| Objective:To investigate the protective effect of TanshinoneⅡA（TSN）on myocardial ischemia/reperfusion（I/R）by upregulating 14-3-3ηto regulate autophagy.Methods:1.Cellular experiments:H9c2 cells were cultured in vitro to establish anoxia/reoxygenation（A/R）model.Cells from the same batch were randomly divided into 7groups for experiments:（1）Control group;（2）A/R group;（3）2.5μM TSN+A/R group;（4）2.5μM TSN+p AD/14-3-3η-sh RNA+A/R group;（5）2.5μM TSN+API-2+A/R（6）2.5μM TSN+3-MA+A/R group;（7）2.5μM TSN+Rapamycin+Anoxia group.Cell viability was detected by CCK-8 method,and lactate dehydrogenase（LDH）.Superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）,catalase（CAT）activities,malondialdehyde（MDA）content,and mitochondrial permeability transition pore（m PTP）opening were measured by multifunctional enzyme marker.Western blotting for the expression of 14-3-3η,P62,LC3,Beclin1,Akt,p-Aktser473.Autophagic lysosomes and autophagic vesicles were observed by fluorescence inverted microscopy.Intracellular reactive oxygen species（ROS）levels and changes in mitochondrial membrane potential（MMP）were measured by flow cytometry.Annexin V-FITC/PI double staining and TUNEL staining method to detect apoptosis.2.Animal experiments:20 healthy adult male Sprague Dawley（SD）rats,weighing about 250-280g,were randomly divided into four groups:（1）Control group;（2）I/R group;（3）TSN+I/R group;（4）TSN+p AD/14-3-3η-sh RNA+I/R group.The rats were injected with sodium TSN sulfonate via intraperitoneal injection according to the grouping.Then an in vivo myocardial I/R injury model was established by myocardial injection of p AD/14-3-3η-sh RNA to construct a low expression 14-3-3ηrat model and ligation of the anterior descending branch of the left coronary artery in rats.Finally,the in vivo myocardial I/R injury model was established.Ejection fraction（EF）and shortening fraction（FS）were measured by small animal echocardiography,and serum lactate dehydrogenase（LDH）,creatine kinase（CK）,and aspartate transaminase（AST）activity were measured by enzyme marker.The hearts were removed,dehydrated,embedded,and sectioned for TTC staining and TUNEL staining.A portion of myocardial tissue was taken to determine caspase-3 activity,and tissue proteins were extracted to detect autophagy-associated proteins by immunoblotting.Results:In vitro experiments showed that TSN increased cell viability and regulated autophagy related proteins during A/R in H9c2 cells,subsequently stabilized mitochondrial function,regulated oxidative stress response and prevented the occurrence of apoptosis.TSN inhibited the expression of autophagy-related proteins by upregulating 14-3-3ηexpression,and application of Akt inhibitor（API-2）did not block the upregulation of 14-3-3ηby TSN,but increased Beclin1 expression and decreased p-Aktser473 expression,and 14-3-3ηwas found to interact with Beclin1.In vivo experiments then found that TSN significantly reduced myocardial infarct size,decreased serum creatine kinase（CK）,lactate dehydrogenase（LDH）,and glutamate aminotransferase（AST）production,regulated cellular autophagy related proteins,and inhibited autophagy,thereby improving cardiac function and reducing acute myocardial injury.Conclusion:TSN up-regulates the expression of 14-3-3ηand Akt phosphorylation,promotes the formation of 14-3-3η/Beclin1,and thus reduces the expression of Beclin1 and the occurrence of autophagy,thus protecting the myocardial ischemia/reperfusion injury. |
