Effects Of Exogenous NAD~+ On Senescent Bone Marrow-derived Mesenchymal Stem Cells And Its Related Molecular Mechanisms

Bone marrow-derived mesenchymal stem cells(BMSCs)are a type of low-immunogenic pluripotent stem cells that have become the most widely used stem cell type in tissue engineering research and clinical cell therapy such as repair and regeneration of damaged tissues.However,during in vitro culture,BMSCs are prone to replicative senescence,which severely restricts their research and application in biomedicine.Nicotinamide adenine dinucleotide(NAD~+),as a cofactor for key enzymes in glycolysis,tricarboxylic acid cycle,and oxidative phosphorylation,is involved in various physiological and pathological processes such as cell metabolism,energy synthesis,and repair the damaged DNA.Many studies have shown that the process of cell senescence is accompanied by a decrease in cellular NAD~+,but whether exogenous NAD~+can effectively delay the senescence of BMSCs?What is its molecular mechanism?These issues are currently unclear.Based on this,this paper mainly used D-galactose(D-gal)to construct the senescent model of rat BMSCs,and studied the effects of exogenous NAD~+on senescent BMSCs,and conducted a preliminary explores on the molecular mechanism.In this paper,rat BMSCs were first extracted and cultured by the whole bone marrow method.Then,explored the effects of exogenous NAD~+on the cell senescence-related?-galactosidase(SA-?-gal)activity,cell viability,and intracellular reactive oxygen species(ROS)in senescent BMSC.On this basis,we further examined the effects of exogenous NAD~+on Sirtuin 1(Sirt1),poly(ADP-ribose)polymerase 1(PARP1),CD38and P53 expression.Finally,small interfering RNA(si-RNA)knockdown Sirt1 was used to explored the key role of Sirt1 in the process of exogenous NAD~+postponed BMSC senescence.The experimental results showed that 10 g/L D-gal significantly increased the positive rate of SA-?-gal staining and reduced the cell viability of BMSCs,suggested that the senescence model of BMSCs has been successfully constructed.On this basis,different concentrations of exogenous NAD~+was used to treat senescence BMSC,SA-?-gal staining and CCK-8 analysis results showed that exogenous NAD~+significantly reduced the positive rate of SA-?-gal staining in and increased the cell viability in senescent BMSCs,indicated that exogenous NAD~+has a positive effect on delaying BMSCs.Further research found that exogenous NAD~+significantly increased the level of NAD~+and the ratio of NAD~+/NADH in senescent BMSCs,and reduced the intracellular ROS.Western blot results confirmed that exogenous NAD~+significantly up-regulated the expression of Sirt1 and inhibited the expression of PARP1,CD38 and P53 in senescent BMSCs.Next,si-RNA knocked down the expression of Sirt1 in senescent BMSC,and analyzed the role of Sirt1 in exogenous NAD~+delay BMSC senescence.The results showed that knockdown of Sirt1 expression significantly up-regulated the expression of PARP1,CD38 and P53 in senescent BMSCs,while exogenous NAD~+significantly inhibited the expression of these three molecules in senescent BMSC.The inhibitory effect of exogenous NAD~+on PARP1,CD38 and P53was weakened,indicating that exogenous NAD~+regulates the expression of PARP1,CD38 and P53 by regulating the expression of Sirt1 in senescent BMSCs.The results of SA-?-gal staining,flow cytometry and CCK-8 analysis showed that knockdown of Sirt1expression in BMSCs significantly increased the positive rate of SA-?-gal staining and intracellular ROS,and reduced the viability of BMSCs and promoted BMSC senescence.After supplementation with exogenous NAD~+,the positive rate of SA-?-gal staining and intracellular ROS in senescent BMSC were significantly decreased,and the viability of BMSC cells was significantly increased,but the effects of exogenous NAD~+postponed BMSC senescence was reduced compared to that of BMSCs without knockdown of Sirt1 expression.These results indicate that Sirt1 plays an important role in exogenous NAD~+postpones the senescence of BMSCs.In conclusion,this study proves that exogenous NAD~+could postpone BMSC senescence,and Sirt1 molecule plays an important signal-mediated role in this process.The results of this study provide a theoretical basis and methodological reference for the widespread application of NAD~+in the field of anti-aging and the acquisition of high-quality BMSCs in vitro and their better clinical application.