| Background Myocardial ischemic disease has become one of the main diseases that harm human health.Thrombolysis,percutaneous coronary angioplasty and coronary artery bypass surgery are used by People to make the ischemic myocardium restore blood supply in a short time,inorder to approve heart function,but still can't avoid aggravating tissue damage,even causing irreversible functional and organic damage with schemia reperfusion.Until now,the mechanism of myocardial ischemia-reperfusion injury(IR)is considered relevant to the role of free radicals,intracellular calcium overload,the activation of leukocytes,endoplasmic reticulum stress and mitochondrial dysfunction and so on.It is always considered that the damage of active oxygen free radical molecules(reactive oxygen species,ROS)and mitochondrial dysfunction are an important segment of the myocardial IR.Autophagy is a kind of special phenomenon retained in the process of evolution,and a lysosome degradation pathway in survival,development,differentiation and cell metabolism.Matter and energy can be updated in the process,which is used to maintain cell function and activity.Hypoxia inducible factor 1(HIF-1)in hypoxia condition can be fully activated,HIF-1 alpha is the active subunits,can combine stably with the special target gene proteins on the hypoxia response element.Through the quick and efficient regulation of transcription and post-transcriptional,HIF-1 alpha participate in cell morphology stability,blood vessel formation,iron and sugar metabolism and cell proliferation,which can produce adaptive response and have important protective effect especially for myocardial cells with large oxygen consumption.Through the study of tumor cells,tumor cells are generally in anoxic state,mitochondria produce large amount of ROS,and ROS can inhibit HIF-1 degradation and stable HIF-1 activity.HIF-1 alpha can activate BNIP-3 and NIX,and activating BNIP-3 and NIX combine with Bcl-2 to promote the release of Beclin-1,which can induce autophagy.Autophagy and hypoxic related molecular signaling pathways are not isolated,but interrelated.Autophagy can help myocardial cell to deal with bad stimulation.In myocardial ischemia phase,autophagy provide nutrients and energy compensation by lysosome degradation of damaged cellular proteins and organelles;during the reperfusion stage of myocardial ischemia,the autophagy is further activated with the accumulation of ROS in myocardial cells which can stabilize HIF-1alpha protein.Autophagy can protect cells by reducing ROS damage toxicity to cells and clearing damage organelles and protein.Autophagy can also lead to cell damage and even death by degradation of normal proteins and organelles.HIF-1? and autophagy have been studied more and more in myocardial IR,but there are few studies on the relationship between them.Zhao Yang etal used oxygen and sugar deprivation model in vitro to observe that HIF-1? had regulation of microglia autophagy,HIF-1? expression quantity increased gradually,autophagy increased also in the cerebral ischemia,and induced the death of microglia,and blocking the expression of HIF-1?,microglia autophagy reduced,the mortality rate reduced too.Research purpose The myocardial IR model in rats was established to observe the autophagy activity and HIF-1? protein expression in ischemia reperfusion.The expression of HIF-1 alpha protein and myocardial injury were observed by regulating autophagy intensity.The expression of HIF-1 alpha protein was modulated to observe autophagy and myocardial injury,so as to explore the effect of HIF-1 alpha on autophagy of myocardial cells and the potential impact on myocardial cell function.In order to provide possible theory for the clinical treatment of myocardial ischemic diseases.Research methods and results 1.The preparation and validation of myocardial ische mia reperfusion injury model in rats 1.1Establishment of myocardial ischemia reperfusion injury model in rats.The healthy male SD(Sprague-Dawley)rats were selected for abrosia 12 h before operation,but water,10% chloral hydrate 30mg/100 g intraperitoneal injection anaesthesia,supine position fixation,mouth intubation,ventilator assisted respiration.Under the sternal incision into the chest,in the rat heart left ear department l-2 mm,seeking to identify the left anterior descending coronary artery(LAD),with double 6-0 no damage polyester suture zone 3mm × 3mm gasket,below the LAD horizontal mattress suture,to wear the same size of gaskets,double knot ligation blocking the coronary blood flow,10# thick silk under each knot to have slipknot effect and restore blood flow perfusion by slow release line knot.1.2Identification of myocardial ischemia reperfusion injury model in rats Experimental group,healthy male SD rats,randomly divided into 5 groups,10 for each group,based on the above model.The experimental group rats survived an average of six to eight,mortality 28%,and six live rats related experimental data were selected eventually in each group for statistical analysis.Control group(a),namely the sham operation group:after the opening of the chest,the suture line was crossed only on the myocardial surface,not to ligate the left coronary artery,and observing for 120 min to obtain the whole blood and cut the left ventricular myocardial tissue;Simple ischemia group(b):ligation for 30 min,no recovering blood;Reperfusion 60 min group(c):ligation LAD for 30 min,recovering blood for 60 min to obtain the whole blood and cut the left ventricular myocardial tissue;Reperfusion 120 min group(d):ligating LAD for 30 min,recovering blood for 120 min to obtain the whole blood and cut the left ventricular myocardial tissue;Reperfusion 180 min group(e): ligating LAD for 30 min,recovering blood for 180 min to obtain the whole blood and cut the left ventricular myocardial tissue.To determine the serum myocardial enzyme cTnT leakage quantity by ELISA,TTC,Venn blue double staining to estimate myocardial ischemia and infarction area,multichannel physiologic recorder monitoring myocardial mechanics index to evaluate myocardial systolic function.Results: Myocardial ischemia reperfusion in rats serum content of cTnT,myocardial ischemia and infarction area increase,further reducing myocardial systolic function,function of 120 min reperfusion injury is the most obvious,180 minutes of reperfusion recovered.2.The expression of HIF-1 alpha protein and autophagy in rats with myocardial ischemia repe rfusion injury 200-300 g adult male SD rats randomly divided into 3 groups,10 of each group,and all surgical operations were in the same chapter 1.Six live rats related experimental data were selected eventually in each group for statistical analysis.Control group(A),namely the sham operation group: after the opening of the chest,the suture line was crossed only on the myocardial surface,not to ligate LAD,and observing for 120 min to obtain the whole blood and cut the left ventricular myocardial tissue;Simple ischemia group(B): ligating LAD,observed 30 min to obtain total blood and cut the left ventricular myocardial tissue;Ischemia reperfusion group(C): ligating LAD for 30 min,loosening the ligation line,and observing 120 minutes to obtain total blood and cut left ventricular myocardium.Using western blot method to detect the expression of myocardial cell HIF-1? protein and autophagy related protein Beclin-1 in each group,indicating autophagy intensity with the ratio of LC3-?/LC3-?,RT-PCR method to detect the expression of myocardial cell HIF-1? mRNA and Beclin 1 mRNA in each group.Results: The ratio of myocardial cells LC3-?/LC3-?and the expression of Beclin-1 and HIF-1? protein in pure ischemia group were higher than that in the control group,the difference was statistically significant(P<0.05).The ratio of myocardial cells LC3-?/LC3-?,Beclin 1 alpha and HIF-1 protein expression in ischemia reperfusion group is higher than that in the pure ischemia group the difference was statistically significant(P<0.05).Pearson correlation analysis showed that the expression of myocardial cells HIF-1? and Beclin-1 protein in ischemia reperfusion group was correlated,with correlation coefficient 0.730 and P=0.000.The expression of Beclin-1 mRNA and HIF-1? mRNA in myocardial cells in ischemia reperfusion group was higher than that in the ischemic group,and the difference was statistically significant(P<0.05).Pearson correlation analysis showed that the expression of myocardial cells HIF-1? and Beclin-1 mRNA in ischemia reperfusion group was correlated,with correlation coefficient 0.630 and P=0.005.3.Inhibiting autophagy,the expression of HIF-1 alpha protein and and injury degree of myocardial cells 200-300 g healthy male SD rats were randomly divided into three groups,10 of each group,Control group,ischemia reperfusion group and autophagy inhibition group and all surgical operations were in the same chapter 1.Six live rats related experimental data were selected eventually in each group for statistical analysis.The rats in autophagy inhibition group had preoperative intraperitoneal injection of autophagy inhibitor 6-amino-3-methylpurine(3-MA),15mg/kg,once a day for 3 days.The same volume of saline was injected into the abdominal cavity of rats in control group and ischemia reperfusion group,once a day for 3 days.Control group(D),namely the sham operation group: after the opening of the chest,the suture line was crossed only on the myocardial surface,not to ligate LAD,and observing for 120 min to obtain the whole blood and cut the left ventricular myocardial tissue;ischemia reperfusion group(E):ligating LAD for 30 min,loosening the ligation line,and observing 120 minutes to obtain total blood and cut left ventricular myocardium;autophagy inhibition group(F):ligating LAD for 30 min,loosening the ligation line and observing 120 minutes to obtain serum and left ventricular myocardial tissue.Using western blot method to detect the expression of myocardial cell HIF-1? protein and autophagy related protein Beclin-1 in each group,indicating autophagy intensity with the ratio of LC3-?/LC3-?,RT-PCR method to detect the expression of myocardial cell HIF-1? mRNA and Beclin 1 mRNA in each group.The area of myocardial ischemia and infarction by TTC and Evans blue double staining,changes of myocardial mechanical index and the measurement of myocardial enzyme cTnT in serum were estimated to assess the degree of myocardial injury.Results: The ratio of myocardial cells LC3-?/LC3-?and the expression of Beclin-1 protein in ischemia reperfusion group was higher than that in the control group,the difference was statistically significant(P<0.05).The ratio of myocardial cells LC3-?/LC3-?and the expression of Beclin-1 protein in the autophagy inhibition group were lower than that in the ischemia reperfusion group,the difference was statistically significant(P<0.05),while Beclin 1 mRNA expression quantity compared with ischemia-reperfusion group was no statistically significant difference(P>0.05).The expression quantity of myocardial cell HIF-1? protein and HIF-1? mRNA was no statistically significant difference between the autophagy suppression group and ischemia-reperfusion group(P>0.05).The serum cTnT content,myocardial ischemia and infarction area of autophagy inhibition group were significantly lower than that in the ischemia reperfusion group,the myocardial mechanical index was better than that in the ischemia reperfusion group,the difference was statistically significant(P<0.05).4.Inhibiting HIF-1 alpha protein expression,autophagy and injury degree of myocardial cells 200-300 g healthy male SD rats were randomly divided into three groups,10 of each group,Control group,ischemia reperfusion group and HIF-1? protein inhibition group and all surgical operations were in the same chapter 1.Six live rats related experimental data were selected eventually in each group for statistical analysis.The rats in HIF-1? protein inhibition group had intraperitoneal injection HIF-1alpha protein inhibitors 3-(5,2-hydroxy methyl furan base)-1-benzyl indazole(YC-1)at 30 minutes before the surgery,the final concentration of 5?M,dosage of 20 mg/kg,the same volume of saline was injected into the abdominal cavity of rats in control group and ischemia reperfusion group at 30 minutes before the surgery.Control group(?),namely the sham operation group: after the opening of the chest,the suture line was crossed only on the myocardial surface,not to ligate LAD,and observing for 120 min to obtain the whole blood and cut the left ventricular myocardial tissue;ischemia reperfusion group(?):ligating LAD for 30 min,loosening the ligation line,and observing 120 minutes to obtain total blood and cut left ventricular myocardium;HIF-1? protein inhibition group(?):ligating LAD for 30 min,loosening the ligation line and observing 120 minutes to obtain serum and left ventricular myocardial tissue.Using western blot method to detect the expression of myocardial cell HIF-1? protein and autophagy related protein Beclin-1 in each group,indicating autophagy intensity with the ratio of LC3-?/LC3-?,RT-PCR method to detect the expression of myocardial cell HIF-1? mRNA and Beclin 1 mRNA in each group.The area of myocardial ischemia and infarction by TTC and Evans blue double staining,changes of myocardial mechanical index and the measurement of myocardial enzyme cTnT in serum were estimated to assess the degree of myocardial injury.Results:The expression of myocardial cells HIF-1? protein in ischemia reperfusion group was higher than that in the control group,and the difference was statistically significant(P<0.05).The expression of HIF-1? protein in the HIF-1? protein inhibition group were lower than that in the ischemia reperfusion group,the difference was statistically significant(P<0.05),while HIF-1? mRNA expression quantity compared with ischemia reperfusion group was no statistically significant difference(P>0.05).The ratio of myocardial cells LC3-?/LC3-?and the expression of Beclin-1 protein in the HIF-1? protein inhibition group were lower than that in the ischemia reperfusion group,the difference was statistically significant(P<0.05),while Beclin-1 mRNA expression quantity compared with ischemia-reperfusion group was no statistically significant difference(P>0.05).The relative myocardial infarction area in HIF-1?protein inhibition group was higher than that in the ischemia reperfusion group,cTnT increased,myocardial contraction function reduced,while the myocardial ischemia area was lower than that in the ischemia reperfusion group,the difference was statistically significant(P<0.05).Conclusion 1.The method of using gasket slipknot ligating rats left anterior descending coronary artery,blocking blood for 30 min and reperfusing for 60-180 min,the injury of reperfusion 120 minutes was the most obvious.The method was easy and safe,could successfully make the model of myocardial ischemia reperfusion injury in rats.2.Autophagy marker protein LC3 significantly increased in rats with myocardial ischemia reperfusion injury,myocardial damage increased,and autophagy induced by myocardial reperfusion could be adverse.The expression of autophagy associated protein Beclin-1and Beclin-1 mRNA in myocardial ischemia reperfusion injury were increased,indicating that autophagy was dependent on Beclin-1 pathway.The expression of HIF-1? protein and HIF-1? mRNA was significantly increased.HIF-1? and Beclin-1 on the protein and gene levels were significantly positive correlation,HIF-1? as central regulatory factors in the anoxic stress reaction may adjust autophagy by Beclin-1 pathway.3.Intraperitoneal injection of autophagy inhibitor 3-MA could effectively inhibit the autophagy strength of myocardial ischemia reperfusion injury at the protein level in rats,and reduce myocardial damage,myocardial ischemia and infarction area.Autophagy had a negative effect on myocardial cells during myocardial ischemia reperfusion injury,and inhibiting autophagy could play a protective role in cardiac function.The expression of HIF-1? protein and HIF-1? mRNA in Myocardial ischemia-reperfusion injury in rats had no obvious change at autophagy level effectively restrain,autophagy strength didn,t affect HIF-1? protein and gene expression.4.The expression of HIF-1?protein was effectively suppressed in protein levels after intraperitoneal injection HIF-1? inhibitors YC-1,autophagy strength induced,HIF-1? may produce a certain regulation effect on autophagy in the myocardial ischemia reperfusion injury in rats.The role of HIF-1? in myocardial ischemia reperfusion injury in rats has two sides.On the one hand,inhibiting HIF-1? protein expression of myocardial ischemia reperfusion injury in rats increased myocardial infarction area,myocardial cell systolic function reduced,myocardial injury aggravated,HIF-1? could promote the activity of the myocyte ischemia anoxic condition,favorable effect on myocardial cells independent of autophagy.On the other hand,HIF-1? induced autophagy,adverse effect on the myocardial cells dependent of autophagy,inhibiting HIF-1? protein expression of myocardial ischemia reperfusion injury in rats could inhibit the hypoxic ischemic area autophagy intensity,reduce myocardial cell autophagy damage,reduce the myocardial ischemia area,play a protective role to cells in the myocardial ischemia area. |
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