Mechanism Of CaMEK Gene Targeting Intervention In The Prevention And Treatment Of Diabetic Heart Ischemia Reperfusion Injury

Study purpose: To explore the mechanism of extracellular signal regulated kinase1/2(ERK1/2)signaling pathway in type II diabetic myocardial ischemia/reperfusion(I/R)injury and targeted intervention effect.To explore whether exogenous targeted transduction of constitutively active MEK1(Ca MEK)gene can reduce type II diabetic myocardial ischemia/reperfusion injury by activating the ERK1/2 signaling pathway,reducing mitochondrial division,improving mitochondrial function,and inhibiting cardiomyocyte apoptosis.Study content: 1)C57BL/6J mice were fed with high-sugar and high-fat diet and injected with Streptozocin(STZ)to establish a type II diabetic mouse model,and observe the effects of post-ischemic adaptation on the myocardium of C57BL/6J mice and type II diabetic mice,and the difference of the protective effect and the influence on the activity of the ERK1/2 signaling pathway.2)At the cellular level,use H9C2 cardiomyocytes to establish a high glucose model of ischemia,hypoxia,and reoxygenation(H/R),to explore the damage of mitochondria and cell apoptosis after activation of ERK1/2 signaling pathway and the mechanism of high glucose myocardial ischemia reperfusion injury.3)Establishing an in vivo myocardial ischemia-reperfusion injury model in type II diabetic mice,and explore the targeted transduction of Ca MEK gene into the heart of type II diabetic mice at the tissue and animal level to combat type II diabetic myocardial ischemia/reperfusion(ischemia/reperfusion,I/R).Study methods: 1)First,select 8-week-old C57BL/6J male mice as the research object,fed with high-fat and high-sugar feed for 4w,and given intraperitoneal injection of streptozotocin(STZ Streptozotocin)at a dose of 50mg/kg to establish a type II diabetic mouse model.The mice in the control group were fed with regular feed.After 8 weeks of feeding,and the type II diabetic model was determined to be successfully constructed by measuring the fasting blood glucose level of type II diabetic mice.The in vivo ischemia-reperfusion and ischemia post conditioning(ischemia post conditioning)IPost C model was established for C57BL/6J and type II diabetic mice to detect myocardium Infarct size,and the expression of related proteins of the ERK1/2 signaling pathway.2)Give ERK activator or transfect Ca MEK gene to H9C2 cardiomyocytes,then establish high glucose model of ischemia,hypoxia,reoxygenation injury.Flow cytometry detection method detected cell apoptosis,Mitotracker detected mitochondrial division and fusion status,JC-1 detected mitochondrial membrane potential,Mito SOX detected cell oxidative stress,Western blot and immunofluorescence detected the expression ERK1/2 of signaling pathway,mitochondrial division related proteins Drp1 and TOMM20,apoptosis related proteins Bcl-2 and Bax.3)For 8-week-old male mice of C57BL/6J fed with regular feed and the type II diabetic mice fed with streptozotocin injection plus high-fat and high-sugar feed at the same time,ds AAV9 was injected separately through the tail vein ds AAV9-e GFP or ds AAV9-Ca MEK viral vectors.Four weeks after transfection,an in vivo myocardial ischemia/reperfusion injury model was established.The main detection indicators are myocardial infarction area,myocardial cell apoptosis index,evaluation of cardiac function,determination of serum myocardial enzymes,oxidative stress level,and expression of key proteins in the ERK1/2 signaling pathway.Study results: 1)Post-ischemic adaptation can significantly reduce the myocardial infarction area of C57BL/6J mice,and has protective effect on C57BL/6J mice,mainly by activating effectivly the ERK1/2 signaling pathway in the myocardium of C57BL/6J mice.However,post-ischemic adaptation has no protective effect on the myocardium of the type II diabetic mice.There is no significant difference in the area of myocardial infarction between the I/R group and the IPost C group(P>0.05),mainly result of post-ischemic adaptation(IPost C)cannot effectively activate ERK1/2 signaling pathway of type II diabetic mice myocardium.2)After simultaneous high glucose stimulation and ischemia hypoxia/reperfusion,the apoptotic rate of H9C2 cardiomyocytes was further increased compared with the high glucose group,and there was a significant difference(P<0.05).However,the phosphorylation level of ERK1/2protein in the high glucose ischemia-reperfusion group did not increase further.After administration of ERK activator or Ca MEK gene transfection,the phosphorylation level of ERK1/2 protein can be significantly activated and the apoptotic rate of cardiomyocytes can be significantly reduced(P<0.05).we found that after administration of ERK activator or transfection of Ca MEK gene,high glucose myocardial ischemia-reperfusion caused a decrease in the expression of pro-apoptotic protein Bax,and an increase in the expression of Bcl-2/Bax ratio.Mitotracker results showed that after transfection of Ca MEK gene,the number of mitochondria punctiform in the high glucose myocardial ischemia-reperfusion group decreased,the average network structure size increased,and the median branch length increased(P<0.01),the median network structure size and the mean branch length increased,and the differences were statistically significant(P<0.05).JC-1 test results showed that the mitochondrial membrane potential increased.Western blotting results indicated that the phosphorylation expression level of the mitochondrial fission-promoting protein Drp1s616 was reduced,and the immunofluorescence results indicated that mitochondrial fission was reduced.Mito SOX results suggested that the level of oxidative stress was reduced.The above results indicated that the administration of ERK activator or Ca MEK gene to activate ERK1/2 signaling pathway can reduce mitochondrial division,improve mitochondrial membrane potential,improve mitochondrial function,reduce oxidative stress,and alleviate high glucose myocardial ischemia-reperfusion injury.3)For in vivo ischemia-reperfusion injury(I/R)studies,it was found that transduction of Ca MEK gene in vivo can effectively activate the myocardial ERK1/2 signaling pathway of myocardial ischemia-reperfusion injury in type II diabetic mice.Myocardial infarction area,myocardial cell apoptosis index,inflammatory factors and myocardial enzyme levels were significantly reduced,and the difference was statistically significant(P<0.01).Moreover,transduction of Ca MEK gene in vivo effectively activated the ERK1/2 signaling pathway of myocardial ischemia-reperfusion injury in type II diabetic mice,and found that it can inhibit myocardial ischemia-reperfusion injury,reduce mitochondrial division,improve hemodynamics,and reduce myocardium Apoptosis.Conclusion: 1)The ineffective activation of ERK1/2 signaling pathway is the main reason that post-ischemic adaptation has no protective effect on myocardial ischemia-reperfusion(I/R)injury in type II diabetic mice.2)By giving ERK activator or transfecting Ca MEK gene,H9C2 cardiomyocyte ERK1/2 signaling pathway can be targeted to activate,reduce the apoptotic rate of cardiomyocytes,reduce mitochondrial division,increase mitochondrial membrane potential,reduce oxidative stress level,and stabilize mitochondria Function,inhibit the expression level of pro-apoptotic protein,increase the expression level of anti-apoptotic protein,thereby reducing myocardial cell apoptosis and improving high glucose myocardial ischemia-reperfusion(I/R)injury.Ca MEK gene targeted transduction is expected to become high glucose myocardial resistance effective method of ischemia/reperfusion injury.3)The administration of Ca MEK gene can significantly activate the ERK1/2 signaling pathway in type II diabetic mice,effectively reduce the area of myocardial infarction and myocardial cell apoptosis,reduce the level of myocardial enzymes,and reduce the level of oxidative stress,thereby reducing the type II diabetic ischemia-reperfusion injury.