| Purpose: Taking fruit flies combined with Caenorhabditis elegans and human diploid fibroblasts(MRC-5)as the research object,the main active ingredient of ginseng ginseng total saponins(TGGR)has been determined to improve the health of the body and prolong healthy life.Based on the composition of total saponins of ginseng,the molecular mechanism is discussed based on transcriptome sequencing technology.Method:1 Pharmacodynamic study of TGGR delaying aging: wild-type nematode N2 is divided into control group,TGGR high(250 μg/m L),medium(125 μg/m L),and low(50 μg/m L)dose groups for lifespan experiment;Drosophila Canton-S was divided into control group,high TGGR(2.5 mg/m L),medium(2.5 mg/m L),and low(0.5 mg/m L)dose groups for lifespan experiments;the continuous passage method was used to construct replicative senescent cells The model used CCK-8 method,cell passage number(PDL)and β-galactosidase staining method as evaluation indicators to screen the safe concentration range of TGGR and detect the effect of TGGR on MRC-5 cell senescence.2 Evaluation of TRRG’s delaying effect on aging and degenerative changes of Drosophila: Establish a comprehensive evaluation system for senescence and degenerative disease including body weight,feeding,reproduction,exercise,autonomous activity and intestinal barrier integrity of Drosophila,and analyze the nature of Drosophila Optimal TGGR intervention time for the aging cycle;wild-type fruit fly Canton-S is divided into control group and TGGR group(5 mg/m L and 2.5 mg/m L),and the above evaluation system is used to evaluate the effect of TGGR on the degenerative changes of aging;Heat stress,oxidative stress and acute toxic stress are used to evaluate the influence of TGGR on the ability of stress stress.3 Discussion on the molecular mechanism of TGGR: HPLC analysis of the composition and content of the main monomer saponins in total saponins;transcriptome sequencing based on the Illumina/Solexa sequencing platform,bioinformatics analysis methods to explore the target group of TGGR to extend the healthy life of fruit flies,combined with q RT-PCR analysis of its regulatory effects on key factors of aging-related signal pathways.Results: 1 TGGR can significantly prolong the survival time of multiple experimental models: Drosophila can be treated with different concentrations(0.5,2.5 and 5 mg/m L)of TGGR throughout their lives.At the concentration of 2.5 and 5 mg/m L,the survival time of female fruit flies can be significantly prolonged(p<0.01,p<0.05);the survival time of male fruit flies was significantly prolonged at concentrations of 0.5 and 2.5 mg/m L(p<0.05,p<0.05).125 μg/m L TGGR can significantly shift the survival curve of nematodes to the right(p<0.05),and the average life span and the maximum life span increase percentage are11.49%(p<0.001)and 9.47%(p<0.01),respectively.In the results of MRC-5 cell viability test,it was found that TGGR had no toxic effect in the range of 0-280 μg/m L.When the concentration was 40 μg/m L,it had a significant effect on cell proliferation(p<0.001),which could significantly increase the transmission of MRC-5 cells.The number of generations(p<0.01),the proportion of galactosidase staining positive cells decreased.2 Based on the Drosophila senescence and degenerative changes evaluation system,22 d(young)and 42 d(middle-aged)are the turning points of Drosophila aging.Taking 22 d and42d as the dividing point,TGGR intervention(0.5,2.5 and 5 mg/m L)was given at different stages.When TGGR was given 3-22 d,2.5 and 5 mg/m L could significantly prolong the lifespan of female and male fruit flies(p< 0.05,p<0.01;p<0.05,p<0.05),the treatment of female fruit flies with 5 mg/m L TGGR had the most significant effect on life extension,the average and maximum life extension rates were 10.14%(p<0.01)and 12.85%,respectively(p<0.05).When TGGR was administered on 22-42 days,the results of observing the effects of life expectancy showed that all doses of TGGR could prolong the lifespan of fruit flies.Among them,the intervention of 5 mg/m L TGGR had the most significant effect on life extension.The average life extension rate and maximum life extension rate of female fruit flies were They were 15.24%(p<0.001)and 15.26%(p<0.05).The average and maximum life extension rates of male fruit flies were 16.59%(p<0.001)and 13.30%(p<0.001),respectively.After comparative analysis,the effect of TGGR 22-42 d intervention to prolong life is better than that of 3-22 d and lifetime intervention.3 TGGR can significantly improve the senescence-like degenerative changes of Drosophila: Choose 22-42 d stage to give TGGR intervention(2.5 and 5 mg/m L),5 mg/m L TGGR can significantly reduce the food intake of Drosophila(p<0.05,p<0.05),while 2.5mg/m L did not have a significant impact on the food intake of fruit flies;the 2.5 mg/m L TGGR intervention fruit flies were tested for triglyceride indicators,and the results showed that TGGR did not significantly reduce the triglyceride of fruit flies.Ester level.TGGR of 2.5and 5 mg/m L can promote the reproductive ability of male fruit flies(p<0.01,p<0.01),and the number of virgin flies mated with them increased significantly;there was no significant effect on the reproductive ability of female fruit flies.TGGR of 2.5 and 5 mg/m L can significantly improve the climbing ability of Drosophila(p<0.05,p<0.05).5 mg/m L TGGR significantly reduced the proportion of "Smurf" fruit flies(p<0.01,p<0.01).TGGR can improve the defects of intestinal barrier function.The TGGR of 5 mg/m L in elderly fruit flies can improve the circadian rhythm activity.4 TGGR can improve the organism’s ability to resist environmental pressure: TGGR intervention can increase the antagonistic ability of fruit flies from starvation.The average survival time of male and female fruit flies has increased by 9.77%(p<0.01)and 15.73%(p<0.01),respectively.TGGR intervention can enhance the ability of hydrogen peroxide stress.After TGGR intervention,the average survival time of male and female fruit flies was prolonged by 21.94%(p<0.001)and 18.57%(p<0.01),respectively.In addition,TGGR intervention can significantly alleviate SDS-induced damage(p<0.05,p<0.01).5 TGGR can increase the activity of antioxidant enzymes in fruit flies: TGGR can significantly increase the activity of CAT and SOD in female fruit flies(p<0.001,p<0.01),and also significantly increase the activity of CAT in male fruit flies(p<0.05).But there is no significant increase in SOD vitality.7 Transcriptome sequencing of female and male fruit flies after TGGR intervention in young,old and elderly TGGR,each sample obtained more than 6.5G of data,the sequence length is 150 bp,the base content distribution is stable,the sequencing quality is good,and the Q20 value is about 95%,Q30 value is about 90%.8 Screened genes with statistically different expressions and examined the regulation of TGGR on differentially expressed genes.A total of 19 down-regulated genes were screened out of the female senescent Drosophila model and recovered to a certain extent after TGGR intervention,of which 11 genes were recovered.With annotation information.In the male aging Drosophila model,the expression levels of 23 down-regulated genes recovered after intervention by TGGR,and 14 of them have annotation information.In female Drosophila,162 genes are up-regulated during aging and down-regulated after TGGR intervention.Among them,27 differential genes are directly related to the regulation of aging.In male Drosophila,232 genes are up-regulated during aging and down-regulated after TGGR intervention.Among them,22 differential genes are directly related to lifespan regulation.9 candidate differential genes are mainly involved in biological processes such as biosynthesis,stress response,protein folding and aggregation,oxidative phosphorylation,apoptosis and aging regulation,as well as the regulation of classic aging-related signal pathways such as PI3K/Akt,m TOR and IIS.Conclusion: In Drosophila,Caenorhabditis elegans and human diploid fibroblasts,TGGR has been observed to have a positive effect on life span and "reverse" aging,confirming the potential applicability of TGGR for aging research.TGGR can improve the health status of aging fruit flies and enhance the stress ability.The possible mechanism of action is achieved by regulating the classic aging-related signal pathways such as PI3K/Akt,m TOR and IIS,regulating biological processes such as biosynthesis and stress response,controlling biological rhythms,and maintaining mitochondria and protein homeostasis. |
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