Regulation Of Nampt On Senescence Of Rat Bone Marrow Mesenchymal Stem Cells

Adult stem cells,which reside in a variety of adult tissues and organs,are the critical factors for homeostatic maintenance and tissue regeneration.Aging of tissues and organs,individual aging,and the occurrence of age-related diseases,such as diabetes and Alzheimer’s disease,have been attributed to the senescence of adult stem cells.Hence,how to activate the senescent adult stem cells in order to rejuvenate,is a key issue need to solve.Adult bone marrow mesenchymal stem cells(MSCs)are ideal seeding cells for tissue engineering and regenerative medicine.However,no matter how old the donors are,MSCs cultured in vitro will inevitably undergo replicative senescence with the increasing passages.It will give rise to the deficiency of seeding sells in tissue engineering and severely restricts their application in tissue repair,autologous transplantation and clinical therapy as well.In 2009,the aging theory “NAD world”proposed that nicotinamide phosphoribosyltransferase(Nampt),as the rate-limited enzyme in NAD biosynthesis,determines NAD level and Sirt1 activity in the metabolism.This regulatory process is termed Nampt/NAD/Sirt1 axis,which plays the essential roles in cell metabolism,cell senescence,cell cycle maintenance and individual aging.Our previous study has revealed that Nampt expression was reduced in a time-dependent manner in rat MSCs(rMSCs)undergoing replicative senescence in vitro.And Nampt expression was obviously lower in rMSCs obtained from aged rats than those from young rats during physiological senescence.These data implied that Nampt may have regulatory effects on r MSC senescence.Wetherefore hypothesize that regulation of Nampt on r MSC senescence could be mediated by Nampt/NAD/Sirt1 axis.In this study,MSCs from 1-2 month-old rats were used.Then the effects of Nampt on r MSC senescence were investigated by gene overexpression,RNA interference(gene silencing)or specific inhibitor.To determine r MSC senescence,cell morphology was observed,cell proliferation and cell cycle were analyzed,osteogenic and adipogenic differentiation were estimated,and senescence-associatedβ-galactosidase(SA-β-gal)activity and the expression of senescence-associated factor pl6INK4 awere measured.Furthermore,Nampt expression was detected by using RT-q PCR and Western blot,and its regulatory mechanism on r MSC senescence was explored by measuring intracellular Sirt1 activity and NAD content.The results are as follows:1.rMSCs at passage 3 and 10 were obtained by serially expanded culture in vitro.Compared with P3 rMSCs(young cells),P10 rMSCs(senescent cells)displayed senescence-like morphology.The cell aspect ratio gradually decreased,while cell areas progressively increased.The cellular SA-β-gal activity in P10 rMSCs was obviously heightened.2.The Nampt inhibitor FK866 or knockdown of Nampt by lentivirus transduction made young P3 rMSCs display senescence-like alterations with enlarged cell areas and reduced cell aspect ratio.Cell proliferation slowed down,while cell population doubling time significantly prolonged.The majority of cells were arrested in G1 phase.Both adipogenic and osteogenic differentiation declined.In addition,SA-β-gal activity was enhanced and the expression of p16INK4 awas elevated.3.Lentivirus-mediated Nampt overexpression led to senescent P10 rMSCs much slender and smaller from senescent morphology,with increased cell aspect ratio and decreased cell areas.The cell growth became faster and cell proliferation capability enhanced,whereas the cell population doubling time shortened markedly.The percentage of cells arrested in G1 phase reduced.Both osteogenesis andadipogenesis were improved.Moreover,SA-β-gal activity and the expression of p16INK4 awere both down-regulated.4.The expression of Nampt was significantly lower in P10 rMSCs as compared to P3 rMSCs.The intracellular NAD concentration and Sirt1 activity in P10 rMSCs were lower than those in P3 rMSCs.Nampt inhibition,either pharmaceutical inhibition or gene silencing,resulted in lower intracellular NAD content and then attenuated Sirt1 activity.While Nampt overexpression gave rise to the elevated intracellular NAD content and increased Sirt1 activity.What’s more,both NAM and NMN,the exogenous small molecules participating in the process of NAD synthesis,attenuated cell senescence induced by FK866.To sum up,Nampt has regulatory effects on r MSC senescence.Specifically,Nampt inhibition induces or accelerates r MSC senescence,while Nampt overexpression inhibits or delays the occurrence of r MSC senescence.The effects can be achieved by Nampt-mediated NAD synthesis and Sirt1 activity.Our findings will not only lay the foundation for exploring the mechanisms underlying stem cell senescence,but also provide novel strategies for addressing the deficient seeding cells,delaying stem cell senescence and preventing age-related diseases.