| Aging is a process of physiological changes that will lead to a gradual decline in the biological functions of various tissues and organs in the organism.Aging is inevitable in living organisms.However,with the development of medicine in recent years,it is effective to develop a certain degree of intervention to delay aging or reduce the risk of various agingrelated diseases and improve the healthy lifespan of all humans,including the elderly.Therefore,it is crucial to search for interventions that can interfere the changes caused by aging and slow down the deterioration of biological functions for the healthy development of human society.Among the many intervention approaches,drug intervention is becoming one of the most commonly used strategies in anti-aging in a time-and dose-dependent manner.Therefore,in order to screen for drugs with anti-aging effects and to further investigate the molecular mechanisms of their anti-aging effects,we conducted such a study by using the model organism Drosophila Melanogaster.First,we screened several drug candidates with potential anti-aging effects,and then we investigated the mechanism of antiaging effects of the best drug,the AKT inhibitor GSK690693.The main results are as follows:1.A variety of drug candidates with potential anti-aging properties,such as earthworm extract(G-90),PDK-1 inhibitor OSU03012,AKT inhibitor GSK690693,allantoin,low concentration combined drugs of dasatinib+quercetin and rapamycin+vitamin E were added in Drosophila food and screened for anti-aging effects.The AKT inhibitor GSK690693 was found to be best to prolong the lifespan of Drosophila.2.GSK690693 at 5μM was able to significantly extend the lifespan of female flies in non DR medium by 8.5%.In spite of partially extension of the lifespan of male Drosophila,it was not statistically significant.In addition,GSK690693 at the concentration of 5μM significantly prolonged lifespan of female Drosophila,effectively whether administered lifelong or mid-life onwards(35 days).3.The addition of GSK690693 did not reduce the female fecundity,nor did it affect the food-intake rate of Drosophila.Beneficially,it improved the survival rate of Drosophila under oxidative stress conditions against hydrogen peroxide,consistent with a significant increase in the expression of antioxidant-related genes.Also,the drug improved gut barrier function in aged Drosophila and did not cause hyperglycemia in flies.It was found that GSK690693 did not improve climbing ability,hunger resistance or triglyceride levels in Drosophila.4.GSK690693 was detected to significantly inhibit the expression levels of phosphorylated AKT and ERK,and significantly increase the expression levels of GSK-3β and FOXO,and FOXO-related target genes by Western blots.However,by silencing key targets in the AKT signaling pathway--AKT,S6 K and FOXO by RNAi or mutation,GSK690693 could not further extend the fly lifespan.The results suggest that the lifeextending effect of GSK690693 is achieved by interacting with key targets in the AKT signaling pathway.5.GSK690693 was able to significantly reduce the number of Acetobacter and Lactobacillus and the expression level of 16 S r RNA in the intestines of aged Drosophila.The result of sequencing of microbial 16 S r RNA in the gut revealed that GSK690693 significantly reduced the abundance,diversity,and homogeneity of fly intestines.This indicates that drug is able to maintain intestinal health to prolong lifespan.6.By metabolome analysis on flies both GSK690693 treated and control group,a total of573 differential metabolites has been identified among the primary substances.To further identification of these differentials in details,15 differential metabolites were classified,of which 9 were up-regulated and 6 were down-regulated.Predicted KEGG pathway enrichment analysis showed that the differential metabolites were mainly enriched in RNA transport,purine metabolism,autophagy and endocytosis,amino acid metabolism.Furthmore,the enriched autophagic pathways were focused in the detail.GSK690693 was found to increase the number of autophagic lysosomes in the fly intestine and stimulate autophagic activity by increasing the expression of autophagyrelated proteins and genes.7.GSK690693 was able to increase the number of apoptotic cells and remove senescent cells to maintain intestinal homeostasis in the aged Drosophila intestine by increasing the expression level of apoptosis-related genes.8.Using hydrogen peroxide to induce senescence in NIH 3T3 cell,GSK690693 treatment was able to reduce the rate of SA-β-Gal positive staining and the number of senescent cells,and did not affect normal cells.GSK690693 was able to reduce the expression levels of AKT,IL6 and p16 in cells,and reduce the expression of senescence-associated secretory phenotype expression,increased the expression levels of apoptosis genes casepase-3 and casepase-6 and increased apoptosis cell number,suggesting its potential as a senolytics drug.In conclusion,our results indicate that the AKT signaling pathway plays an important role in aging and anti-aging,and treatment with the AKT inhibitor GSK690693 can extend the lifespan of Drosophila,improve physiological functions and achieve healthy aging.This is the first report on the effect of AKT-specific inhibitors on anti-aging.This study provides experimental data and scientific basis for further clinical practice and theoretical studies... |
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