| Objective: To investigate the regulator y effect of micro RNA-34a(mi R-34a)on Alginate oligosaccharide(AOS)delaying D-galactose(D-gal)-induced cardiomyocytes senescence and its possi ble mechanism.Methods: The experiment was divided into six groups: Control(Control)group,model(D-gal)group,D-gal actose+Alginate oligosaccharide(D-gal +AOS)group,Alginat e oli gosaccharide(AOS)group,D-gal actose+Alginat e oligosaccharide+micro RNA-34 a mimi c(D-gal +AOS+mi R-34 a mimic)group,D-galactose+Algi nate oligosaccharide+micro RNA-Negative Control(D-gal+AOS+mi R-NC)group.D-gal+AOS+mi R-34 a mimic group and D-gal+AOS +mi R-NC group were transfected with Lipofectamine TM 3000 for mi R-34 a mimic(50n M)and mi R-NC(50n M),respectively,and then these groups should be change d the medium after 4-6 hour s.T hen,D-gal,D-gal +AOS,D-gal +AOS+mi R-34 a mimic and D-gal+AOS+mi R-NC groups were treated with D-gal(200m M)for 48 hours to induce cardiomyocytes senescence model.After that,D-gal+AOS,AOS,D-gal+AOS+mi R-34 a mimic and D-gal +AOS+mi R-NC groups were treated with AOS(100?g/m L)for 48 hours.After AOS treatment for 48 h,the number of senescent cardiomyocytes was detected by ?-galactosidase staining.The expression of mi R-34 a was detect ed by RT-P CR.The change of mitochondrial m embrane potential was det ected by mitochondri al membrane potential detection.The expression of aging-relat ed proteins p16,p21 and p53,as well as,the proteins of Sirtuin1(SIRT1)and peroxisome proliferator activated receptor gamma coactivator 1 alpha(PGC1?)and oxidative stress-related proteins p47 phox and gp91 phox was detect ed by Western blot.The interaction between SIRT1 and PGC1? was detected by Co-imm unoprecipit ation.The expression of mitochondrial function-r elated gene SIRT1 and PGC1? m RNA was detected by RT-PCR.Results: 1.The results of ?-galactosidase staining showed that,compared with the Control group,the positive rate of ?-galactosidase staining was significantl y increased in the D-gal group(p<0.01).Compared with the D-gal group,the positive rate of ?-galactosidase staining was significantly decreased after the AOS intervention(p<0.01).Compared with D-gal +AOS+mi R-NC group,the positive rate of ?-galactosidase staining was significantly increased in the D-gal+AOS+miR-34 a mimic group(p<0.01).2.The expression of mi R-34 a showed that compared with Control group,the expression of miR-34 a in the D-gal group was significantly increased(p<0.01).Compared with D-gal group,the expression of mi R-34 a in the D-gal+AOS group was significantly reduced(p<0.01).Compared with D-gal+AOS+mi R-NC group,the expression of miR-34 a in the D-gal+AOS+miR-34 a mimic group was significantly increased(p<0.01)3.The results of mitochondrial membrane potential detection showed that compared with Control group,the mitochondrial membrane potential was significantly decreased in the D-gal group(p<0.01).Compared with D-gal group,AOS could increase the mitochondrial mem b rane potentia(p<0.01)l.Compared with D-gal +AOS+mi R-NC group,the mitochondrial membrane potential was significantly decreased in the D-gal+AOS+miR-34 a mimic group(p<0.01).4.West ern blot showed that the expression of senescence-relat ed proteins p16,p21 and p53 in D-gal group were significantly higher than that in Control group(p<0.01).Compared with D-gal group,AOS could del ay the process of cardiomyocyt es senescence and significantly decreased the expression of senescence-relat ed proteins p16,p21 and p53(p<0.01).Compared with D-gal +AOS+mi R-NC group,the expression of senescence-r elated proteins p16,p21 and p53 in D-gal group were significantly increased in the D-gal+AOS+miR-34 a mimic group(p<0.01).5.West ern blot showed that compared with Control group,the expression of SIRT1 and PGC1? proteins in D-gal group were significantly decreased(p<0.01).Compared with D-gal group,AOS could improve the mitochondrial function of cardiomyocytes senescence and upregulate the expression of SIRT1 and PGC1? proteins(p<0.01).Compared with D-gal +AOS+mi R-NC group,the expression of SIRT1 and PGC1? proteins in D-gal+AOS+miR-34 a mimic group were significantly decreased(p<0.05).6.The results of Co-immunoprecipitation showed that compar ed with Control group,the binding of SIRT1 and PGC1? was decreased and the function of mitochondrial biosynthesi s was decreased in D-gal group.Compared with D-gal group,the binding of SIRT1 and PGC1? was increased after AOS intervention,which improved the function of mitochondrial biosynthesi s.Compar ed with D-gal+AOS +mi R-NC group,the binding of SIRT1 and PGC1? was decreased and the function of mitochondrial biosynthesi s was decreased in D-gal+AOS+miR-34 a mimic group.7.West ern blot showed that compared with Control group,the expression of oxidative stress-related proteins p47 phox and gp91 phox was si gnificantl y increased in D-gal group(p<0.05).Compared with D-gal group,AOS could decrease the expression of oxi dative stress-relat ed proteins p47 phox and gp91phox(p<0.01).Compared with D-gal +AOS+mi R-NC group,the expression of oxidative stress-r elated proteins p47 phox and gp91 phox was significantl y increased in D-gal+AOS+mi R-34 a mimic group(p<0.05).8.RT-PCR showed that compared with Control group,the expression of SIRT1 and PGC1? m RNA was significantly decreased in D-gal group(p<0.01).Compared with D-gal group,the expression of SIRT1 and PGC1? m RNA was increased in AOS group(p<0.01).Compared with D-gal+AOS+mi R-NC group,the expression of SIRT1 and PGC1? m RNA was significantly decreased in D-gal+AOS+miR-34 a mimic group(p<0.01).Conclusion: AOS delayed D-gal-induced cardiomyocytes senescence by regulating mi R-34 a to improve mitochondri al function and oxi dative stress. |
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