Nicotinamide Mononucleotide Up-regulates SIRT3 Through The NAD~+ Metabolic Pathway To Antagonize D-galactose-induced HT22 Cell Senescence

Objective:To investigate the antagonistic effect of nicotinamide mononucleotide(NMN)on D-galactose-induced cell senescence of HT22 cells and the specific mechanism,whether it can delay cell senescence by upregulation of SIRT3 through NAD~+metabolic pathway.Methods:Firstly,D-galactose with different concentration gradients(10 mM,50 mM,100 mM)was used to conduct senescence model on HT22 cells.Cell activity was detected and the cell growth curve was drawn by MTT method.The concentration of senescent cells were determined by senescence associated acidic-?-Galactosidase method(SA-?-Gal).The expression of senescence related proteins and genes was detected by Western Blot(WB)and RT-PCR to evaluate the senescence degree of HT22cells.Then,the aging HT22 cell model was saved by 200?M NMN administration,and the rescue effect was also evaluated by MTT,SA-?-Gal,WB and RT-PCR.After that,the specific molecular mechanism of NMN antagonizing D-galactose-induced HT22cell aging was preliminarily explored.The expression of NAD~+gene,total antioxidant capacity,SOD enzyme activity,mitochondrial membrane potential and silent mating type information regulator 3(SIRT3)were detected.Finally,a combination of SIRT3inhibitor which is 20?M 3-TYP and NMN was administered in the senescence cell model,and the role of SIRT3 in the process of cell aging and reversal was evaluated again by MTT,SA-?-Gal,WB,etc.Results:1.The D-galactose-induced cell senescence model of HT22 was successfully established.2.Nicotinamide mononucleotide has an antagonistic effect on D-galactose-induced HT22 cell senescence model.3.Nicotinamide mononucleotide up-regulates SIRT3 through the NAD~+metabolic pathway antagonizing D-galactose-induced HT22cell senescence.Conclusion:Nicotinamide mononucleotide up-regulates SIRT3 through the NAD~+metabolic pathway to antagonize D-galactose-induced HT22 cell senescence.