Resveratrol Inhibits Senescence-associated Secretory Phenotype By SIRT1/NF-κB Pathway In Gut Of The Annual Fish Nothobranchius Guentheri

As the animal ages,senescent cells accumulate in a variety of tissues and secrete a series of inflammatory cytokines,chemokines,growth factors,and proteases,called senescence associated secretory phenotypes,referred to as SASP.Senescent cells also undergo cell cycle arrest,irreversible loss of proliferation potential and senescence-associated?-galactosidase activity.Many components of SASP affect the surrounding cells and their microenvironment,and SASP may have a role in cell senescence,individual aging,and aging-related diseases.Resveratrol,an activator of SIRT1,has many pharmacological effects such as anti-aging,anti-inflammatory,anti-oxidant,and anti-free radicals.Nothobranchius guentheri is a kind of teleost that grows in East Africa.It has the advantages of shorter life span and faster breeding speed.It has become an emerging animal model for studying aging.Previous studies in our laboratory have shown that resveratrol significantly prolongs the life span of N.guentheri^[1,2].NF-?B is an important regulator of SASP.RelA/p65 is a subunit of NF-?B,and I?B?is an inhibitor of NF-?B,and phosphorylation of I?B?activates NF-?B.Previous studies have shown that SIRT1 inhibits transcription of NF-?B by deacetylating the K310 of RelA/p65^[3].In this experiment,the annual fish N.guentheri were used as research object,a total of 144fish.The fish grew to sexual maturity at 4 months old and they were randomly separated into two groups:the experimental group was fed with resveratrol-supplemented food（200μg/g food）,and the control group was fed with standard food（Totally,72 fish were fed with resveratrol and another 72 fish for controls）.The fish were killed as rapidly as possible minimize suffering when they were 6-,9-and 12-month-old.At three different ages,the activity of SA-β-gal was tested to reflect the aging of the gut.ELISA,qPCR,immunohistochemistry,immunblot,immunoprecipitation and co-immunoprecipitation were employed to investigate the effect of aging on SASP and the underlying mechanism that resveratrol inhibited SASP in gut of the fish.The experimental results showed:1.We found that?-galactosidase activity showed a rising trend with aging in gut of N.guentheri,and activity of?-galactosidase was decreased after resveratrol feeding.2.In order to explore the effect of aging and resveratrol on SASP in gut of the fish,we used qPCR and ELISA to detect the changes of SASP-related secretory factors.Levels of pro-inflammatory cytokines IL-8 and TNF?gradually increased with age,and the level of anti-inflammatory cytokine IL-10 gradually decreased with age,while resveratrol significantly reversed these cytokine changes.3.We used qPCR and immunohistochemistry to detect levels of SIRT1 in gut of the fish.It was found that mRNA and protein levels of SIRT1 gradually decreased with the age of fish,and resveratrol increased SIRT1 expression significantly.4.We used qPCR to detect the expression of NF-?B in gut of the fish.The result showed that,mRNA level of NF-?B increased significantly with age,while resveratrol induced a statistically significant reduction of NF-?B.Protein was extracted from 9-and 12-month-old fish for immunblot.It was found that protein levels of Rel A/p65 and p-I?B?increased dramatically with age,and resveratrol significantly decreased expression of these proteins.The acetylation degree of RelA/p65 was detected using Ac-Lys antibody with immunoprecipitating equivalent amount of RelA/p65 protein.The result showed that resveratrol reduced acetylation of RelA/p65 protein in fish at 9-month-old.The interaction between SIRT1 and RelA/p65 was checked using Co-IP assay then.Result showed that resveratrol increased interaction between SIRT1 and RelA/p65,thereby inhibiting the activity of NF-?B,ultimately inhibited SASP.5.The cell proliferation marker PCNA was detected by immunohistochemistry,and the stem cell marker LGR5 was detected by immunoblot assay.It was found that the number of intestinal epithelial cells（IECs）and intestinal stem cells（ISCs）decreased significantly in fish with age.Resveratrol significantly reversed this decrease.In conclusion,resveratrol activated SIRT1,decreased p-I?B?and Ac-RelA/p65,inhibited the transcriptional activity of NF-?B,and reduced levels of SASP-related pro-inflammatory cytokines IL-8 and TNF?,increased the level of anti-inflammatory cytokine IL-10,ultimately inhibited SASP.Resveratrol also increased the number of intestinal epithelial cells and intestinal stem cells.Finally,resveratrol reversed the decline of IECs and ISCs caused by aging,and delayed aging and prolonged lifespan of the annual fish N.guentheri.