein-protein interaction(PPI)network is constructed by using STRING 11.0 protein interaction platform,and the core targets of PPI network are screened based on topology analysis and cluster analysis by using Cytoscape 3.8.0 network topology attribute analysis software.The network model of "traditional Chinese medicine-component-target-disease" is constructed by using the network topology attribute analysis software of Cytoscape 3.8.0,and the core components are analyzed;With r language and Bioconductor bioinformatics software,Gene ontology(GO)enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis were carried out to summarize the biological processes and major metabolic pathways involved in common targets.3 Based on PI3K/AKT signaling pathway,the mechanism of VAP improving cognitive dysfunction in rats with different MCI models was studiedDifferent MCI rat models were induced by intraperitoneal injection of scopolamine and unilateral ligation of common carotid artery.Open field experiment and Morris water maze experiment were used to observe the influence of VAP on behavioral changes of different MCI model rats Hematoxylin-eosin staining(HE)was used to observe the effect of VAP on pathological changes in hippocampus of rats with different MCI models.ELISA was used to detect the effects of VAP on SOD,MDA,brain-derived neurotrophic factor(BDNF),Nerve growth factor(nerve growth factor),NGF),vascular endothelial growth factor(VEGF),Testosterone(T)and Cortisol;Western Blot was used to detect the effect of VAP on the expression level of PI3K/AKT signaling pathway related proteins in hippocampus of different MCI model rats.4 Based on metabonomics analysis,VAP improves cognitive dysfunction in MCI model ratsUltra-high performance liquid chromatography coupled with tandem quadrupole time-of-flight secondary mass spectrometry(UPLC-Q/TOF-MS)metabonomics technology was used to detect the brain tissue and kidney of MCI model rats.The dynamic changes of differential metabolites and metabolic pathways among the groups were analyzed.5 The mechanism of VAP antagonizing damaged HT22 cells induced by Okadaic acid(OA)was discussed based on PI3K/AKT signaling pathwayThe model of HT22 cell injury induced by OA was established.after administration,the activity of HT22 cells was detected by CCK-8 method,and the Superoxide dismutase(SOD),Malondialdehyde(malondialdehyde),MDA)and Glutathione peroxidase(GSH-PX).Western Blot was used to detect the expression level of PI3K/AKT signaling pathway related proteins in HT22 cells.Result:1 VAP identification and detectionThe physical and chemical properties of VAP selected in the experiment are protein,with a molecular weight of about 3.2k Da,which is composed of 17 kinds of amino acids,among which there are 7 kinds of essential amino acids,the content is 393.39±1.65g/kg,the total content of amino acids is 826.55±10.75g/kg,and the content of different components is stable,which can be used in this experimental study.2 Study on the mechanism of antler improving MCI based on network pharmacologyMost active ingredients in velvet antler are important building materials in VAP.Using interactive network,it is found that active ingredients in velvet antler can improve MCI through PI3K/AKT signaling pathway,and the mechanism may be related to nerve signal transduction,neuron injury,oxidative stress process and sex hormones in vivo.This study provides methods and ideas for exploring the improvement of MCI by VAP.3 Based on PI3K/AKT signaling pathway,the mechanism of VAP improving cognitive dysfunction in rats with different MCI models was studiedThe VAP can improve the learning and memory abilities of rats with different MCI models,improve the constriction of hippocampus,increase the contents of SOD,BDNF,NGF,VEGF,T and Cortisol in serum of rats with different MCI models(P < 0.05 or P <0.01),and decrease the content of MDA(P < 0.05 or P < 0.01).The expressions of Bcl-2and m TOR were increased(P < 0.05 or P < 0.01),while the expressions of Bad,Bax and Caspase-3 were decreased(P < 0.05 or P < 0.01),and the expression of PI3K/AKT signaling pathway was up-regulated(P < 0.05 or P < 0.01).4 Based on metabonomics analysis,VAP improves cognitive dysfunction in MCI model ratsThe mechanism of VAP on improving cognitive dysfunction in MCI model rats may be related to regulation of folate and lipid metabolism in brain,cyclic adenosine monophosphate energy cycle,amino acid biosynthesis,and kidney neutral hormone secretion.5 The mechanism of VAP antagonizing damaged HT22 cells induced by Okadaic acid(OA)was discussed based on PI3K/AKT signaling pathwayVAP can improve the survival rate of damaged HT22 cells(P < 0.01),enhance the activities of SOD and GSH-PX(P < 0.01),reduce the content of MDA(P < 0.01),and decrease Bad,Bax and Caspase-3(P < 0.01),and the expression of PI3K/AKT signaling pathway was up-regulated(P < 0.01).Conclusion: VAP may regulate PI3K/AKT signaling pathway by "replenishing essence and replenishing marrow",antagonize damaged HT22 cells induced by OA,improve learning and memory ability of different MCI model rats,regulate kidney function,promote the growth,proliferation and differentiation of neuron cells,protect central nervous system,and its mechanism may be related to regulating folic acid and lipid metabolism in brain,cyclic adenosine monophosphate energy cycle,amino acid biosynthesis,and kidney neutral hormone secretion. |
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