Adiponectin Modulates Oxidative Stress-induced Mitophagy And Protects C2C12 Myoblasts Against Apoptosis

Background and Objevtive:Adiponectin(APN),also known as ap M1,Acrp30,GBP28 and adipo Q,is a circulating hormone that is predominantly produced by adipose tissue.Many pharmacological studies have demonstrated that this protein possesses potent anti-diabetic,anti-atherogenic and anti-inflammatory properties.Although several studies have demonstrated the antioxidative activity of this protein,the regulatory mechanisms have not yet been defined in skeletal muscles.The aim of the present study was to examine the cytoprotective effects of APN against damage induced by oxidative stress in mouse-derived C2C12 myoblasts and its molecular mechanism.Methods:In this study,hydrogen peroxide(H2O2),a classical model of oxidative stress,was used to simulate oxidative damage.After pretreatment with APN,C2C12 myoblasts cells were treated with hydrogen peroxide to simulate oxidative damage.Cell viability was performed to confirm the optimum concentration and time of hydrogen peroxide treatment and the appropriate concentration of APN.Reactive oxygen species(ROS)assay was performed to analyze the effect of APN on the level of oxidative stress.Western blot was performed to analyze the effect of APN on antioxidant substances.Annexin V/PI double staining was performed to detect the effect of APN on cell apoptosis.The protective effect of APN on mitochondria was demonstrated by detecting the changes of mitochondrial membrane potential.Bongkrekic acid(BA),a mitochondrial permeability transition pore inhibitor,was performed to investigate the mechanism of the changes of mitochondrial membrane potential.Confocal microscopy was performed to examine the effects of APN on autophagy induced by oxidative damage,mitophagy and the colocalization of mitochondria and lysosomes.Chloroquine diphosphate(CQ)was used to further verify the effect of APN on mitophagy.The effect of adiponectin on mitochondrial DNA was detected by mitochondrial DNA copy number.Western blot and real-time quantitative polymerase chain reaction(RT-PCR)were performed to investigate the molecular mechanism of the effects of adiponectin on mitophagy and apoptosis.Results:APN treatment at concentrations up to 30 ?g/m L did not result in any cytotoxic effects,whereas cell viability increased at the concentration of 30 ?g/m L. Cell viability dose-dependently decreased and time-dependently decreased.Pretreatment with APN significantly protected cells against the H2O2-induced reduction in cell viability.ROS levels increased in H2O2-treated cells compared with untreated cells.However,ROS levels were significantly inhibited in the presence of APN.The results showed that the treatment of C2C12 cells with APN prior to H2O2 exposure strongly protected the cells against apoptosis.Adiponectin enhanced the expression of antioxidant Nrf2 and HO-1.H2O2 treatment resulted in a significant reduction in the membrane potential in C2C12 cells.However,APN pretreatment caused a marked increase in the membrane potential in H2O2-treated cells.Bongkrekic acid(BA)is an inhibitor of adenine nucleotide translocase,which is a component of the MPTP complex.BA pretreatment increased cell viability in response to H2O2 treatment.We observed a markedly increased number of autophagosomes and autolysosomes after H2O2 treatment,whereas puncta accumulation was decreased in C2C12 cells upon APN pretreatment.APN decreased mitophagy and the colocalization of mitochondria with lysosomes in response to H2O2 treatment.According to the TIM23 levels which are indicators of mitophagy,we found a dramatic decrease in mitophagy after H2O2 treatment in C2C12 cells.The level of LC3 increased over time.Immunoblotting revealed that pretreatment with various concentrations APN suppressed H2O2-induced degradation of TIM23 without affecting the LC3-I to LC3-II transition.Adiponectin pretreatment inhibited the expression of Pink1 and Parkin induced by hydrogen peroxide.The addition of autophagy inhibitor chloroquine could significantly reverse the hydrogen peroxide-induced TIM23 degradation,but had no significant effect on adiponectin pretreatment of TIM23.Adiponectin inhibited the decrease in mitochondrial DNA induced by hydrogen peroxide.APN pretreatment significantly reduced the transcript levels of Bax and other apoptosis-related proteins in H2O2-induced cells compared with controls.Immunoblotting analysis revealed that various concentrations of APN resulted in potent downregulation of Bax/Bcl-2 protein expression,which also determined the apoptotic potential in C2C12 cells.Conclusion:These findings suggest that APN has cytoprotective effects on damage induced by oxidative stress in mouse-derived C2C12 myoblasts.APN attenuatedH2O2-induced growth inhibition,increased antioxidant capacity and exhibited scavenging activity against intracellular reactive oxygen species that were induced by H2O2.APN also suppressed increase of Pink1 and Parkin,H2O2-induced mitophagy and partially inhibited the colocalization of mitochondria with autophagosomes/lysosomes.Moreover,APN protected C2C12 myoblasts against oxidative stress-induced apoptosis.Furthermore,APN significantly reduced the m RNA and protein expression levels of Bax and the transcription level of apoptosis-related proteins.These data suggest that APN has a moderate regulatory role in oxidative stress-induced mitophagy and suppresses apoptosis.Because of its antioxidant effect,APN can be considered as a prevention and treatment in oxidative stress-associated skeletal muscle diseases.