| Objective:To study the molecular mechanism of the 14-3-3? protein was involved in endotoxin tolerance against hypoxia/reoxygenation injury in cardiomyocytes at the cellular level,whether 14-3-3? protein targeted phospho-GSK3? in the cytoplasm to complete the regulation of ?-catenin,which affected the transcription of inflammatory factors,and thus played a role in endotoxin tolerance to protect hypoxic/reoxygenation injury of cardiomyocytes.Methods:We used H9c2 cardiomyocytes and constructed an in vitro model of hypoxia/reoxygenation injury.H9c2 cells were divided into 5 groups for experimental operation,Control(normal control group),A/R(hypoxia/reoxygenation group),LPS + A/R(lipopolysaccharide + A/R group),NC + LPS + A/R(negative control + lipopolysaccharide + A/R group),siRNA14-3-3? + LPS + A/R(siRNA14-3-3? + lipopolysaccharide + A/R group).1.Three RNAi-14-3-3? lentiviruses with high gene sequence were constructed by biotechnology company,and H9c2 cells were infected with fluorescent(GFP)negative control(NC)virus,using fluorescence inversion.The expression of green fluorescence was observed by microscope,and the reinfection index(MOI value)of the virus and the infection system were screened out.The virus sequence with the best silencing effect was screened out from the three sequences by Western Blotting method.In order to subsequent experiments,then we put it into production.2.LPS concentration was screened by CCK-8 kit and cytometry,that was the establishment of endotoxin tolerance protection model,and Western Blotting method to verify 14-3-3? protein endotoxin tolerance protection against A/R whether the expression was up-regulated during the injury process.3.Co-IP was used to detect the recognition of ligand protein of 14-3-3?,that was GSK3 protein subtype,which interacted with 14-3-3? during endotoxin tolerance to protect myocardial hypoxia/reoxygenation injury.Immunofluorescence(IF)verified whether the 14-3-3? protein was consistent with the subcellular localization of its ligand protein during endotoxin tolerance protection against myocardial hypoxia/reoxygenation injury.4.By Western Blotting method to observe the expression of ?-catenin protein under different interventions,CCK-8 kit was used to determine the survival rate of myocardial-like cells in 5 experimental groups,and the degree of apoptosis and activity of each group Oxygen(ROS)level and how the mitochondrial membrane potential changes(??m)were measured by purchasing a corresponding kit and following the instructions in the manual using a flow cytometer,the degree of opening of the mitochondrial permeability transition hole(mPTP).The activities of creatine kinase(CK)and lactate dehydrogenase(LDH)were detected by using a microplate reader after purchasing the corresponding kits and following the instructions in the instruction.Finally,the secretion level of the TNF-? and IL-6 were detected by enzyme-linked immunosorbent assay(Elisa).Results:1.The best sequence for screening RNAi-14-3-3? lentivirus was Ywhag-RNAi(44876-1),and the negative Control lentivirus had no significant effect on 14-3-3? protein.The optimal conditions for lentiviral infection were that multiply of infection index(MOI)was 20,and the infection system was DMEM medium with 10% FBS and HiTransG P,the infection time was 72 h.2.The cell viability measured by the CCK-8 kit and the degree of apoptosis measured by the cytometer showed that the LPS concentration was 200 ng/mL,it appeared better in the hypoxia/reoxygenation injury of cardiomyocytes.The protective effect namely the endotoxin tolerance protection model and the hypoxia/reoxygenation injury model were successfully established.Western Blotting result showed that the expression of 14-3-3? protein in LPS + A/R and NC + LPS + A/R was up-regulated,which was consistented with our previous experimental results.3.Using the Co-immunoprecipitation(Co-IP)technique,IP GSK3,IB 14-3-3? detected the subtype of GSK3 protein interacting with 14-3-3? protein as phospho-GSK3?,and the interaction only occur that it pretreated in LPS.The result of Immunofluorescence showed that the subcellular localization of phospho-GSK3? was consistented with the 14-3-3? trend during endotoxin tolerance to protect cardiomyocytes from hypoxia/reoxygenation injury,they all transfer from the nucleus to the cytoplasm.4.Comparing with the AR group,the result of Western Blotting showed that the expression level of ?-catenin protein was higher in LPS + A/R and NC + LPS + A/R,indicating that the concentration of ?-catenin was significantly increased after LPS pretreatment.And the accumulation of ?-catenin protein would transfere the cytoplasm into the nucleus,which affected the transcription of inflammatory factors,CCK-8 kit assay result showed that the cell viability of LPS + A/R and NC + LPS + A/R were significantly increased.The degree of apoptosis decreased significantly,the level of reactive oxygen species(ROS)production was significantly reduced,the mitochondrial membrane potential change(??m)was stable,the absorbance method was used to determine the opening of mitochondrial permeability transition pore(mPTP),the activity of creatine kinase(CK)and lactate dehydrogenase(LDH)were decreased.Finally,the secretion level of the TNF-? and IL-6 was detected by enzyme-linked immunosorbent assay(Elisa),which were significantly reduced.Conclusion:Endotoxin tolerance had cross-protection against hypoxia/reoxygenation injury in cardiomyocytes by low-dose LPS phosphorylation of GSK3 viaing the TLR4/PI3K/Akt pathway.The extensive expression of 14-3-3? assisting phospho-GSK3? to locate in the cytoplasm,and then the intracytoplasmic regulation of ?-catenin in the Wnt pathway,which could allow ?-catenin to be aggregated in the cytoplasm without degradation by the ubiquitin-protease system.This accumulation promoted that ?-catenin transfered into the nucleus and it could bind to the transcription factor TCF/LEFs in the nucleus,which to regulate the transcription of a large number of inflammatory related genes,thereby playing a protective role. |
PDF Full Text Request