| With the intensification of the aging issue,age-related functional decline and geriatric diseases have garnered widespread attention.Among various studies on aging,gut homeostasis is considered a key factor,as the health of the gut directly impacts overall physiological functions and the immune system.During aging,changes in the gut microenvironment,adjustments in the composition of the microbiome,and alterations in the proliferation and differentiation patterns of stem cells are closely associated with the development of age-related diseases.Considering the limitations of existing aging-delaying interventions and treatments for geriatric diseases,such as adverse side effects and specificity for certain symptoms or diseases,there has been a growing focus on finding more safe,lowtoxic,and widely effective anti-aging strategies.Ginseng(Panax ginseng C.A.Mey.),known for its long-term consumption benefits of body lightening and lifespan extension,has demonstrated significant potential in modern pharmacology for delaying aging and extending healthy life expectancy.Additionally,ginseng has shown positive effects in maintaining and promoting gut health.However,current research on the anti-aging effects of ginseng based on the maintenance of gut homeostasis is not sufficiently in-depth,especially in terms of the pharmacological basis and mechanisms of action.Therefore,our research systematically explored the anti-aging pharmacological basis of ginseng,focusing on maintaining gut homeostasis to elucidate the mechanisms and targets of ginseng’s anti-aging effects.This would contribute to scientifically interpreting its benefits for body lightening and lifespan extension and provide a scientific basis for developing new anti-aging treatment strategies based on gut health.Objective: To construct an in vitro aging cell model and a Drosophila model organism evaluation system,in order to screen for ginseng’s active components that delay aging;and to use the maintenance of gut homeostasis as a starting point to comprehensively evaluate the indicators of intestinal function in living intestinal organs,thereby clarifying the pharmacological basis of ginseng’s gut-protective and aging-delaying effects.On this basis,by doubly focusing on the intestinal microbiome and intestinal stem cells,we aim to explore the main pathways of ginseng’s aging-delaying effects and reveal the mechanism by which ginseng improves gut homeostasis in aging Drosophila.Methods:1 Study of the Pharmacological Basis of Ginseng for Aging Delay through Gut ProtectionPrepare three main active ginseng components,including total saponins,total polysaccharides,and total proteins.Initially,use an aging cell model combined with MTT and CCK-8 assays to assess the effects of ginseng components on cell viability and proliferative capacity.Employ propidium iodide staining to evaluate the cell cycle and select ginseng components with effective anti-aging activity in vitro.In vivo assessment of ginseng components’ effectiveness,using a Drosophila model to measure lifespan extension and Xgal staining to evaluate the impact of β-galactosidase activity,an aging marker in intestinal epithelial cells.Utilize laboratory-improved preparation processes to obtain ginseng total saponins,and compare their content with pharmacopoeia standards and commercial products using UV analysis.Assess anti-aging activity by examining Drosophila lifespan extension and β-galactosidase activity,and compare the composition of saponin components using HPLC analysis.Further,in Drosophila intestinal organs,analyze intestinal length through microscopic photography,assess intestinal barrier function using Smurf assays,monitor feeding volume using spectrophotometry and CAFE methods,and observe the length of intestinal villi using transmission electron microscopy.Additionally,employ a luciferase reporter system and specific reporter lines for intestinal stem cells and their progeny,combined with immunofluorescence staining of stem cell and mitotic markers,to determine the number of intestinal stem cells and progeny cells.Finally,quantify intestinal bacterial load using quantitative bacterial PCR.2 Study of the Influence and Mechanism of Ginseng’s Effective Components on the Intestinal Microbiome of Aging DrosophilaBased on 16 s intestinal microbiome sequencing,analyze α-diversity using the Shannon index and β-diversity using PCA.Conduct microbial multivariate statistical analysis to differentiate bacterial species abundance at various subclass levels and use Bugbase to analyze microbial phenotypes.Through the reassociation of microbiota,specific dominant bacterial genera from the microbiome of aged Drosophila melanogaster were introduced.Survival experiments were conducted to assess lifespan,Smurf assays were performed to evaluate gut integrity,q PCR was employed to detect the expression of IMD pathway target genes,and immunofluorescence was used to examine intestinal stem cell proliferation.Additionally,under the condition of IMD-specific RNAi,immunofluorescence was employed to assess intestinal stem cell proliferation.These experiments aimed to further identify the association between specific dominant bacterial genera in aged fruit flies and the IMD pathway.The study also aimed to elucidate their impact on intestinal function and contribute to intestinal stem cell proliferation.3 Study of the Mechanism of Ginseng’s Effective Components in Maintaining Intestinal Stem Cell Homeostasis in Aging DrosophilaUsing specific reporter lines for Drosophila intestinal stem cells and immunofluorescence staining,detect stem cell numbers and proliferative capacity under oxidative stress conditions induced by paraquat and bleomycin,combined with survival experiments to test survival rates under stress.In naturally aging Drosophila,assess antioxidant enzyme activity and oxidative stress product activity,measure ROS levels using DHE,observe mitochondrial morphology using electron microscopy,characterize mitochondrial turnover with mito Timer,evaluate autophagy activity with Lysotracker and p62 expression,detect mitochondrial quality control gene expression with q PCR,and determine marf protein expression using western blotting.In gene-edited states overexpressing/deletion marf,observe intestinal stem cell numbers,mitochondrial morphology,turnover,and autophagy.Additionally,use Xbp1 and p-eif2 a to report endoplasmic reticulum stress levels,combine gene editing to inhibit/activate endoplasmic reticulum stress protein expression in old/young Drosophila with immunofluorescence staining to observe intestinal stem cell numbers and proliferation,and use p-eif2 a staining to observe endoplasmic reticulum stress in marf overexpression states.Validate the direct interaction between marf and key endoplasmic reticulum stress proteins through coimmunoprecipitation.Report JNK activity with puc-lac Z,detect JNK and p-JNK expression using western blotting,and add endoplasmic reticulum stress inhibitors to verify the association between JNK and endoplasmic reticulum stress.Combine gene editing to inhibit/activate JNK pathway expression in old/young Drosophila with immunofluorescence staining to observe intestinal stem cell numbers and proliferation.The exact role of JNK contribution was verified through RNAi of endoplasmic reticulum stress key protein.In marf overexpression states,use electron microscopy to observe intestinal villi,microscopic observation to measure excretion,Smurf assays to assess intestinal integrity,and survival experiments to determine lifespan extension.Results:1 Pharmacological Basis of Ginseng for Aging Delay through Gut ProtectionThis study,utilizing both in vitro aging cell models and in vivo natural aging Drosophila models,successfully identified that total ginseng saponins possess superior lifespan extension and anti-aging activities,significantly outperforming total ginseng polysaccharides and proteins.Laboratory-extracted total ginseng saponins,using ultrasonication,exceeded pharmacopoeia standard saponins in yield and anti-aging activity and showed significantly increased content after purification with D301 anion exchange resin.The total saponins prepared in the laboratory were similar to commercial products in terms of monomer composition and content,validating the effectiveness of the preparation process.The study also focused on Drosophila intestinal organs,evaluating the intervention effects of total ginseng saponins on normal physiological functions and stem cell proliferation and differentiation under natural aging conditions.Post-intervention with total ginseng saponins effectively alleviated aging in Drosophila intestinal tissues,characterized by inhibiting intestinal shortening,promoting the restoration of intestinal barrier function,alleviating digestive and absorption dysfunction,rebalancing the proliferation and differentiation of intestinal ISCs,and reducing excessive bacterial load.Therefore,total ginseng saponins were identified as a key pharmacologically active anti-aging substance in ginseng.2 Influence and Mechanism of Ginseng’s Effective Components on the Intestinal Microbiome of Aging DrosophilaTotal ginseng saponins improved the intestinal microbiome of aging Drosophila,specifically restoring microbial diversity,adjusting microbial composition and structure,reducing the abundance of Proteobacteria and Acetobacteraceae,and increasing the abundance of Actinobacteria.They played a key role in reshaping the gut microbiome by inhibiting the increase in aging-related microbial phenotypes such as aerobic,Gram-negative,potentially pathogenic,and stress-tolerant types,thereby alleviating disorder in aging-related microbial phenotypes.The mechanism by which total ginsenoside improve the intestinal microbiota of aging Drosophila depends on inhibiting the accumulation of the Acetobacter genus SLV-7,and the intestinal barrier function of aging fruit flies was restored,consequently extending lifespan.This restoration relied on the selective depletion of the Acetobacter genus SLV-7,which suppressed the excessive activation of the IMD signaling pathway in the intestinal tract of aged fruit flies.As a result,the overproliferation of intestinal stem cells(ISCs)was inhibited,alleviating tissue hyperplasia defects.3 Mechanism of Ginseng’s Effective Components in Maintaining Intestinal Stem Cell Homeostasis in Aging DrosophilaTotal ginseng saponins enhanced oxidative stress tolerance in stressed aging Drosophila and boosted the activity of antioxidant enzymes and clearance of ROS accumulation,effectively inhibiting oxidative stress in the intestinal stem cells of aging Drosophila.The primary mechanism involved the restoration of marf expression in the intestines of aging Drosophila,improving mitochondrial morphology,restoring mitochondrial numbers,and enhancing mitochondrial turnover,thereby maintaining mitochondrial quality control in aging Drosophila intestinal stem cells.Moreover,marf was a crucial mediator in the regulation of endoplasmic reticulum stress in the intestinal stem cells of aging Drosophila by ginseng total saponins.Ginseng total saponins acted by intervening in the direct physical interaction between marf and endoplasmic reticulum stress proteins IRE1/PERK,inhibiting the activation of endoplasmic reticulum stress and IRE1 downstream JNK pathway,thus maintaining the proliferative homeostasis of intestinal stem cells in aging Drosophila.Conclusion:1.Through in vitro aging cell models and the Drosophila model organism,total ginseng saponins were successfully identified as key components in ginseng for delaying aging.It was confirmed that the total ginseng saponins prepared in the laboratory are significantly superior in content and anti-aging activity compared to pharmacopoeia standards,and there is no significant difference in content,anti-aging activity,and monomer composition compared to commercialized industrial products.In a living intestinal organ evaluation system,total ginseng saponins were confirmed as the pharmacological basis for delaying aging through gut protection in ginseng.2.The mechanism of action of total ginseng saponins in extending the healthy lifespan of aging Drosophila through intestinal microbiome regulation is not limited to improving the composition and diversity of gut microbiota.It also relies on the synergistic inhibition of the IMD and JNK pathways associated with Acetobacteraceae SLV-7 in aging Drosophila,as well as the repair of intestinal barrier function and strict control over the proliferation of intestinal stem cells.3.Total ginseng saponins maintain mitochondrial quality control by regulating marf,and limit aging-induced intestinal stem cell excess proliferation by inhibiting endoplasmic reticulum stress and excessive activation of JNK downstream of IRE1 through interaction with the endoplasmic reticulum stress-related protein IRE1/PERK.This study,from the perspective of maintaining gut homeostasis,delves into the regulatory effects and biological mechanisms of ginseng on the microbial and stem cell homeostasis of aging Drosophila’s intestines.It provides data support for ginseng’s contribution to delaying aging and extending lifespan,offers a scientific basis for developing targeted anti-aging products. |
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