Effect Of Annexin-A1 Short Peptide On Hypoxia/reoxygenation Injury Of Cardiac Myocytes In Diabetic Rats And Related Research

Objective:To investigate the effect and mechanism of Annexin-A1sp on hypoxia/reoxygenation injury of cardiac myocytes in diabetic rats.Methods:Isolated adult diabetic rat heart by MPA isolated heart perfusion device.Myocytes after being continuously cultivated 4h were randomly divided into five groups:Diabetic group（DM group）、Ddiabetic hypoxia/reoxygenation group（H/R group）、Annexin-A1sp+diabetic hypoxia/reoxygenation group（ANX+H/Rgroup）、5-hydroxyap-atinic acid+diabetic hypoxia/reoxygenation group（5-HD+H/Rgroup）、Annexin-A1sp+5-hydroxyapatinicacid+diabetichypoxia/reoxygenationgroup（ANX+5-HD+H/R group）.The treatment for each group was:the DM group:continuous culture in a normal incubator（95%O₂+5%CO₂）for 105 min;the H/R group:45 min of hypoxia（5%CO₂+1%O₂+94%N₂）and then 60 min of reoxygenation（95%O₂+5%CO₂）;the ANX+H/R group:the same procedure as the H/R group,after reoxygenation immediately added Annexin-A1sp（final concentration 10μM）to the medium;the 5-HD+H/R group:the same procedure as the H/R group,after reoxygenation immediately added 5-hydroxysunic acid solution（final concentration 10μM）to the medium;the ANX+5-HD+H/R group:the same procedure as the H/R group,after reoxygenation immediately added both Annexin-A1sp and the 5-hydroxyapatinic acid solution the final（both final concentration was 10μM）to the medium.After reoxygenation,all the cells in each group were compared and observed using:（1）Fluo-4 probe,the laser confocal method to detect calcium ion level;（2）DCFH-DA fluorescent probe,a multi-functional enzyme label instrument to detect the reactive oxygen free radical level;（3）ATP kit,a multi-functional enzyme label instrument to detect the ATP content;（4）CCK-8 kit,a multi-functional enzyme label instrument to detect the viability of myocardial cells in each group;（5）0.4%trypan blue staining,the myocardial cell viability was calculated under inverted phase contrast microscopy.Results:1、The content of reactive oxygen species（ROS）and calcium ion in cardiac myocytes of each group:the DM group were lower than the other groups（P<0.05）,and the two indices in ANX+H/R group were lower than those in the H/R group,the ANX+5-HD+H/R group,and 5-HD+H/R group（P<0.05）.The two indices in the H/R group and 5-HD+H/R group were higher than those in ANX+5-HD+H/R group（P<0.05）.The concentration of calcium ion in the 5-HD+H/R group was lower than that in group H/R（P<0.05）.There was no significant difference in the amount of reactive oxygen species free radicals between group 5-HD+H/R and the H/R group（P>0.05）.2、ATP content of cardiac myocytes in each group:the DM group was the highest（P<0.05）;the ATP content in ANX+H/R group was lower than that in the DM group but higher than that in H/R group and 5-HD+H/R group（P<0.05）;the ATP content in ANX+5-HD+H/R group was lower than that in ANX+H/R group but higher than that in the H/R group and the 5-HD+H/R group（P<0.05）;There was no significant difference in the ATP content between the 5-HD+H/R group and the H/R group.（P>0.05）.3、The viability and the survival rate of myocardial cells:the DM group were higher than the other groups（P<0.05）;those in the ANX+H/R group were higher than those in the H/R group and 5-HD+H/R group（P<0.05）;while those in ANX+5-HD+H/R group were lower than those in the ANX+H/R group,but higher than those in the H/R group and the5-HD+H/R group（P<0.05）;there was no significant difference between the 5-HD+H/R group and the H/R group（P>0.05）.Conclusion:1、Annexin-A1sp has protective effect on myocardial cells of diabetic rats with hypoxia/reoxygenation injury.2、The protective mechanism of the Annexin-A1spon diabetic rat cardiomyocytes may be related to its ability to open the mitoKATP channels,thus inhibiting the ROS outbreak,reducing the intracellular calcium overload and preserving the intracellular ATP content.