Relationship Between Nur77-GRP78 Pathway And Increased Sensitivity To Diabetic Myocardial Ischemia-reperfusion Injury

Objective:To study the role of Nur77 and GRP78 in the increased sensitivity of diabetic myocardial ischemia-reperfusion injury,and the potential molecular pathways of the above。Methods:H9C2 cardiomyocytes cultured in normal and high glucose medium were cultured under anaerobic conditions for 2 hours to establish myocardial ischemia-reperfusion(I /R)model,and the normal culture + hypoxia induced group(NC + I Group,n = 9)and high glucose culture + hypoxia induction group(H / H + I group,n = 9);normal cells served as blank control group(NC group,n = 9).Annexin-V FITC / PI double staining method,spectrophotometric method were used to measure Caspase3 activity and JC-1staining method to measure mitochondrial membrane potential,evaluating the difference of apoptosis in each group;Fluorescence staining and Western blot to detect the distribution and relative expression of Nur77 and GRP78 in the cell,the Co-IP method to determine the intracellular binding of Nur77 and GRP78.Results:The difference in apoptosis of cardiomyocytes in each group: Compared with the NC group,the apoptosis in the other two groups(NC,H / H + I)increased significantly(P <0.01);The H / H + I group,compared with the NC + I group,was significantly increased(P <0.01).The distribution of Nur77 and GRP78 subcells in each group was compared: Compared with the NC group,Nur77 distribution around the nucleus in the NC+I group and H/H+I group increased significantly(P<0.01).Compared with the NC+I group,Nur77 distribution around the nucleus in the H/H+I group increased significantly(P<0.05).The distribution of Nur77 around the mitochondria in the NC + I group and H /H + I group was significantly increased(P <0.01),compared with the NC group;The distribution of Nur77 around the mitochondria in the H / H + I group was significantly increased(P <0.01),compared with the NC + I group.Compared with the NC group,the distribution of GRP78 around the mitochondria in the other two groups was relatively increased(P <0.05);Compared with the NC + I group,the distribution of GRP78 around the mitochondria in the H / H + I group was slightly increased(P> 0.05).Compared with the NC group,the distribution of GRP78 around the endoplasmic reticulum in the other two groups increased significantly(P <0.01);The distribution of GRP78 around the endoplasmic reticulum in the H / H + I group increased significantly(P <0.01),compared with the NC + I group.The relative expression levels of Nur77 and GRP78 in each group can be obtained: Compared with the NC group,the expression of Nur77 in the other two groups(NC,H / H + I)cells was significantly increased(P <0.01);Compared with the NC + I group,the expression of Nur77 in the H / H + I group was significantly increased(P <0.05).Compared with the NC group,the other two groups has significantly increased in the GRP78 expression(P <0.05);Compared with NC + I group,the H / H + I group was also significantly increased too(P <0.01).Detect the content of Nur77 and GRP78 in the lysate of each group after IP reaction: Compared with NC group,Nur77 content in NC+I group and H/H+I group did not increase significantly after IP reaction(P>,0.05).Compared with NC+I group,Nur77 content in the H/H+I group did not increase significantly after IP reaction(P>,0.05).Compared with NC group,GRP78 content in NC+I group and H/H+I group increased significantly after IP reaction(P<0.01).Compared with NC+I group,GRP78 content in H/H+I group increased significantly after IP reaction(P<0.01).Conclusion:Normal cardiomyocyte I / R and increased myocardial cell I / R sensitivity in high glucose culture are closely related to apoptosis;The occurrence of cardiomyocyte apoptosis in the above process may be closely related to the increased expression and intracellular displacement of Nur77 and GRP78;Nur77 can bind to GRP78.Based on the aforementioned relevant conclusions,it is speculated that Nur77 and GRP78 can simultaneously shift to mitochondria and interact,when the apoptosis signal is triggered in the above process,and the above process eventually lead to apoptosis.Myocardial cell I / R and increased myocardial cell I / R sensitivity in high glucose culture may be related to the Nur77-GRP78 apoptosis pathway。...