Study On The Effect Of Centaulin-3-O-Glucoside On Myocardial Senescence Through CD38-Sirt6 Pathway

Objective:The onset of aging and many age-related diseases include myocardial damage,and cardiovascular injury caused by aging mainly includes myocardial injury,atherosclerosis（AS）and hypertension.Senescent myocardial injury often results in reduced nicotinamide adenine dinucleotide（NAD+）levels.CD38,as the main NADase in cells,plays a central role in age-related NAD+decline.Cyanidin-3-o-glucoside（C3G）,as a natural inhibitor of CD38,has an anti-aging effect,and it has been found in the preliminary study of our group that C3G can inhibit the expression of CD38.Sirt6,as a member of the Sirtuins protein family,is considered to be the star gene of anti-aging.Therefore,this study explored the mechanism of CD38 participation in aging,the anti-aging effect of C3G,a natural inhibitor of CD38,and the role of CD38-SIRT6 in anti-aging pathway.Materials and methods:D-galactose（D-gal）induced senescence of myocardial H9C2 cells.H9c2 cells were also respectively treated with C3G,CD38 specific si RNA and Sirt6 specific si RNA OSS＿128167Sirt6 inhibitors and Sirt6 activator UBCS039 processing,testing each cell proliferation activity（CCK-8）,the levels of apoptosis,intracellular reactive oxygen species（ROS）level in order to determine phenotypic changes in aging.SA-β-Gal staining kit was used to determine the expression of SA-β-gal and determine the senescence level of cells.The m RNA expression levels of CD38,Sirt6,HK2 and TERT were detected by fluorescence real-time quantitative PCR（q PCR）,and the protein expression levels of CD38 and Sirt6 were detected by Western blot.The levels of NAD+and NAD+/NADH ratio of CD38 si RNA and Sirt6si RNA after H9c2 cell intervention were detected.Subacute aging model of C57BL/6 mice was established by injecting D-gal（100 mg/kg）with D-galactose daily for 8 weeks.Hematoxylin-eosin staining（HE）and MASSON were used to observe myocardial pathology and fibrosis.The routine changes of peripheral blood were detected by automatic blood analyzer,and the changes of serum biochemical indexes were detected by automatic biochemical analyzer.Mice were placed in a metabolic measuring cage for 24 hours.Oxygen consumption（VO₂）and carbon dioxide emission（VCO₂）of different groups of mice were detected by Oxylet system.Respiratory entropy（RQ）was calculated as the ratio of carbon dioxide production to oxygen consumption.Energy consumption（EE）was calculated by Weir formula（EE=1.44×VO₂×（3.815+1.23×RQ））.The expression of CD38 and Sirt6protein in myocardium was detected intuitively by immunohistochemistry.The cytokine levels in the blood of mice were determined by flow cytology using the Mouse Inflammation kit（13-PLEX）.Finally,the expression of Sirt6 and CD38 in myocardial tissue was detected by Western blot and q PCR.Results:D-gal induced apoptosis of H9c2 cells,inhibited cell proliferation,telomerase reverse transcriptase（TERT）expression and energy metabolism,and promoted ROS,SA-β-galactosidase（SA-β-Gal）and hexokinase 2（HK2）levels,proving that D-gal successfully constructed subacute aging model.C3G and CD38-specific si RNA reversed D-gal-induced cell senescence by inhibiting apoptosis and ROS,HK2 andβ-gal levels and stimulating cell proliferation and TERT expression in treated cells.Although UBCS039 could not promote cell proliferation alone,its other effects were similar to those of C3G and CD38 specific si RNA,while OSS＿128167 had similar effects to D-gal.H9c2 cells that underwent D-gal-induced aging showed increased CD38 expression and decreased Sirt6 expression.CD38-specific si RNA and C3G not only inhibited CD38 expression and stimulated Sirt6 expression but also relieved the inhibitory effect of Sirt6-specific si RNA on Sirt6 expression.Sirt6-specific si RNA,OSS＿128167 and UBCS039 did not change CD38 expression.D-gal-treated aging mice showed increased levels of IL-1β,IL-6,IL-17A,TNF-α,glutamic-pyruvic transaminase（ALT）,lactate dehydrogenase（LDH）,creatine kinase（CK）,and lactate dehydrogenase（LDH）and decreased NAD+levels,energy consumption and the NK cell proportion in peripheral blood.C3G treatment reduced the levels of these cytokines and metabolites and restored the NK cell proportion and energy consumption,C3G can play the role of anti-aging.CD38 expression was increased,while Sirt6 expression was decreased in the myocardial tissues of aging mice.C3G treatment reduced CD38 expression and increased Sirt6 expression in these tissues.CONCLUSION:（1）D-gal stimulated the expression of CD38,which inhibited Sirt6 expression to induce cell senescence in mouse cardiomyocytes.（2）C3G,as a classical flavonoid,has an anti-senescence effect induced by D-Gal,and plays a central role through the CD38-SIRT6 pathway.（3）SiRNA interferes with the expression of Sirt6 and CD38 in H9c2 cells,suggesting that CD38 plays an important role in the aging process by inhibiting Sirt6 expression in myocardial tissue.