| Background and Aims:Aging is caused by a combination of many factors,which is characterized by the declining function of organ and tissues.With the improvement in living standard and progressively increasing aging population,the mobility of aging and age-related diseases has increased significantly.There are a variety of factors causing senescence and the excessive oxidative stress is the leading cause of aging.NAD~+,a coenzyme of redox reaction,plays an important role in the regulation of cell metabolism.Many studies showed that the intracellular content of NAD~+were remarkably decreased with aging.In mammalian cells,CD38 is a major hydrolase for degradation of NAD~+,which is extremely important in regulating the intracellular level of NAD~+.Our previous work showed that CD38 knockout mice were resistant to AngII-induced cardiac hypertrophy and significantly protected heart from ischemia/reperfusion injury and high fat diet induced oxidative stress.However,the mechanism of CD38 in aging heart remains unclear.In this proposal,we will further study the role and molecular mechanism of CD38-mediated NAD~+metabolic pathways in heart aging with CD38 knockdown H9c2 stable cell line.Obviously,this study will provide new insights in elucidating the mechanism of heart aging and finding the therapeutic targets for delaying or preventing heart aging.Methods:1.Preparation and analysis of the model of senescence induced by D-galactose(D-gal)in vitro:H9c2 myocardial cells with or without CD38 knockdown were treated with D-gal(10 g/L)for 48 h.SA-?-Gal Staining was used to evaluate myocardial cell senescence.The intracellular ROS production was examined using H2DCF-DA.The content of MDA was detected with MDA Assay Kit according to the manufacturer's instructions.2.Supplement NAD~+under D-gal treatment:When the cells were about 80% confluence,the cells were treated with D-gal(10 g/L)for 24h.Then cells were incubated with both NAD~+(1.0 mM)and D-gal(10 g/L)for another 24 h before analysis.The H2DCF-DA and MDA Assay Kits were used to detect the intracellular ROS level and the content of MDA separately.Also,we evaluate myocardial cell senescence with SA-?-Gal Staining.3.Mechanism analysis:To determine the protective mechanisms of CD38 knockdown on senescence model induced by D-gal,we examined the level of total protein acetylation,protein and mRNA level of p16,p21,CD38,as well as genes which is associated with oxidative stress,including NOX4 and SOD2.Results:1.The expressions of senescence marker p16,p21 and the senescence were significantly increased in myocardial cells treated with D-gal.The expression of CD38 was markedly increased in H9c2 cells after D-gal treatment.2.The expression of p16 was increased in heart tissue in old mice compared with young mice.On the contrary,the expressions of Sirt1 and NAMPT were significantly downregulated in old mice.3.CD38 knockdown attenuated D-gal induced myocardial cells senescence and decreased the expression of senescence marker p16 and p21.4.D-gal increased the level of protein acetylation while CD38 knockdown reduced it compared with control with or without D-gal stimulation,suggesting that the Sirtuins activity might be increased.Furthermore,ROS production and the content of MDA were increased in H9c2 cells under D-gal treatment,but knockdown of CD38significantly decreased it.In addition,CD38 knockdown decreased the expression of NOX4 but increased the expression of SOD2 in H9c2 cells after D-gal treatment.5.Supplement of NAD~+markedly alleviated D-gal induced the senescence of myocardial cells and decreased the content of ROS and MDA.In addition,it can also increase the expression of SOD2 but decreased the expression of NOX4.Conclusion:1.The expression of CD38 was increased in myocardial cells treated with D-gal,but the expressions of Sirt1 and NAMPT,which are key enzymes involving in NAD~+ metabolic pathway,were downregulated in heart tissues of old mice.2.CD38 knockdown attenuated D-gal induced myocardial cells senescence and oxidative stress.3.Supplement NAD~+partially alleviated D-gal induced myocardial cells senescence and oxidative stress. |
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