Study On Screening Of Pharmacodynamic Substances And Mechanism Of Anti-brain Aging In Red Ginseng

Aging is an irreversible process in which the function of molecules,cells,tissues,organs and even the whole-body changes with age and tends to die.Aging is closely related to many common geriatric diseases,including Alzheimer’s disease,coronary heart disease,diabetes,cancer and osteoporosis,which seriously affect the quality of life in old age.The process of aging is regulated by many factors inside and outside the body,mainly related to the excessive production of reactive oxygen species（ROS）in vivo,which is the free radical theory.The brain is highly susceptible to oxidative damage due to its high demand for oxygen and unsaturated lipids and relatively inadequate levels of antioxidant defense systems.Therefore,brain aging is one of the most typical manifestations of the aging process.Although the process of aging is irreversible,appropriate drug intervention can delay the aging process and prevent the occurrence and development of related diseases.In the research field of anti-aging drugs,natural drugs have been widely concerned because of their long history and relatively few side effects.Among these drugs,Panax ginseng.C.A Meyer as the traditional Chinese medicine with the most national characteristics in China,ranks first in the top grade of Shen Nong’s Herbal Classic because of its functions of"tonifying the five internal organs,calming the spirit,calming the soul,relieving palpitation,eliminating evil,improving eyesight and intelligence,and prolonging life after long-term use".During the processing of panax ginseng,some metabolic enzymes are decomposed and inactivated by heating and steaming,resulting in secondary ginsenoside conversion and Maillard reaction,which converts unstable components into stable components,thus preserving the active components and enhancing the drug activity,of which red ginseng is the most representative.Red Ginseng（RG）is the processed product obtained by steaming the fresh root of panax ginseng.Ginsenosides are the most important active components of red ginseng.Modern pharmacological studies have shown that non-saponins,especially amino acids,are also one of the important material bases for red ginseng to exert its efficacy.In addition,a number of studies suggested that red ginseng might play the anti-aging effect by improving oxidative stress response,but the specific efficacy and mechanism have not yet been confirmed.Due to the active ingredients in red ginseng are rich and complex,it is not clear which active ingredients play a leading role in the"anti-aging"efficacy of red ginseng.Therefore,based on the free radical theory of aging,this study scientifically and comprehensively verified the pharmacodynamic material basis and molecular mechanism of action of red ginseng in delaying brain aging,which is of great significance for interpreting the modern scientific connotation of"taking ginseng for a long time to lighten the body and prolong life"recorded in ancient books and for the subsequent development of anti-aging drugs for red ginseng.In this study,a variety of in vitro and in vivo models of brain aging were constructed,with oxidative stress as the central point,from the animal level,cellular level,molecular level and other dimensions,the pharmacodynamic material basis of red ginseng for delaying brain aging and the specific molecular mechanism of its efficacy were revealed,and the modern medicinal value of red ginseng and its active ingredients was further excavated.It also provided a potential target for the clinical exploration of brain aging and the treatment of related chronic diseases.The main research contents are as follows:1.Pharmacodynamic verification of red ginseng extracts for delaying brain aging induced by D-gal in miceIn this study,the GEO database combined with KEGG and GO analysis were first used to analyze the differential genes between normal rats and aging rats in order to find the potential molecular targets of aging.The results showed that the biological process（BP）of gene differences between normal rats and aging rats was closely related to the production of reactive oxygen species,cell proliferation,neuronal apoptosis and long-term memory,which indicated that the excessive production of reactive oxygen species might be positively correlated with the aging process,and further supported the statement that brain aging was one of the most typical manifestations of the aging process.Based on the above information,the D-gal-induced subacute aging mouse model was established in this study.Immunofluorescence double-staining was used to verify the specific expression sites of aging protein p21 in hippocampal neurons by using MAP2 as a neuronal marker,so as to confirm the successful establishment of the D-gal induced brain aging model.At the same time,the learning and memory ability of mice was monitored by water maze and cholinergic kit after the intervention of different extracts of red ginseng（water extract and alcohol extract）.The results showed that D-gal（800 mg/kg）could significantly promote the expression of aging marker p21 in hippocampal neurons,indicating the successful establishment of D-gal induced brain aging model in mice.Both alcohol extract of red ginseng（RGAE,200 mg/kg）and water extract of red ginseng（RGWE,200 mg/kg）could effectively alleviate D-gal induced the decrease of basic metabolic indexes and learning and memory function in mice.Further histopathological staining and Nissl staining showed that red ginseng extract could significantly improve the pathological changes of hippocampal neurons and the decrease of neuronal density caused by neuronal loss.Western blot（WB）was used to monitor the expression of aging markers p53,p21,p16 proteins,senescence secretion phenotype（SASP）protein（IL-6）and cell cycle protein CDK4 in mouse hippocampal neurons to verify the anti-aging effect of red ginseng extract on D-gal induced brain aging.The results showed that the expression of senescence markers p53,p21,p16 and inflammatory senescence secretion phenotype IL-6 protein were increased after D-gal treatment,while the expression of cell cycle protein CDK4 was decreased.The expression levels of these proteins could be reversed in different degrees after treatment with different red ginseng extracts,showing good anti-aging activity.On the basis of verifying the anti-aging efficacy of red ginseng,Liquid Chromatograph Mass Spectrometer（LC-MS）was used to identify the main active components of the two red ginseng extracts,which provided a material basis for the later screening of effective substances in red ginseng.2.Establishment of brain senescence cell models and screening of anti-senescence activities of pharmacodynamic substances from red ginsengBased on the free radical theory,the subacute aging model of mouse hippocampal neuron（HT22）was established by using hydrogen peroxide（H₂O₂）and D-gal.Western blot was used to detect the expression changes of aging markers p53,p21 and p16,the inflammatory senescence secretion phenotype IL-6,and the cyclin CDK4 to verify whether the model was established successfully.The results showed that H₂O₂（3 h,40μM）or D-gal（3 days,55 m M）could significantly promote the protein expression of p53,p21,p16,IL-6 and MMP3 while reducing the protein expression of CDK4 in HT22 cells,which indicated the successful establishment of subacute cell senescence model.According to the division of ginsenosides and main products of ginseng Maillard reaction,the main active components in the identified extracts of red ginseng were classified and screened,and the potential active substances that could resist the decrease of cell viability were screened using MTT and lactate dehydrogenase（LDH）kits.The expression changes of six related proteins（p53,p21,p16,IL-6,MMP3,and CDK4）were detected by western blot to re-verify the screened active substances.The optimal active components of each model were initially screened for preliminary cross-fitting,and six potential active substances were obtained,namely ginsenoside Rg2（S type and R type）,20（S）-protopanaxadiol（PPD）,20（S）-protopanaxatriol（PPT）,Arginyl-fructosyl-glucose（AFG）and maltol.Then,cell apoptosis and cell cycle were detected by flow cytometry,and the six active ingredients（20μM）screened from the two models were compared and screened.Finally,the three best potential active substances were determined,namely 20（S）-ginsenoside Rg2（S-Rg2）,PPT,and AFG.The above results indicated that these three active substances might be the pharmacodynamic material basis of red ginseng for delaying brain aging,which laid a foundation for subsequent research.3.Study on pharmacodynamic effect of potential active substances from red ginseng in delaying brain aging induced by D-gal in miceBased on the protective effect of potential active substances（S-Rg2,PPT,AFG）on the neuronal cell senescence in mice induced by active oxygen inducers,this chapter further explored the delaying effect of these substances on the brain aging of subacute aging mice in vivo.The results show that potential active substances（S-Rg2（10 mg/kg,20 mg/kg）,PPT（10mg/kg,20 mg/kg）,AFG（40 mg/kg,80 mg/kg））could significantly restore D-gal-induced memory impairment,choline dysfunction,and redox system imbalance in mice.Simultaneously,the potential active substances also observably reduced the D-gal induced the loosening of hippocampal neuronal structure,the decrease of neuronal density,the rupture of nucleus,the loss of Nissl bodies,and other pathological changes.At high doses（S-Rg2（20 mg/kg）,PPT（20mg/kg）,AFG（80 mg/kg））,potential active substances had more significant inhibitory effects on age-related proteins（p53,p21,p16,IL-6）.Furthermore,the number of mitochondria,the degree of mitochondrial damage and the number of autolysosomes in hippocampal neurons of mice were analyzed by transmission electron microscopy,and autophagy substrates（ATG3,ATG5,ATG7）were detected by western blot.The key proteins（LC3,p62）of autolysosome formation and lysosomal functional proteins（TFEB,LAMP2）indicated that the delay of brain aging in D-gal induced subacute senescence mice by potential active substances was related to ROS level,mitochondrial autophagy and lysosome biogenesis.The results fully supported the previous results in vitro,and preliminarily revealed that the molecular mechanism of potential active substances might be related to the mitochondria-lysosome damage mechanism induced by high throughput reactive oxygen levels,which provided a new idea for systematically exploring the molecular mechanism of red ginseng and its active substances in delaying brain aging.4.Study on the molecular mechanism of anti-aging effect of potential active substances from red ginsengThe natural senescence model of mouse embryonic fibroblast（ICR-MEF）cells with highly restricted passage characteristics was constructed.The senile ICR-MEF cells were identified as P4 generation by morphological analysis,cytoskeleton（F-actin）,western blot and real-time Quantitative polymerase chain reaction（q PCR）.Intervention with potential active substances（S-Rg2,PPT,AFG,20μM）could markedly inhibit the retention of senile ICR-MEF cells in G1phase and increase the expression of cell cyclin protein CDK4,thus promoting the operation of cell cycle.Meanwhile,the protein expression of p53,p21,p16 and IL-6 were decreased,which further proved the protective effect of potential active substances on senile ICR-MEF cells.The result of Mito SOX Red（for the detection of mitochondrial ROS）further revealed the protective effect of potential reactive substances on mitochondrial oxidative damage.The degree of mitochondrial damage was monitored by transmission electron microscopy（TEM）analysis.The results showed that the intervention of potential active substances could markedly improve the degree of mitochondrial swelling,alleviate the dissolution of mitochondrial membrane and matrix overflow in senile ICR-MEF cells.The results of DQ Red BSA detection showed that the potential active substance could observably promote the lysosomal digestion function of senile ICR-MEF cells.Confocal laser scanning microscopy combined with Mito-Tracker Green and Lyso-Tracker Red probes were used to detect the effects of potential active substances on the functional interaction between mitochondria and lysosomes in senile ICR-MEF cells.The results of the probe showed that the potential active substance could dramatically increase the fluorescence intensity of the mitochondria-lysosome probe,indicating that the potential active substance could significantly improve the functional interaction between mitochondria and lysosomes in senile ICR-MEF cells.Moreover,the results of protein expression of autophagy substrates（ATG3,ATG5,ATG7）,key proteins of autolysosome formation（LC3,p62）,and lysosomal functional proteins（TFEB,LAMP2,Cathepsin B）revealed that the treatment of potential active substances could increase autophagy flux and promote autolysosome digestion.Combined with the expression of aging marker proteins and the results of mitochondria-lysosome function verification,it was further confirmed that the anti-aging effect of potential active substances was related to the level of mitochondrial ROS（mt ROS）（key point 1）and mitochondria-lysosome-mediated autophagy flux（key point 2）.In order to verify the critical role of ROS levels,the above cell model was combined with the intervention of N-Acetyl-L-cysteine（NAC）,a ROS inhibitor,on the basis of potential active substance treatment.It is clear that correcting the dysfunction of mitochondria-lysosome functional interaction caused by mt ROS is the key to the anti-aging effect of potential active substances.5.Mechanism verification of potential active substances in red ginseng for delaying brain aging based on mitochondria-lysosome interactionSince mitochondrial autophagy requires the continuous consumption of a large number of lysosomes,TFEB,as an important nuclear transcription factor regulating mitochondrial autophagy and lysosome biogenesis,plays a key role in maintaining mitophagy-lysosome functional interaction.Therefore,in this chapter,based on the established natural senescent cell model,combined intervention of potential active substances and TFEB activator 1 was given to evaluate the effects of potential active substances on the nuclear translocation ability of TFEB and the effect of the nuclear translocation ability of TFEB on the level of mt ROS and the functional interaction between mitochondria and lysosome.The purpose of this chapter is to clarify that the promotion of TFEB nuclear translocation is the key for potential active substances to increase mitochondria-lysosome functional interaction,thereby increasing mitophagy flux and lysosome biogenesis,and thus down-regulating ROS levels to play the anti-aging role.The results showed that TFEB activator 1（1μM）combined with potential active substances（S-Rg2,PPT,AFG,20μM）had a stronger effect on delaying senile ICR-MEF cells than ICR-MEF-P4-potential active substance individual treatment group.It is confirmed that the promotion of TFEB nuclear translocation is the key to the anti-aging effect of potential active substances.Meanwhile,compared with the ICR-MEF-P4-potential active substance individual treatment group,the ICR-MEF-P4-potential active substance-TFEB activator 1 combined treatment group had stronger ability to promote the increase of autophagy flux（the expression level of p62 protein was lower,the expression levels of LAMP2,Cathepsin B protein and TFEB nuclear protein were higher）,suggesting that potential active substances could increase autophagy flux by promoting TFEB nuclear translocation,thereby clearing damaged mitochondria and promoting lysosome biogenesis.TEM analysis,Mito Tracker Green probe,Lyso Tracker Red probe and DQ Red BSA staining analysis confirmed the above conclusion.Besides,Mito SOX Red results indicated that potential active substances could down-regulate mitochondrial ROS levels by promoting TFEB nuclear translocation.On the basis of the in vitro experiment,3-month-old SAMP8 mice were selected as the research object（wild-type SAMR1mice as the control group）to verify the results of the in vitro experiment.The results showed that the potential active substances（S-Rg2（20 mg/kg）,PPT（20 mg/kg）,AFG（80 mg/kg））could significantly increase the expression level of TFEB protein in the nucleus of hippocampal neurons of mice,and reduce the fluorescence expression level of p21 protein in hippocampal neurons.Moreover,potential active substances could promote the functional interaction between mitochondria and lysosomes by increasing the nuclear protein expression of TFEB,thereby reducing the level of oxidative stress,and thus delaying the brain aging in SAMP8 mice.These results suggested that TFEB nuclear translocation mediated mitochondria-lysosome functional interaction was the ultimate potential molecular target for red ginseng and its pharmacodynamic substances to delay brain aging.To sum up,this study revealed that the specific mechanism of action of red ginseng and its pharmacodynamic substances in delaying brain aging was to promote the nuclear translocation of TFEB protein by targeting,thereby increasing mitochondrial autophagy flux to remove damaged mitochondria and promote lysosomal biogenesis,and thus correcting the mitochondria-lysosome functional interaction disorder mediated by mitochondrial oxidative damage.The above conclusions provided theoretical references and data references for the development of red ginseng as an anti-aging drug.