The Molecular Biology Research Of FOXO1 In Reducing Oxidative Stress-induced Cell Injury By Inducing Cardiomyocyte Autophagy

Background:Myocardial ischemia/reperfusion (I/R) injury is on the top list of mortality cause worldwide. Cardiac oxidative injury during I/R causes damage to cardiomyocytes or even results in cell death followed by such physic-pathological process (Murphy & Steenbergen 2008) as fibrosis, hypertrophy, and ventricular chamber dilation, and ultimately leads to heart failure. Overloaded reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide and hydroxyl radical in oxidative stress deregulates the stress-signaling pathways can compromise cell viability and trigger apoptosis, via inducing damage to DNA, protein and lipids. On the other side, there are multiple markers exerting protective roles against the ROS-mediated cardiac injury, including AMP-activated protein kinase (AMPK), sirtuins (Sirts) and the activated phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling. The moderate Sirtl expression induces resistance to oxidative stress and apoptosis in heart 2007). And it was indicated that AMPK deficiency significantly increases MI/R injury in an AMPK-defective mouse model. Therefore, it is promising to identify anti-oxidative stress pathway for preventing the progression of heart disease.Forkhead box O (FOXO) transcription factors such as FOXO1 and FOXO3 regulate cell proliferation, metabolism, and aging in a variety of cell types (Huang & Tindall 2007), including cardiomyocytes. FOXO1-or FOXO3-deficient mice are embryonic lethal due to impaired vasculogenesis or develop cardiac hypertrophy as adults. In particular, FOXO1 is crucial for sustaining cardiomyocyte metabolism and cell survival, and mediate the oxidative stress response interacting with Hippo-YAP signaling. And multiple signaling pathways or markers have been indicated to be implicated in the anti-oxidative stress. SIRT1 activation induced by resveratrol posed marked effect on the Foxol-associated apoptotic signaling in heart. (Sin et al.2014). Activation of AMPK. inhibits cardiomyocyte hypertrophy by modulating of the FOXO1/MuRF 1 signaling pathway in vitro (Chen et al.2010). Moreover, FOXO1 is the most well-recognized member of FOXOs family, posing a key mediator of autphagy. However, the specific functions of FOXO1 in protection from I/R injury in the heart are not well understood.Here, we demonstrate that FOXO1 are both induced and protect cultured cardiomyocytes from oxidative injury. Likewise, combined deficiency of FOXO1 specifically in cardiomyocytes leads to increased oxidative stress-induced apoptosis; whereas the FOXO1 overexoression reduced such apoptosis via promoting autophagy. The present study implies the key protective role of FOXO1 in the anti-oxidative stress response via mediating the autophagy.Objective1. By hydrogen peroxide cultured myocardial cell,we simulated superoxide free radicals to injured on myocardial cell, the effects of hydrogen peroxide on myocardial cell apoptosis were tested, FOXO1 protein and mRNA level and change laws of FOXO1 phosphorylation level were detected. In order to investigate the function of FOXO1 to oxidative stress injury of myocardial cells.2. Gene transfection technique was used to knockout FOXO1 gene, FOXO1 silence were made, using different concentration of FOXO1-specific siRNA transfected myocardial cells, the content of FOXOlmRNA and cell apoptosis rate were tested, the caspase 3 content, content of PARP, caspase 3 activity, the formation of autophagic vacuoles and and LC3II LC3I ratio also were detected in order to investigate the relationship of FOXO1 with the mechanism of hydrogen peroxide function on myocardial cell apoptosis and autophagy.Methods1.Cell culture with H2O2 and Apoptosis assayH9c2 cells with more than were subject to added with 0 or 200 μM H2O2 for 24and 48 hours, The H2O2-induced apoptosis of H9c2 cells was assayed with an Annexin V-FITC apoptosis detection kit. cells were detected by a FACScan flow cytometer. Induced cell apoptosis was presented as percent apoptotic cells to total cells.2.Detected caspase 3, PARP expression and caspase 3 activity of Hydrogen peroxide induced myocardial cellH9c2 cells with more than were subject to added with 0 or 200 μM H2O2 for 24and 48 hours, expression of Caspase 3, PARP expression and caspase 3 activity were detected with W-B, use spectrophotometer meter under 342/441 nm wavelength absorbance measured caspase 3 activity.3.Detected FOXO1 protein expression and phosphorylation of Hydrogen peroxide induced myocardial cellH9c2 cells with more than were subject to added with 0,50,100,200uM H2O2 for 24 hours, expression of FOXO1 protein and phosphorylation were detected with W-B.4.Detected FOXO1 mRNA of Hydrogen peroxide induced myocardial cellH9c2 cells with more than were subject to added with 0,50,100,200uM H2O2 for 24 hours, expression of FOXO1 mRNA were detected with RT-PCR.5.Knockout FOXO1 with FOXO1-specific siRNAto transfected into H9c2 cellsTo knockout FOXO1, the FOXO1-specific siRNA oligonucleotides (25 or 50 nM) or the scrambler oligonucleotides as control (25 or 50 nM) were transfected into H9c2 cells with Lipofectamine RNAiMax.6 h post transfection, cells were updated with fresh DMEM medium with 2% FBS, and were assayed for the knockout efficiency post another inoculation of 24 h.6. Detected FOXO1 mRNA of FOXO1-specific siRNA transfected H9c2 cells.FOXO1-specific siRNA oligonucleotides (25 or 50 nM) or the scrambler oligonucleotides as control (25 or 50 nM) were transfected into H9c2 cells with Lipofectamine RNAiMax.6 h post transfection, the FOXOIRNA were detected with Rt-PCR.7.apoptosis of H9c2 cells after FOXO1-specific siRNA transfected into H9c2 cells FOXO1-specific siRNA oligonucleotides (25,50nM) or the scrambler oligonucleotides as control (25,50nM) were transfected into H9c2 cells with Lipofectamine RNAiMax.cells were detected by a FACScan flow cytometer. Induced cell apoptosis was presented as percent apoptotic cells to total cells.8.Detected caspase 3, PARP expression and caspase 3 activity of Hydrogen peroxide induced FOXO1-specific siRNA transfected into H9c2 cellsFOXO1-specific siRNA transfected into H9c2 cells with more than were subject to added with 200uM H2O2 for 24 hours, eexpression of Caspase 3, PARP expression and caspase 3 activity were detected with W-B, use spectrophotometer meter under 342/441 nm wavelength absorbance measured caspase 3 activity.9.Quantify the H202-promoted autophagic of Hydrogen peroxide induced FOXO1-specific siRNA transfected into H9c2 cellsFOXO1-specific siRNA transfected into H9c2 cells with more than were subject to added with 200uM H2O2 for 24 hours, Quantify the H2O2-promoted autophagic (GFP-LC3、LC3II/LC3I) were detected with green fluorescent proteinResults1.Apoptosis assay of H9c2 cells cultured with H202With the increase of concentration of hydrogen peroxide to cultivate, the extension of incubation time, cell apoptosis increased.2.Detection of caspase 3 and its metabolites PARP protein expression and caspase 3 activity.after Hydrogen peroxide to cultivate H9c2 cardiac muscle cellsWith the increase of concentration of hydrogen peroxide cultivation and the extension of incubation time, caspase 3 and its metabolites PARP protein expression and caspase 3 activity were increased3.Detection of FOXO1, FOXO1mRNA and FOXO1 phosphorylation after Hydrogen peroxide to cultivate H9c2 cardiac muscle cellsFOXO1 protein and mRNA level, as shown, with 50,100, or 200 uM under the disturbance of hydrogen peroxide concentration, myocardial H9c2 cells cells FOXO1mRNA level and there was no statistically significant difference between the blank control group, and the change rule of FOXO1mRNA, with 0,50,100, or 200 uM under the disturbance of hydrogen peroxide concentration, myocardial H9c2 cells cells FOXO1 protein level and there was no statistically significant difference between the blank control group, and also no statistically significant difference between the different concentration groups.Contrary to the above rules, the myocardial H9c2 cells cells with 0,50,100, or 200 uM under the disturbance of hydrogen peroxide concentration, the differences were statistically significant, the degree of phosphorylation and metering dependence to the concentration of hydrogen peroxide.(a),hydrogen peroxide to promote myocardial cell H9c2 cells of FOXO1 phosphorylation.4.Detected FOXO1 mRNA of Hydrogen peroxide induced myocardial cellH9c2 cells with more than were subject to added with 0,50,100,200uM H2O2 for 24 hours, expression of FOXO1 mRNA were detected with RT-PCR. FOXO1-specific siRNA between the expression of FOXO1mRNA transfection cell group was lower than the blank control group, there was statistically significant difference between the blank control group, As transfection FOXO1-specific siRNA concentration increased, FOXO1mRNA content decreased, and also statistically significant difference between the different concentration groups.5.Apoptosis of H9c2 cells after FOXO1-specific siRNA transfected into H9c2 cellsFOXO1-specific siRNA transfection cell apoptosis group was higher than the blank control group, there was statistically significant difference between the blank control group (P< 0.05), with the transfection FOXO1-specific siRNA the increase of the concentration, the cell apoptosis rate, difference between groups was statistically significant (P< 0.05).6.Detected caspase 3, PARP expression and caspase 3 activity of Hydrogen peroxide induced FOXO1-specific siRNA transfected into H9c2 cells200 uM hydrogen peroxide cultivate FOXO1-specific siRNA transfection the caspase 3 activity of myocardial cells, and caspase 3 PARP protein expression and its metabolites were higher than blank control group (P< 0.05).As the FOXO1-specific siRNA transfection concentration increased and increased, caspase 3 activity and heighten of caspase 3 PARP protein expression and its metabolites, differences between groups with statistical significance (P< 0.05).7.Hydrogen peroxide cultured FOXO1-specific siRNA after transfection myocardial cells and control, detection cell autophagy200 uM hydrogen peroxide could significantly induce the control cell autophagy vesicle formation (green fluorescent protein negative point),200 uM hydrogen peroxide under cultivation,50 nM FOXO1-specific siRNA autophagy vesicle cell group was lower than the control group (green fluorescent protein negative point), 200 uM hydrogen peroxide under cultivation,50 nM FOXO1-specific siRNA GFP cell groups-LC3, LC3Ⅱ/LC3I was lower than the control group (P< 0.05).The transfection FOXO1-specific siRNA H9c2 cardiomyocytes group was lower hydrogen peroxide induced autophagy (p< 0.01).Conclusion1.The model that hydrogen peroxide injuries myocardial cells is successful, and FOXO1 has been silent in myocardial cells (with FOXO1-specific siRNA transfection H9c2 cells).2. With the increase of concentration of hydrogen peroxide and the extension of incubation time, myocardial cell apoptosis, caspase 3, PARP protein levels and activities of caspase 3, were dose dependent.Tip hydrogen peroxide induced apoptosis.3. After hydrogen peroxide interference myocardial cell, FOXO1 protein and mRNA level unchanged, with higher concentration of hydrogen peroxide, FOXO1 phosphorylation levels, tip hydrogen peroxide induced myocardial cell apoptosis may be through promoting the FOXO1 phosphorylation.4. After the 200 uM hydrogen peroxide culture, as the FOXO1-specific siRNA transfection concentration increased, the proportion of apoptosis, caspase 3 content, PARP and caspase 3 activity increased and Prompt FOXO1 silent promote the apoptosis induced by hydrogen peroxide, we also summed up that FOXO1 suppress the myocardial cell apoptosis induced by hydrogen peroxide.5.200 uM hydrogen peroxide induced cell autophagy the formation of vesicles, hydrogen peroxide induced transfection FOXOl-specific siRNA H9c2 myocardial cell, LC3II/LC3I reduced. Prompt FOXOl silence reduce the autophagy induced by hydrogen peroxide, we also summed up that FOXOIZ enhance autophagy induced by hydrogen peroxide.