Senescence Of Bone Marrow Mesenchymal Stem Cells From Aged Rat And The Expression Of Nampt And Its Significance

The essence of individual aging is the senescence of stem cells. How to regulate the stem cells from senescence to youth in order to delay aging and prevent and treat age-related diseases, is the emergency to be solved. Due to the merits, mesenchymal stem cells(MSCs) have been the ideal seeding cells in autologous stem cell transplantation. However, the function of MSCs obtained from aged individual will be diminished because of the physiological aging, which severely restricts the therapeutic effect during the autologous stem cell transplantation. It is also the main reason for the declined organ functions in elderly people. In 2009, Imai at first time proposed the aging theory “NAD world ”. Nampt, NAD+ and Sirt1 comprised the regulatory network of mammal aging, which plays the critical roles in energy metabolism, cell senescence and maintenance of life span. However, the research on the “NAD world ” theory mainly focused on somatic cells. It is still unknown whether Nampt could influence stem cell senescence via the regulation of NAD+ and Sirt1. Our previous study found that the expression of Nampt was reduced time-dependently in MSCs replicative senescence in vitro, which implied that Nampt could have regulatory effects on MSCs senescence. Therefore, we hypothesize that Nampt may play a vital role in the regulation on natural senescence of MSCs from aged rat.In the present study, MSCs from aged rat(15-18 months old) and young rat(1-2 months old) were used. To detect the biological characteristics of MSCs in the process of physiological aging, cell morphology were observed, and cell proliferation and cell cycle were analyzed. The natural senescence of MSCs from aged rat was testified by investigation of SA-β-gal activity, oxidation and antioxidant levels, DNA damage, telomerase activity and the expression of senescence-associated factors. In addition, the expression and significance of Nampt in natural senescence of MSCs were further explored by using quantitative RT-PCR, Western blot and immunofluorescent staining, and then Sirt1 expression and activity, as well as intracellular NAD+ concentration were determined.The results are as follows:1. MSCs from aged rat showed senescent morphology. The cell aspect ratio was reduced, while cell areas increased. The growth of MSCs slowed down and the majority of cells were arrested in G0/G1 phase.2. In the process of physiological aging, MSCs from aged rat displayed the natural senescence. SA-β-gal activity was elevated, the balance of intracellular oxidation and antioxidant was disrupted, cellular DNA was severely damaged, telomerase activity was declined, and the expression of senescence-associated factors pl6INK4 Aand p21WAF1/ CIP were upregulated.3. The expression of Nampt was significantly lower in MSCs from aged rat as compared to young rat. The decreased Nampt resulted in lower intracellular NAD+ concentration, and further gave rise to the downregulation of Sirt1 expression and activity.Taken together, our results indicate that Nampt plays a regulatory role in natural senescence of MSCs. The mechanism may lie in the reduced intracellular NAD+ concentration by the decrease of Nampt, which subsequently inhibits Sirt1 expression and activity. Our findings will provide not only a new way to elucidate the mechanisms of stem cell senescence, but also a novel target for delaying stem cells senescence and prevention and treatment of age-related diseases.