The Effect And Mechanism Of Cervi Cornu Pantotrichum Extract On Improving Learning And Memory Ability In Aging Mice

Objective: Aging is a natural physiological phenomenon in the body.During the aging process,the brain development is hindered and neurons shrink,leading to various neurodegenerative diseases.The most prominent manifestation is the decline in learning and memory abilities.Learning and memory belong to the advanced neural activity of the brain,and the mechanism of learning and memory decline mainly revolves around aspects such as neuroinflammation,oxidative stress,and synaptic plasticity.Deer antler has pharmacological effects such as anti-aging,enhancing immunity,promoting cell differentiation,and promoting nervous system regeneration.The research on the effect and mechanism of deer antler on the learning and memory ability caused by aging is relatively weak.Based on this,this article conducts relevant research:1.Extract different active parts of deer antler,study the improvement effect of different extracts of deer antler on the learning and memory ability of D-gal-induced aging mice,and screen the activity of deer antler to improve learning and memory ability;2.Using UHPLC-MS and GC-MS techniques to analyze the components of deer antler extract,and combining network pharmacology and molecular docking to explore the potential targets and mechanisms of deer antler extract in improving learning and memory ability;3.Using BDNF(+/-)gene mice as controls,the mechanism of action of deer antler extract on the learning and memory abilities of aging mice was verified from the perspectives of neurotrophic factor signaling pathways,synaptic plasticity,and neuroinflammation;4.Study the improvement of deer antler extract on D-galactose-induced HT22 cell damage and verifying the results of in vivo experimental mechanism research.Methods:1.Extract different extracts of deer antler using ultrasound and colloid grinding methods;The aging model was replicated by intraperitoneal injection of D-galactose into C57BL/6 wild-type mice.Different extracts of deer antler were orally administered,and Morris water maze experiment,ELISA enzyme-linked immunosorbent assay,and HE staining were used to screen the active sites of deer antler that improve the learning and memory ability of aging mice.2.Use UHPLC-MS and GC-MS techniques to analyze the components of alcohol extracts from deer antler;Data platforms such as Swiss ADME and Swiss Target Prediction screen for major chemical components and targets,and use the Metascape data platform for KEGG pathway enrichment analysis and GO enrichment analysis.Cytoscape 3.7.1 software constructs a multi-level network of "component target disease pathway" to identify key targets and obtain relevant signaling pathways;Using Auto Dock software for molecular docking validation,analyze the binding ability of components to target proteins.3.The aging mouse model was induced by intraperitoneal injection of D-gal into C57BL/6 wild-type mice,with BDNF(+/-)mice as the control.The aging model was constructed using the same method,and the same dose of deer antler extract was given.The Morris water maze experiment investigated the effect of deer antler extract on improving learning and memory abilities.ELISA was used to detect the levels of inflammatory factors in brain tissue,HE staining was used to observe hippocampal tissue damage,RT-q PCR was used to detect m RNA transcription levels of BDNF,Trk B,and SYN in brain tissue,IHC and Western blot were used to detect protein expression of BDNF,Trk B,and SYN,to explore the possible mechanism of deer antler alcohol extract in improving learning and memory in aging mice.4.Using HT22 cells as the research object,a aging injury model of HT22 neurons was constructed using D-galactose,and the CCK8 method was used to determine the dosage and duration of administration.Adopt β-Galactosidase staining was used to detect the degree of cell aging,flow cytometry was used to detect cell apoptosis,RT-q PCR was used to analyze m RNA transcription levels of BDNF,Trk B,SYN,et al.Western blot technology was used to detect protein expression of BDNF,Trk B,SYN,et al.Consistent with in vivo experimental results.Results:1.Extracted deer antler alcohol extract and deer antler water extract were used to evaluate the effects of deer antler alcohol extract,deer antler water extract,and deer antler powder on the learning and memory abilities of aging mice using the D-gal aging model.The Morris water maze experiment showed that deer antler powder could increase the number of times aging mice crossed quadrants(P<0.01),deer antler water extract could shorten the latency period of aging mice,and increase the number of times crossing quadrants(P<0.05,P<0.01),and deer antler alcohol extract could shorten the latency period of aging mice,increase the number of times crossing the platform,and crossing quadrants(P<0.01).ELISA showed that deer antler powder could enhance the activity of SOD in mouse brain tissue and serum,reduce MDA content,increase GSH-Px content in serum,while reducing the content of IL-1β in mouse brain tissue and serum,and reducing the content of TNF-α in brain tissue(P<0.05 or P<0.01);deer antler water extract could enhance the SOD activity in the brain tissue of aging mice,reduce the MDA content in brain tissue and serum,increase the GSH-Px content in serum,while reducing the inflammatory factors such as IL-1β,IL-6,and TNF-α in the brain tissue and serum of aging mice(P<0.05 or P<0.01);deer antler alcohol extract could enhance the SOD activity in mouse brain tissue and serum,reduce MDA content,increase GSH-Px content,while reducing the content of IL-6,TNF-α in brain tissue and serum,and decreasing the content of IL-1β in brain tissue(P<0.05 or P<0.01),exerting anti-inflammatory effects.HE staining confirmed that deer antler powder,deer antler water extract,and alcohol extract could improve the number of hippocampal neurons in aging mice,and the cellular structure was relatively intact.Based on the above studies,deer antler alcohol extract was selected as the pharmacologically active substance basis for deer antler,and subsequent studies were conducted on this basis.2.83 components in deer antler alcohol extract were identified through UHPLC-MS,and 71 chemical components were identified through GC-MS analysis,mainly including acyl lipids,carboxylic acids and derivatives,steroids and steroid derivatives,etc.Compound target prediction resulted in 661 targets,639 targets related to learning and memory ability diseases,132 intersecting targets,and PPI analysis identified core targets such as BDNF,SRC,AKT1,TNF,TP53.Pathway enrichment analysis results showed that deer antler alcohol extract may improve learning and memory ability by regulating the neurotrophic factor signaling pathway,the pathway of neurodegenerative diseases,long-term potentiation,and the PI3K/AKT signaling pathway.Molecular docking verified that β-Estradiol,Estrone,and BDNF,AKT1 have good binding ability.3.BDNF(+/+)mice and BDNF(+/-)mice aging models were constructed,and the same dose of deer antler alcohol extract was administered.The Morris water maze experiment showed that deer antler alcohol extract could shorten the latency period of aging model mice,significantly increase the number of times crossing the platform and crossing quadrants(P<0.05 or P<0.01).Except for the number of times crossing the platform,mice in the BDNF(+/-)dosing group showed opposite performance under the same conditions(P<0.05).ELISA found that deer antler alcohol extract could down-regulate the levels of inflammatory factors such as TNF-α,IL-6,and IL-1β in the brain tissue of aging model mice(P<0.05 or P<0.01);HE staining also confirmed that deer antler alcohol extract improved the number and morphology of hippocampal neurons in aging mice.RT-q PCR,IHC,and Western blot found that deer antler alcohol extract could up-regulate the m RNA transcription levels and protein expression of BDNF,Trk B,SYN,and PSD-95,and significantly down-regulate the m RNA transcription levels and protein expression of PI3 K and AKT1(P<0.05 or P<0.01).The dosing group of BDNF(+/-)mice showed the opposite trend compared to normal mice under the same conditions(P<0.05 or P<0.01),and the dosing group of BDNF(+/-)mice showed improvement compared to the model group of BDNF(+/-)mice,but the difference in results was not significant.4.An HT22 cell damage model was established using 100 mmol/L D-gal,and the deer antler alcohol extract solution was administered after modeling for 48 hours.β-galactosidase staining showed that deer antler alcohol extract could inhibit HT22 cell aging,and flow cytometry experiments found that deer antler alcohol extract could reduce the apoptosis rate of HT22 cells(P<0.01).RT-q PCR and immunoblot experiments found that deer antler alcohol extract could up-regulate the m RNA transcription levels and protein expression of BDNF,Trk B,SYN,and PSD-95,and significantly down-regulate the m RNA transcription levels and protein expression of PI3 K and AKT1(P<0.05 or P<0.01),which was basically consistent with in vivo experiments.Conclusion: Different extracts of deer antler were obtained through research,and after screening,it was determined that the alcohol extract of deer antler is the pharmacological material basis for improving the learning and memory ability of aging mice with deer antler.UHPLC-MS identified 83 components in the alcohol extract of deer antler,while GC-MS analysis identified 71 chemical components,mainly including fatty acyls,carboxylic acids and their derivatives,steroids,and steroid derivatives.Network pharmacology and molecular docking have found that the alcohol extract of deer antler mainly acts on core targets such as BDNF and AKT1,and improves learning and memory ability through multiple signaling pathways such as neurotrophic factor signaling pathway,neurodegenerative pathway-multiple diseases,long-term enhancement,PI3K/AKT signaling pathway,etc.It has the characteristics of multi-component,multi target,and multi pathway.Animal and cell experiments have shown that the alcohol extract of deer antler may regulate the neurotrophic factor signaling pathway through BDNF/Trk B,enhance synaptic plasticity,and have an impact on the PI3K/AKT pathway,reducing neuroinflammatory responses,protecting stress-induced neuronal damage,and thereby improving the learning and memory abilities of aging mice.