Effect Of Apelin On Mitochondrial Structure And Energy Metabolism In Aged Cardiomyocytes Induced By D-Galactose

ObjectiveTo explore the effects of Apelin on D-galactose(D-Gal)-induced cardiomyocyte senescence and mitochondrial energy metabolism.Method1.H9c2 cells were intervened with D-galactose to establish aging myocardial model,using cell counting kit-8(CCK8),β-galactosidase staining,RT-PCR detect P16 gene expression to test whether the aging myocardial model was established successfully.Using concentration gradient method to explore the best working concentration of D-Gal and Apelin.2.Through electroporation transfection technique,to screen APJ over-expressing cells(h-APJ)and APJ low-expressing cells(Si-APJ).3.Experimental grouping:(1)Control group(Group C): H9c2 cells were cultured without special treatment;(2)Senescence group(Group D): H9c2 cells were cultured with D-Gal at a concentration of 20 g /L;(3)Group Si-APJ D: Si-APJ H9c2 cells were cultured with D-Gal at a concentration of 20 g /L;(4)Group Si-APJ D+A: Si-APJ H9c2 cells were cultured with D-Gal+ Apelin;(5)Group h-APJ D: h-APJ H9c2 cells were cultured with D-Gal at a concentration of 20 g /L;(6)Group h-APJ D+A: h-APJ H9c2 cells were cultured with D-Gal+ Apelin;4.Using the high-content imaging analysis system to detect the fluorescence intensity,then analyze the mitochondrial membrane potential of cells,the mitochondrial density,mitochondrial size,and mitochondrial texture of the cells.The citrate synthase detection kit detects cell citrate synthase activity.Western-Blotting detects protein expression of aging marker P16,PGC-1α,CPT1 A,GLUT4.RT-PCR detects gene expression of P16,CPT1 A,GLUT4,TFAM,NRF1.Result1.The optimal working concentration of D-Gal is 20 g / L,and the optimal working concentration of Apelin is 10-7mol / L.2.H9c2 cells in the aging group compared with the blank control group,the relative expression levels of cell aging markers P16 increased;H9c2 cells in h-APJ D+A group compared with the h-APJ D group,the relative expression of P16 decreased;there was no significant difference in the expression of P16 between the Si-APJ D + A group and the Si-APJ D group.3.H9c2 cells in the aging group compared with the blank control group,the citrate synthase activity was significantly decreased(P <0.01);the citrate synthase activity in the h-APJ D+A group was higher than that in the h-APJ D group(P <0.01);the citric acid synthase activity of Si-APJ D+A group was only slightly higher than that of H9c2 cells in Si-APJ D group.4.H9c2 cells in the aging group compared with the blank control group,the relative expression of PGC-1α decreased,the relative expression of glucose metabolism markers GLUT4,fatty acid metabolism related markers CPT1 A were reduced;H9c2cells in h-APJ D+A group compared with h-APJ D group,the relative expression of PGC-1α increased,the relative expression of glucose metabolism marker GLUT4 and fatty acid metabolism related marker CPT1 A increased;There was no significant difference in the expression of PGC-1α,GLUT4,CPT1 A between Si-APJ D+A group and Si-APJ D group.5.H9c2 cells in the aging group compared with the blank control group,the mitochondrial membrane potential decreased(P <0.01)and the mitochondrial biosynthesis decreased,but the mitochondrial size and mitochondrial texture parameters did not change significantly;H9c2 cells in h-APJ D+A group Compared with h-APJ D group,there was no significant difference in cell mitochondrial membrane potential,mitochondrial biosynthesis,mitochondrial size and mitochondrial texture parameters;H9c2 cells in Si-APJ D+A group compared with Si-APJ D group,there were no significant differences in cell mitochondrial membrane potential,mitochondrial biosynthesis,mitochondrial size and mitochondrial texture parameters.ConclusionApelin has a protective effect on D-Gal induced cardiomyocyte senescence.Apelin has a protective effect on mitochondrial energy metabolism in senescent cardiomyocytes induced by D-Gal,but has no significant effect on mitochondrial structural damage and mitochondrial biosynthesis.