NMN Improves Mitochondrial Function And Rescues Cellular Senescence By NAD~+/Sirt3 Pathway In Mesenchymal Stem Cells

The senescence of adult stem cells has contributed to tissue and organ aging,organism aging and age-relatetd diseases.Due to earlier and deeper investigations,bone marrow mesenchymal stem cells?MSCs?,as one of adult stem cells,have extensive prospects for basic research and clinical applications.However,during the long-term cultivation and expansion in vitro,MSC replicative senescence has severely limited its applications in tissue injury repair and clinical therapies.Therefore,exploiting the effective anti-aging drugs to delay stem cell senescence is a crucial issue that we need to solve urgently.Nicotinamide adenine dinucleotide?NAD⁺?is a key coenzyme in cellular energy metabolism and oxidative stress adaptation,which can participate in a variety of metabolic pathways and affect mitochondrial function.Mitochondrial dysfunction is one of the hallmarks of aging,and NAD⁺depletion is closely related to cellular senescence and energy metabolism disorders.As a NAD⁺dependent deacetylase in mitochondria,Sirt3 plays an important role in the regulation of mitochondrial function and cellular senescence.Accumulating studies suggest that nicotinamide mononucleotide?NMN?,a precursor of NAD⁺,can increase mitochondrial NAD⁺level,normalize NAD⁺/NADH ratio,and thus inhibit the senescence of muscle stem cells from aged mice and improve the mitochondrial homeostasis.Combined with our previous data,the intracellular NAD⁺content decreases in senescencet MSCs,accompanied by down-regulated Sirt3 expression,and NMN can repress MSC senescence induced by Nampt inhibitor FK866.We thus propose that NMN might improve mitochondrial function and further inhibit cellular senescence through NAD⁺/Sirt3 pathway during the process of MSC senescence.However,are there any abnormalities in mitochondrial function of senescent MSCs?Whether can NMN improve the mitochondrial function in senescent MSCs?How does NMN influence mitochondrial function and modulate MSC senescence?None of the above issues is clear.Objectives:To study the effects of NMN on mitochondrial function and MSC senescence,and explore the mechanisms of NMN regulating stem cell senescence,so as to provide experimental basis for exploiting the effective anti-aging drugs and solving deficient seed cells caused by stem cell senescence.Methods:1.P3MSCs?early passage,EP?and P10MSCs?late passage,LP?were obtained from male,1-2-month-old Wistar rats by whole bone marrow adherent method and serial expansion in vitro.To establish MSC replicative senescence model,cellular senescence was evaluated by morphological observation,detection of senescence-related?-galactosidase?SA-?-gal?activity and mRNA expression of the senescence-associated factor P16^INK4a.EPMSCs are used as young cells and LPMSCs are as senescent cells in subsequent experiments.2.The alterations in mitochondrial function of senescent MSCs were determined by the observation of mitochondrial morphology and structure,and measurement of ATP content,ROS level,mitochondrial membrane potential and oxygen consumption rate?OCR?.3.Intracellular NAD⁺content and NAD⁺/NADH ratio were determined by NAD⁺/NADH quantification kit,and Sirt3 expression at mRNA and protein levels were detected by RT-qPCR and Western Blot.4.Senescent MSCs were treated with NAD⁺precursor NMN,and its effects on mitochondrial function and MSC senescence were discussed.5.The effects of Sirt3 on mitochondrial function and MSC senescence were explored by using Sirt3 lentiviral transduction or Sirt3 inhibitor 3-TYP,and the molecular mechanisms underlying NMN roles were further investigated.Results:1.Compared with young MSCs,senescent MSCs displayed obvious morphological differences with irregular and flattened cell bodies,unclear boundaries,as well as increased cell areas and decreased aspect ratio.The ratios of SA-?-gal-positive cells were obviously elevated,and P16^INK4a expression at mRNA level was significantly up-regulated.2.Mitochondrial dysfunction occurred in senescent MSCs.Mitochondria displayed scattered distribution and obvious fragmentation,and mitochondrial volume increased with vacuoles,as well as disrupted double-layer membrane and ridges.ATP content,mitochondrial membrane potential and OCR were significantly reduced in senescent MSCs,while ROS level was enhanced.3.Senescent MSCs manifested apparent morphological changes after NMN treatment with reduced cell area and increased aspect ratio.The ratios of SA-?-gal-positive cells as well as p16^INK4a mRNA expression were markedly diminished.The mitochondrial function of senescent MSCs was remarkably improved in response to the addition of NMN,including concentrated mitochondrial distribution,augmented ATP content and mitochondrial membrane potential,and declined ROS level as well.4.NAD⁺content,NAD⁺/NADH ratio and Sirt3 expression were all lower in senescent MSCs compared to those in young MSCs.NMN strikingly up-regulated the reduction in NAD⁺content,as well as NAD⁺/NADH ratio and Sirt3 expression in senescent MSCs.5.Sirt3 overexpression in senescent MSCs effectively ameliorated the mitochondrial dysfunction,including relatively normalizd mitochondrial morphology,elevated intracellular ATP content and OCR,and reduced mitochondrial ROS level.Cellular senescence in LPMSCs was markedly rescued by Sirt3 overexpression.Both the positive ratios of senescent cells and p16^INK4a expression in Sirt3 over-expressed MSCs were substantially alleviated.In addition,Sirt3 inhibitor 3-TYP resulted in mitochondrial dysfunction and promoted cellluar senescence in young MSCs.6.In senescent MSCs,3-TYP led to abnormal mitochondrial morphology which originally tended to be normal due to NMN treatment,and down-regulated the upward trend in intracellular ATP content and mitochondrial membrane potential caused by NMN,and reversed the reduction in mitochondrial ROS level.In addition,3-TYP could also up-regulate NMN-mediated decline in the positive ratios of senescent cells and P16^INK4a mRNA expression in senescent MSCs.Furthermore,NMN significantly attenuated 3-TYP-induced mitochondrial dysfunction and cellular senescence in young MSCs.Conclusions:1.Mitochondrial dysfunction occurs in senescent MSCs,accompanied by the reduction in NAD⁺content and Sirt3 expression.2.Sirt3 overexpression in senescent MSCs effectively ameliorates the mitochondrial dysfunction and markedly rescues cellular senescence,while Sirt3 inhibitor 3-TYP has the opposite effects.3.NMN can improve mitochondrial function and rescue cellular senescence in senescent MSCs,which is achieved by up-regulating NAD⁺content and Sirt3expression.