Dual Regulative Effect On Autophagy Of 14-3-3? At Different Stages Of Myocardial Anoxia/reoxygenation Injury

Chapter 1 IntroductionIschemic heart disease(IHD)has been one of the severe diseases threatening human health,and its incidence in the world have increased year by year.Myocardial ischemia/hypoxia is the direct cause of ischemic heart disease,and timely reperfusion is needed to protect myocardium,but reperfusion per se contributes to injury and ultimate infarct size.In this study,transgenic cardiomyocytes with p AD/Flag-14-3-3? or p AD/sh-14-3-3? were constructed,and cardiomyocytes and its transgenic cells were damaged by acute anoxia or acute anoxia/reoxygenation(A/R).Based on this,we explored the dual regulative effects on autophagy of 14-3-3? at the different stages of cardiomyocytes acute A/R injury,and further elucidated the relevant underlying mechanism by western blotting,co-immunoprecipitation and immunofluorescence.To confirm the protective effects of 14-3-3? on cardiomyocytes,the intracellular oxidative stress levels were measured by flow cytometer,confocal microscope and microplate reader,and the indexes related to mitochondrial function were detected.Furthermore,compared and confirmed the results through isolated rat hearts experiments.Finally,pretreated with ginsenoside Rg1 in cardiomyocytes to preliminarily explore its role on cardiomyocytes protection.To lay a theoretical and experimental foundation for in-depth study on the significant of 14-3-3? and ginsenoside Rg1 on cardiovascular diseases.Chapter 2 The dual regulative effects on autophagy of 14-3-3? at the different stages of cardiomyocytes acute A/R injuryObjective: To investigate the dual regulative effects on autophagy of 14-3-3? at different stages of cardiomyocytes acute A/R injury.Methods: The cardiomyocytes protective effect of 14-3-3? was determined by the measurement of cell viability and lactate dehydrogenase(LDH)activities,the expression of 14-3-3? and LC3? was detected by Western blot,moreover,the activities of autophagy and autolysosomes were observed under the transmission electron microscope(TEM)or laser confocal microscope.Results: Overexpression of 14-3-3? presented a protective effect on cardiomyocytes by increasing cell viability and decreasing lactate dehydrogenase activities.Furthermore,overexpression of 14-3-3? induced autophagy levels at acute anoxia injury stage,while overexpression of 14-3-3? inhibited excessive autophagy at acute anoxia/reoxygenation injury stage.Conclusion: The dual regulative effects on autophagy of 14-3-3? at different stages of cardiomyocytes acute anoxia/reoxygenation injury.Chapter 3 The dual regulative mechanism on autophagy of 14-3-3? at the different stages of cardiomyocytes acute A/R injuryObjective: To investigate the dual regulative mechanism on autophagy of14-3-3? at the different stages of cardiomyocytes anoxia/reoxygenation injury.Methods: The expressions of 14-3-3?,LC3?,AMPK?,m TOR,ULK1,p70S6 K,Raptor,Akt and beclin1 were detected by Western blot;the interaction between14-3-3? and beclin1 was detected by co-immunoprecipitation;the co-localization of14-3-3?,beclin1 and vimentin was observed by immunofluorescence.Results: During the acute anoxia stage,overexpression of 14-3-3? triggered autophagy by activating AMPK?,ULK1,and inhibiting m TOR,simultaneously,decrease in 14-3-3? expression elevated Raptor,p70S6 K and m TOR,which partially inhibited the effects of rapamycin on autophagy.While,overexpression of 14-3-3?suppressed autophagy by activating Akt and inhibiting beclin1 after the acute anoxia/reoxygenation injury.Conclusion: Overexpression of 14-3-3? induced autophagy by activating AMPK?-m TORC1/ULK1 pathway at acute anoxia injury stage,and inhibited excessive autophagy by activating Akt-beclin1 pathway at acute anoxia/reoxygenation stage.Chapter 4 The effects of 14-3-3? on oxidative stress at the different stage of cardiomyocytes acute A/R injuryObjective: To investigate the effects of 14-3-3? on oxidative stress at the different stages of cardiomyocytes acute anoxia/reoxygenation injury.Methods: Intracellular/mitochondrial ROS levels were detected by flow cytometry or observed by laser confocal microscope.The activities of antioxidant enzymes(GSH-Px,SOD and catalase),and the content of MDA,GSH and GSSG were measured by a microplate reader.Results: Overexpression of 14-3-3? decreased ROS generation,which were induced by acute anoxia or reoxygenation.Moreover,it increased the activities of GSH-Px,SOD and catalase,reduced the content of MDA,and elevated the ratio of GSH/GSSG.Conclusion: Overexpression of 14-3-3? protect cardiomyocytes at different stages of acute hypoxia/reoxygenation injury by reducing the intracellular/mitochondrial oxidative stress and these protective effects were related to the regulation of autophagy.Chapter 5 The effects of 14-3-3? on mitochondrial function at the different stages of cardiomyocytes acute A/R injuryObjective: To investigate the effects of 14-3-3? on mitochondrial function at the different stages of cardiomyocytes acute anoxia/reoxygenation injury.Methods: The morphological changes of mitochondria were observed under transmission electron microscope.The expression of mitochondrial complex I and complex III were detected by Western blotting.The oxygen consumption rate and extracellular acidification rate were detected by Seahorse XFe24 Extracellular Flux analyzer.The disturbance of mitochondrial membrane potential was detected by flow cytometry,the m PTP opening and caspase-3 activities were detected by microplate reader,the apoptosis were detected by flow cytometry and observed under the microscope.Results: Overexpression of 14-3-3? maintained mitochondrial morphology,increased the expression of NDUFB8 and UQCRC2 protein,increased ATP production,maintained mitochondrial aerobic respiration,decreased glycolysis,inhibited lactic acid accumulation,sustained stable mitochondrial membrane potential and reduced m PTP opening.Conclusion: Overexpression of 14-3-3? protect cardiomyocytes by maintaining the mitochondrial function at different stages of acute anoxia/reoxygenation injury and these protective effects were related to the regulation of autophagy.Chapter 6 The mechanism of 14-3-3? protect isolated rat heart against acute anoxia or A/R injuryObjective: To compared and confirmed the results through isolated rat hearts experiments.Methods: Establishment of the over/under expression 14-3-3? rat model,and then the isolated rat heart was subjected to acute anoxia or anoxia/reoxygenation to construct the vitro model.Based on this,collection the perfusate for measurement the LDH activity.After the perfusion,myocardial tissue was dehydrated,embedded and sectioned for triphenyltetrazolium chlorid(TTC)and TUNEL staining.The left ventricular papillary muscle was taken for observation of ultrastructure under transmission electron microscope,and the remaining myocardial tissue was taken for detecting the expression of 14-3-3? and LC3?.Results: At the different stages of acute anoxia/reoxygenation injury,overexpression of 14-3-3? decreased LDH activity,decreased infarct size,inhibited myocardial fibrosis,maintained mitochondrial morphology and myocardial structure.Furthermore,overexpression of 14-3-3? increased the expression of LC3? at acute anoxia stage,while it suppressed the expression of LC3 ? at acute anoxia/reoxygenation stage.Conclusion: Confirmed that 14-3-3? presented a positive role at the different stages of isolated rat heart acute anoxia/reoxygenation injury.Chapter 7 Ginsenoside Rg1 presents a dual regulative effect on autophagy at different stages of cardiomyocytes acute A/R injury via up-regulating 14-3-3?Objective: To investigate the cardiomyocyte protective mechanism of Ginsenoside Rg1.Methods: Cardiomyocytes were pretreated with ginsenoside Rg1,and then establishment of acute anoxia and anoxia/reoxygenation models.Measurement of the cell viability,LDH activities and caspase-3 activities by microplate reader,detection of the 14-3-3? and LC3? expression,observation of apoptosis dot under the microscope.Results: Ginsenoside Rg1 up-regulated the expression of 14-3-3? protein,increased the cell viability,decreased the LDH activities.Moreover,Rg1 elevated LC3? levels after undergoing anoxia injury,while it reduced LC3? levels at acute A/R injury stage,inhibited caspase-3 activities and apoptosis.Conclusion: Ginsenoside Rg1 presents a positive role at the different stages of cardiomyocytes acute anoxia/reoxygenation injury via up-regulating 14-3-3?.