| Senescent cells accumulate in tissues with aging,preventing tissues regeneration and resulting in aging-related diseases,including chronic inflammation and cancer,etc.The characteristics of senescent cells include cell cycle arrest,resistance to apoptosis,activation of senescence-associated β-galactosidase(SA-β-gal)and accumulation of senescence-associated secretory phenotype(SASP).In recent years,drugs which promote apoptosis of senescent cells and inhibit SASP can delay occurrence and development of aging-related diseases,thereby improving the lifespan and healthspan of mammals.Therefore,studying drugs with the function of reducing senescent cells and inhibiting SASP and the relationship between SASP and aging-related diseases has become the focus in the field of aging research.Metformin is an oral biguanide drug that is mainly used to treat type 2 diabetes,and has potential anti-aging and anti-cancer effects.The annual fish Nothobranchius guentheri has relatively shorter lifespan,and exhibits aging-related biological markers in histology and biochemistry,making it an emerging model organism for aging studies in recent years.Previous studies in our laboratory show that metformin delays aging with cognitive ability improvement,reduction of lipofuscin accumulation in liver and resistance of mitochondrial oxidative damage,and prolongs the lifespan in N.guentheri.In this study,when the fish grew to 4-month-old(sexual maturity),they were randomly divided into two groups: the control group was fed standard food,and the experimental group was fed with metformin-supplemented food(2mg/g).The fish were killed as rapidly as possible to minimize suffering when they were 6-,8-,10-and 12-month-old.A total of 148 fish(74 fish in control group and 74 fish in metformin-fed group)were used to investigate effects of metformin on senescent cells,SASP and age-dependent spontaneous tumors,as well as mechanism by which metformin acted in liver.To further demonstrate that metformin may play a role in anti-aging by reducing senescent cells,when the fish grew to 9-month-old(which were used as a model of aged fish),they were randomly divided into control group and metformin-fed group.A total of 12fish(6 fish in control group and 6 fish in metformin-fed group)were used to prove effects of metformin on senescent cells and SASP.The experimental results are as following:1.To explore the effect of metformin on longevity,we recorded natural survival days of the fish and found that metformin significantly extended the mean lifespan by 22.5%and the maximum lifespan by 13.7% compared with control group.2.SA-β-gal activity increased with age,and metformin-fed group showed a marked decrease in SA-β-gal staining compared with control group in liver of the fish at 6-and8-month-old.To demonstrate the effect of metformin on senescent cells,we detected SA-β-gal activity in liver of aged fish which started feeding metformin from 9-monthold to 10-month-old,and the results showed that metformin reduced SA-β-gal positive cells in liver of the fish.To further explore the effect of metformin on anti-apoptotic characteristics of senescent cells,we found that expression of anti-apoptotic protein Bcl-2 increased with age,and metformin inhibited expression of Bcl-2 by immunoblot assay in liver of the fish at 6-and 10-month-old.Metformin significantly reduced Bcl-2 m RNA level by q PCR assay in aged fish which were fed metformin started from 9-month-old for one month.3.To investigate the effect of metformin on SASP,q PCR assay was used to detect expression of SASP factors.Our results showed that metformin significantly reduced expression of pro-inflammatory cytokines(IL-8,TNF-α,IL-1β and TGF-β),and upregulated anti-inflammatory cytokine IL-10 m RNA level in liver of the fish at 6-monthold.Metformin significantly inhibited SASP in aged fish which were fed metformin started from 9-month-old for one month.4.To explore the mechanism of metformin on inhibition of SASP,we used q PCR assay to detect m RNA levels of SIRT1,DLC1 and NF-κB in liver of 6-month-old fish,and found that metformin significantly up-regulated expression of SIRT1 and DLC1 and down-regulated NF-κB m RNA level.Immunoblot assay results showed that expression of SIRT1 and DLC1 attenuated and NF-κB activity increased with age,and metformin reversed expression of SIRT1,DLC1 and NF-κB in liver of the fish at 6-and 10-monthold.Immunohistochemical staining results showed that metformin increased protein expression of SIRT1,and inhibited the nuclear translocation of NF-κB in liver of the fish at 6-month-old.5.To investigate the effect of metformin on age-dependent spontaneous tumors,we used H&E staining and tumor marker TP53 staining to detect tumorigenesis in liver of6-,9-and 12-month-old fish,and found that number and volume of spontaneous tumors gradually increased with age.Metformin alleviated spontaneous tumorigenesis.In order to detect the effect of epithelial-mesenchymal transition(EMT)on tumorigenesis,we detected expressions of E-cadherin,β-catenin and slug in liver of the fish at 6-month-old,and found that metformin attenuated EMT and inhibited early tumorigenesis.In conclusion,metformin promoted apoptosis of senescent cells by downregulating expression of Bcl-2,and reduced the number of SA-β-gal positive cells,thereby delayed aging in liver of the fish.Metformin significantly up-regulated expression of SIRT1 and DLC1,down-regulated expression of NF-κB,reduced levels of SASP pro-inflammatory cytokines(IL-8,TNF-α,IL-1β and TGF-β),and increased anti-inflammatory cytokine IL-10 m RNA level,ultimately inhibited SASP.Metformin inhibited age-dependent liver spontaneous tumorigenesis by alleviating EMT and extended the lifespan of the annual fish N.guentheri. |
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