| Objective:It is a natural process of growth,development,maturation,aging and death of an organism.With the development of social economy and medical technology,the acceleration of population aging and the increase of people’s life expectancy,the research on aging biology and the prevention and treatment of geriatric diseases has become a crucial research field with high attention in natural sciences and social sciences.Among these,brain aging is a key component of aging biology research and an important direction for the prevention and treatment of geriatric diseases.Brain aging is usually characterized by a decline in learning ability,memory,attention,decision-making speed,sensory perception（vision,hearing,touch,smell and taste）,and motor coordination.Recent studies have identified the loss of neurons in the brain and spinal cord and functional decline as the main pathophysiological mechanisms of brain aging.The gradual onset and development of neurophysiological dysfunction of the nervous system over time is called Neurodegenerative disease（NDD）,including Alzheimer’s disease,Parkinson’s disease,Huntington’s chorea,and multiple sclerosis.The mechanisms of the occurrence and development of NDD are still unclear,and recent studies suggest that NDD may be closely related to oxidative stress in neurons,abnormal protein aggregation,mitochondrial dysfunction,and inflammatory response.Studies have suggested that human aging is manifested at multiple levels,including systems,organs,tissues,cells,and biomolecules.The cell is the fundamental unit that constitutes the structure,development and function of an organism,and it is evident that cellular aging is an important basis for the aging of the organism.Therefore,it is believed that the essence of brain aging is the aging of various cells in the brain.Among them,neural stem cells（NSCs）are undifferentiated cells with high self-renewal capacity and multi-directional differentiation potential in specific regions of the nervous system,and NSCs can proliferate and differentiate into neurons,oligodendrocytes and astrocytes.NSCs perform important regulatory roles in CNS development,maintenance and damage repair through complex biological processes.In recent years,activation of own NSCs or transplantation of NSCs has become a new strategy for the treatment of various NDDs with the aim of replacing,repairing or enhancing the biological functions of damaged neurons,which is a new therapeutic way for the treatment of NDDs.Recent studies suggest that the mechanism of NDD occurrence and development is associated with the aging of NSCs in the organism,thus,exploring the mechanism of activating the proliferation and differentiation of aging NSCs in depth is the most critical for the search of delaying the aging pathway of NSCs and the prevention and treatment of NDD.According to traditional Chinese medicine theory,positive energy is the body’s internal natural defense system,which protects the body from external invasion of illness and infection,keeps the body healthy.As the body grows older,the body’s internal positive qi gradually weakens,making it vulnerable to the invasion of external disease and evil,leading to the decline and aging of the body’s functions.According to the theory of positive energy in Chinese medicine,the positive energy inside the human body encourages self-repair and regeneration,while stem cells are one of the important cell types for self-repair and regeneration inside the human body.Therefore,by enhancing the body’s internal positive qi,the proliferation and differentiation of stem cells can be promoted and the self-repair and regeneration ability of tissues can be improved,thus restoring the body’s health and vitality.Ginseng（Panax ginseng C.A.Meyer）is an important clinical"Qi tonic"in Chinese medicine and has a history of clinical use for thousands of years.The effective pharmacological components of ginseng include ginsenosides,ginsenosides polysaccharides,proteins and phenolic compounds,etc.Different components have different biological activities,and they may interact with multiple therapeutic targets,and this multi-component,multi-target and multi-pathway regulation may be an important strategy for the treatment of complex clinical diseases.Therefore,elucidating the molecular targets and interactions of various components and their mechanisms of action is a great challenge.Network Pharmacology is a new approach in pharmacological research and is now widely used in the study of traditional Chinese medicine and herbal compounding.Relying on major network databases,network Pharmacology deciphers the action of complex drug components and their related mechanisms at the molecular level by constructing interactions networks.It provides new ideas and methods for the modernization research of TCM,and is prospective to bring new breakthroughs for the development and application of TCM.Decades of research by our group have demonstrated that ginsenoside Rg1 is an important anti-aging monomeric component of ginseng,with clear effects of delaying stem cells and antagonizing the decay-causing effects of decay-causing agents on various organs,but the mechanism of action is still unclear.The basic research idea of this study to explore the active ingredients of ginseng against NDD using network pharmacology is to construct a complex biological network based on a massive database,find the key nodes in the network through network analysis,and predict the key biological processes.To guarantee the scientific validity of the prediction results,we used experimental mice and NSCs to construct in vivo and in vitro aging models,which were validated by molecular biology experiments.By exploring the effects of Rg1,the anti-aging active ingredient of ginseng,on the biology of aging,we elucidated its related mechanism of delaying brain aging and regulating aging in NSCs,which provides theoretical and experimental basis for its application in the prevention and treatment of NDD.Methods1.Web-based database exploration and analysis1)Searching"RENSHEN"based on TCMSP database and BATMAN-TCM database for the active ingredients and predicted targets of ginseng.2)Predicting the possible biological effects of ginseng active ingredients based on GO functional enrichment and KEGG pathway enrichment analysis.3)Searching Neurodegenerative disease（NDD）related targets and predicted targets of ginseng based on Gene Cards,OMIM,TTD,Drug Bank databases,and obtaining"intersecting genes"by intersection.4)Analyze the possible mechanism of ginseng against NDD based on GO functional enrichment and KEGG pathway enrichment.5)Using PUBCHEM database and STITCH data platform for the basic information of ginsenoside Rg1 and protein interactions network nodes.2.Construction of an in vivo aging model in mice to verify the results of network pharmacological analysis Twenty 6-8-week-old C57BL/6J mice purchased at the Experimental Animal Center of Chongqing Medical University were randomly divided into four groups of five mice each.Modeling was started after 3 days of observation.Control group:intraperitoneal injection of saline,10 ml/kg/d,42 d;D-gal group:intraperitoneal injection of D-galactose,120 mg/kg/d,42 d;Rg1+D-gal group:intraperitoneal injection of D-galactose,120mg/kg/d,42 d,from the 16th d,ginsenoside Rg1,40 mg/kg/d,26 d;Rg1group:intraperitoneal Diet,water intake and body weight of mice were recorded daily during the modeling.The modeling experiments were repeated three times.The following experiments were completed after modeling.1)Tail-hanging experiment for assessing the mood of mice.2)The pole climbing experiment for assessing mice’s locomotor and balance abilities.3)Morris water maze for assessing the spatial learning memory ability of each group of mice.4)Blood biochemical analysis was performed to detect liver function and blood lipids in mice.5)Observation of brain morphology,weighing and calculation of organ indices.6)Preparation of paraffin sections and HE staining for the observation of hippocampal tissue structure and cell number changes in mice.7)Nissl staining was performed to analyze the changes in the number of Nissl vesicles in the hippocampal neurons of mice.8)Transmission electron microscopy for observing microstructural changes in mouse hippocampal neurons.9)Enzymatic chemical assay for the detection of superoxide dismutase（SOD）and catalase（CAT）activities and nitric oxide（NO）and malondialdehyde（MDA）contents in brain tissue.10)RT-q PCR for the detection of aging-related gene expression in mouse brain tissues.11)Immunohistochemical assay for the determination of BDNF protein levels in mouse hippocampal tissues.12)Immunofluorescence assay for the determination of Sirt1 protein level in mouse hippocampal tissue.13)Western blot was performed to analyze the expression levels of aging marker proteins,Sirt1-Nrf2-BDNF pathway proteins and mitochondrial marker proteins in mouse brain tissues.3.Construction of the aging model in vitro of NSCs to explore the biological mechanism of agingWe extracted NSCs from the hippocampus of C57BL/6J mammary mice.NSCs that were obtained by passing to the 3rd generation were divided into 4 groups.Control group:incubated in NSCs complete medium for 24 h;D-gal group:incubated in NSCs complete medium with D-gal（final concentration of 10 mg/ml）for 24 h;Rg1 group:incubated in NSCs complete medium with Rg1（final concentration of 20μg/ml）for24 h;Rg1+D-gal group:incubated in NSCs complete medium with Rg1（final concentration as in the Rg1 group）and D-gal（final concentration as in the D-gal group）were incubated together for 24 h,and the following experiments were performed.1)The isolation,culture and identification of NSCs.2)CCK-8 assay for the inhibition or promotion of the growth of NSCs by drug intervention.3)Observation of neurosphere morphology under inverted light microscopy and counting of the number of neurospheres in each group.4)Aging-relatedβ-galactosidase（SA-β-Gal）staining for detecting the level of aging in each group of NSCs.5)DCFH-DA method for detecting ROS content in NSCs.6)Mito-Tracter assay for the detecting the mitochondrial activity in NSCs.7)Enzyme chemistry assay for the detection of superoxide dismutase（SOD）and catalase（CAT）activities and nitric oxide（NO）and malondialdehyde（MDA）contents in NSCs.8)RT-q PCR for the detection of aging-related gene expression in NSCs.9)Immunofluorescence assay for the determination of nuclear translocation of Nrf2 protein and the expression of Sirt1 protein.10)Western blot was performed to analyze the expression levels of Sirt1-Nrf2-BDNF pathway proteins and mitochondrial marker proteins in NSCs.11)Reagent kit for the detection of NAD+and NADH in NSCs.12)Seahorse assay for the detection of cellular respiration rate in NSCs.Results1.Results of web-based database mining and analysis1)Searching"REN SHEN"based on TCMSP database and BATMAN-TCM database to obtain 192 ginseng active ingredients and 1198predicted targets.2)Based on GO functional enrichment and KEGG pathway enrichment analysis,we predicted that the active ingredients of ginseng may have the functions of regulating hormone levels,promoting glandular development and regulating membrane potential levels;participating in promoting the composition of presynaptic membrane,postsynaptic membrane and ion channel complexes;regulating the activity of neurotransmitter receptors,organic acid binding and carboxylic acid binding.3)Based on Gene Cards,OMIM,TTD and Drug Bank databases,132neurodegenerative disease targets were collected by searching"Neurodegenerative disease",and the predicted targets of ginseng were intersected to obtain"52 intersecting genes".4)Based on GO functional enrichment,we analyzed the possible biological mechanisms of ginseng in neurodegenerative diseases related to inflammation,oxidative stress,cell proliferation and metabolic regulation;KEGG pathways were enriched to analyze the possible MAPK signaling pathway,lipid and atherosclerosis related pathway and neurotrophin signaling pathway.5)Using the PUBCHEM database and STITCH data platform,we obtained the basic compound information and protein interaction network nodes of ginsenoside Rg1,and found that ginsenoside Rg1 can regulate cell proliferation,intracellular nutrient levels and metabolism,of which BDNF is an important node of action.2.Results of in vivo aging model1)During the modeling process,the mice in the D-gal group lost weight,decreased diet and increased water consumption;the mice in the Rg1anti-decay group had higher body weight,increased diet and increased water consumption compared with the D-gal group;the mice in the Rg1group had no significant changes compared with the control group.2)The results of tail hanging experiment showed that the immobility time of mice in the D-gal group was prolonged compared with the control group;the immobility time of mice in the Rg1 anti-decay group was shortened compared with that of mice in the D-gal group;there was no significant difference between the Rg1 group and the control group.3)The results of the pole-climbing experiment showed that the pole-climbing time of the mice in the D-gal group was prolonged and the mice had slipped down the pole compared with the control group;the pole-climbing time of the mice in the Rg1-resistant group was shorter than that of the D-gal group,and the mice did not slip down the pole.4)The results of Morris water maze showed that compared with the control group,the mice in the D-gal group had longer escape latency,less time to cross the original platform and less time to stay in the quadrant where the original platform was located;the mice in the Rg1-resistant group had shorter escape latency,more time to stay in the quadrant of the original platform and more times to cross the original platform than those in the D-gal group.5)No significant changes in liver and kidney function in mice by biochemical analysis.6)Observation of brain gross morphology and weighing:compared with the control group,the brains of mice in the D-gal group were whitish in color,smaller in size and lighter in weight;the brain volume and weight of mice in the Rg1 anti-decay group were larger than those in the D-gal group.7)The results of HE staining showed that the brain tissues of mice in the control group and Rg1 group were normal in structure with neat and orderly cell arrangement,while the cells in the hippocampal region of the brain of mice in the D-gal group were disordered with a slight decrease in cell number.Compared with the D-gal group,the cells in the hippocampal region of the brain in the Rg1 anti-decay group were more neatly arranged,and the number of cells was not significantly reduced.8)Nissl staining results showed that the number of Nissl vesicles in the hippocampal neurons of mice in the D-gal group was reduced compared with the control group;the number of Nissl vesicles in the neurons of the hippocampal region of mice in the Rg1 anti-decay group was increased compared with that of the D-gal group.9)The results of transmission electron microscopy showed that the cytosolic membranes of hippocampal neurons in the control group and Rg1 group were intact,the nuclei were homogeneous,and the mitochondrial structures were not significantly abnormal;in the cytoplasmic nuclei of hippocampal neurons in the D-gal group,there were multiple distributions of electron-dense granules,a large number of mitochondria were swollen,and the internal cristae disappeared;in the hippocampal neurons of mice in the Rg1 anti-failure group,mitochondrial swelling was rare.10)The results of antioxidant enzymes and lipid peroxidation products assay showed that the activity of SOD and CAT in the brain tissue of D-gal group was decreased and the content of MDA and NO was increased compared with the control group;the activity of SOD and CAT in the brain tissue of Rg1 anti-decay group was significantly increased and the content of MDA and NO was decreased compared with the D-gal group.11)RT-q PCR detected the expression of aging-related genes in mouse brain tissues:compared with the control group,the expression of Sirt1,Foxo1 and PCNA in brain tissues of the D-gal group was significantly decreased,while the expression of IL-1α,IL-6α,CCL5,TNF-α,TGF-β,p53,p16 and p21 was increased;compared with the D-gal group,the expression of Rg1 expression of Sirt1,Foxo1,PCNA was significantly higher in brain tissue of the anti-decay group,while the expression of IL-1α,IL-6α,CCL5,TNF-α,TGF-β,p53,p16,p21 was decreased.12)Immunohistochemical results showed that the expression of BDNF in the hippocampal region of the brain of mice in the D-gal group was decreased compared to the control group,and the expression of BDNF in the hippocampal region of mice in the Rg1 anti-decay group was increased compared to the D-gal group.13)Immunofluorescence results showed that the expression of Sirt1 in the hippocampal region of the brain of mice in the D-gal group was decreased compared with that in the control group,and the expression of Sirt1 in the hippocampal region of mice in the Rg1 anti-depletion group was increased compared with that in the D-gal group.14)Western blot results showed that compared with the control group,the expression levels of p53 and p21 proteins in the hippocampal tissue of mice in the D-gal group were increased,and the expression levels of Sirt1-Nrf2-BDNF pathway proteins and mitochondrial marker proteins CS,COX IV,TOMM20 were decreased;compared with the D-gal group,the expression levels of p53 and p21 proteins in the hippocampal tissue of mice in the Rg1 anti-decay group were The expression levels of p53 and p21 proteins decreased and the expression levels of Sirt1-Nrf2-BDNF pathway proteins and mitochondrial marker proteins CS,COXⅣand TOMM20 increased in the hippocampal tissues of mice in the Rg1 anti-decay group.3.Results of in vitro aging model1)Isolation and in vitro culture of neural stem cells showed that the isolated neural stem cells still have the ability to grow into neurospheres.2)Identification of neural stem cells:the in vitro cultured cells were identified as neural stem cells by using Nestin and Sox2 for fluorescent double-labeling.3)The results of CCK-8 experiment showed that the cell proliferation rate of each group increased with the increase of Rg1 concentration,while there was no significant difference between the 20μg/ml and 40μg/ml groups;the cell inhibition rate of each group increased with the increase of D-gal concentration,and the IC10of D-gal was calculated to be about 10 mg/ml.4)The growth of neurospheres in each group was observed and detected under light microscope:the growth diameter of neurospheres in the D-gal group was significantly shorter compared to the control group;the growth diameter of neurospheres in the Rg1 anti-aging group was increased compared to the D-gal group.5)Aging-relatedβ-galactosidase（SA-β-Gal）staining showed that the neurospheres in the D-gal group were stained dark blue-green and strongly positive for senescence,while only a small number of cells in the Rg1 anti-decay group were seen to be blue-green,and the proportion of senescent cells was significantly decreased compared with that in the D-gal group.6)DCFH-DA method to detect the ROS content in cells:compared with the control group,the ROS content in neural stem cells was significantly higher in the D-gal group;compared with the D-gal group,the ROS content in neural stem cells was significantly lower in the Rg1 anti-aging group.7)Mito-tracter assay of mitochondrial activity in cells:mitochondrial activity was significantly higher in neural stem cells in the D-gal group compared with the control group.Significantly lower mitochondrial activity in neural stem cells in the Rg1 anti-decay group compared to the D-gal group.8)Corresponding kits to detect antioxidant enzymes and lipid peroxidation products in cells:compared with the control group,the activities of SOD and CAT in neural stem cells in the D-gal group were decreased,and the contents of MDA and NO were increased;compared with the D-gal group,the activities of SOD and CAT in neural stem cells in the Rg1 anti-aging group were significantly increased,and the contents of MDA and NO were decreased.9)RT-q PCR to detect the expression of senescence-related genes in neural stem cells:compared with the control group,the expression of Sirt1,Foxo1 and PCNA in neural stem cells in the D-gal group was significantly decreased,while the expression of IL-1α,IL-6α,CCL5,TNF-α,TGF-β,p53,p16 and p21 was increased;compared with the D-gal group,the the expression of Sirt1,Foxo1 and PCNA in neural stem cells in the Rg1 anti-decay group was significantly higher,while the expression of IL-1α,IL-6α,CCL5,TNF-α,TGF-β,p53,p16 and p21 was decreased.10)Immunofluorescence results showed that the expression of Nrf2 in the nucleus of neural stem cells was significantly decreased in the D-gal group compared with the control group;the expression of Nrf2 in the nucleus of neural stem cells was significantly increased in the Rg1 anti-decay group compared with the D-gal group;the expression level of Sirt1protein was decreased in the D-gal group compared with the control group;compared with the D-gal group,the Sirt1 protein expression level in neural stem cells was increased in the Rg1 anti-decay group compared to the D-gal group.11)Western blot results showed that the expression levels of Sirt1-Nrf2-BDNF pathway protein and mitochondrial marker proteins CS,COX IV and TOMM20 were decreased in neural stem cells in the D-gal group compared with the control group;compared with the D-gal group,the expression levels of Sirt1-Nrf2-BDNF pathway protein and mitochondrial marker proteins CS,COX IV,and TOMM20 in small neural stem cells in the Rg1 anti-degeneration group were increased.12)The reagent kit detected the levels of NAD+and NADH in neural stem cells:compared with the control group,the levels of NAD+in neural stem cells in the D-gal group decreased,the levels of NADH increased,and the NAD+/NADH ratio decreased;compared with the D-gal group,the levels of NAD+in neural stem cells in the Rg1 anti-degeneration group increased,the levels of NADH decreased,and the NAD+/NADH ratio elevated.13)Seahorse method to detect the cellular respiration rate of neural stem cells:compared with the control group,the basal respiration rate of neural stem cells in the D-gal group decreased,ATP production capacity decreased,and maximum respiration rate decreased;compared with the D-gal group,the basal respiration rate of neural stem cells in the Rg1anti-degeneration group increased,ATP production capacity increased,and maximum respiration rate increased.Conclusion1.Ginsenoside Rg1 may prevent and treatment of NDD by regulating intracellular nutrient levels and metabolism and regulating cell proliferation,in which Sirt1 and BDNF are important nodes of action.2.D-gal has been shown to caused structural and functional damage to brain tissues and NSCs,thus it can be used in the construction of brain aging model and the study of its prevention and control mechanism.3.Rg1 is an effective anti-aging component in ginseng,which can significantly reduce D-gal-induced brain aging and NSCs aging,which can be used to prevention and treatment of NDD and it has further research and development value.4.The mechanism of Rg1 reducing D-gal-induced brain aging may be related to the activation of Sirt1-Nrf2-BDNF signaling pathway,enhancing cellular energy metabolism and promoting the proliferation of NSCs. |
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