Effects And Mechanism Of Adiponectin On Oxidative Stress-Induced Cardiomyocytes Senescence

Objective:To investigate the effect of adiponectin on oxidative stress-induced cardiomyocytes senescence and the possible relationship with mitochondrial autophagy by culturing the myocardial cells in neonatal SD rats and establishing the H2O2induced senescence model.Methods:1. Primary cultured cardiomyocytes were obtained from neonatal Sprague-Dawley rats by trypsin and collagenase digestion, and purified by treatment with the differential adhesion technique and the chemical inhibitor5-brdu. then cultured in the Dulbecco’s modified eagle culture medium consisting of20%fetal bovine serum. The morphology of cardiomyocytes and spontaneous beatings were observed under the inverted microscope. The molecular markers were identified by a-actin immunofluorescent technique.2. Primary cultured cardiomyocytes were divided into5groups at random:control group, the cells of which were cultured in routine condition; H2O2group, the cells of which were induced to be senescent by incubating with30μmol/L H2O2for2hours; H2O2+APN group, the cells of which were incubated with30mg/L APN for48hours before senescent induction; H2O2+APN+AraA group, the cells of which were incubated with30mg/L APN and1mmol/L AraA for48hours before senescent induction; H2O2+AraA group, the cells of which were incubated with lmmol/L AraA for48hours before senescent induction.3. Senescence associated β-galactosidase (SA-P-gal) staining was used to identify the senescent myocardial cells. 4. Western blot was used to detect the expression of LC3, the mitochondrial autophagy-related proteins.Results:1. Identification of cardiomyocytes:the microscope showed that the cells grew into clusters and beat regularly, the frequency of which were between90to120beats per minute. When the primary antibodies of myocardial sarcomeric actin were used, the cells emitted green fluorescence under the fluorescence microscope.2. The results of senescence associated (3-galactosidase staining:the positive rate of SA-β-gal staining in the H2O2group was significantly increased compared with the control group (P<0.05). The positive rate of SA-β-gal staining in the H2O2+APN group was obviously lower than that of the H2O2group (P<0.05). Compared with the H2O2+APN group, the H2O2+APN+AraA group had a higher positive rate of SA-p-gal staining (P<0.05). Adiponectin can inhibits the oxidative stress-induced cardiomyocytes senescence, and this effect can be reversed by AraA, the specific inhibitor of AMPK (AMP-activated protein kinase).3. The results of Western blot:The expression of LC3Ⅱ/Ⅰ in H2O2+APN group were significantly higher than that of the H2O2group (P<0.05). Compared with the H2O2+APN group, the expression of LC3Ⅱ/Ⅰ in H2O2+APN+AraA group decreased significantly (P<0.05). Adiponectin can increase the expression of mitochondrial autophagy-related proteins, which can be partly blocked by AraA.Conclusions:1. H2O2can induce the cardiomyocytes to be senescent.2. Adiponectin can inhibits the oxidative stress-induced cardiomyocytes senescence, and promote the mitochondrial autophagy.3. The inhibition of senescence of adiponectin on cardiomyocytes can be partly weakened after the mitochondrial autophagy is blocked by AraA, the inhibitor of AMPK.4. Adiponectin inhibits oxidative stress-induced cardiomyocytes senescence by promoting the mitochondrial autophagy related with AMPK.