Calcitonin Gene-related Peptide Inhibits Anoxia/reoxygenation Induced Increases In The Enzymatic Activity Of Caspase-3 And Caspase-9 Through The PKC-mitoK_ATP Pathway

Objective:1.To observe the effect of pre-administration Calcitonin Gene Related Peptide（CGRP）on myocardial injury induced by anoxia/reoxygenation（A/R）in rats.2.To investigate the effect of mitoK_ATP channel activation on anoxia/reoxygenation cardiomyocyte injury in rats.3.To observe the changes of Caspase-3 and Caspase-9 enzyme activities before and after anoxia-reoxygenation,to study the effect of CGRP on cardiomyocyte-protective effect on sarcK_ATP/mitoK_ATPTP channel effects.Methods:1.Neonatal rat cardiomyocytes is in vitro culture and identification.Selection of healthy SD rats 1-3 days old,male or female,for the establishment of primary cultured cardiomyocytes in vitro model.The cultured cardiomyocytes were identified by immunocytochemical SABC method.2.Cardiomyocytes anoxia/reoxygenation（A/R）model establishment.The anoxia solution was replaced by cultured cardiomyocytes cultured at 72 hours and pulsating hole.The cells were placed in a closed oxygen-deficient box which was continuously filled with high-purity nitrogen for 3 hours to simulate anoxia.After rehydration,the cells were cultured in a normal incubator for 2 hours Simulate reoxygenation.3.The effect of CGRP on neonatal rat cardiomyocytes anoxia-reoxygenation（A/R）injury.The 24-hour culture of cardiomyocytes cultured for 72h was selected and randomly divided into 4 groups（n=6）:（1）control group:no A/R treatment,continuous culture under normal conditions;（2）anoxia/reoxygenation group:A/R treatment;（3）CGRP+A/R group:CGRP(10^-8mol/L)was given 30 minutes before anoxia,then began A/R treatment;（4）CGRP_8-37+CGRP+A/R group:CGRP_8-37(10^-6mol/L)was given 30minutes before anoxia,CGRP was given 10 minutes later,and then same as A/R group.After reoxygenation,the activity of Caspase-3 and Caspase-9 were detected by spectrophotometry.4.Effect of mitoK_ATP channel activation on anoxia/reoxygenation cardiomyocyte injury in rats.Six-hole cells were cultured for 72 h.DZ+A/R group:DZ(10^-4mol/L)was given 20 minutes before anoxia,then same as A/R group.After reoxygenation,the activity of Caspase-3 and Caspase-9 were detected by spectrophotometry.5.The effect of CGRP on sarcK_ATP/mitoK_ATP channel in cardiomyocyte-protective effect.The cultured cells were cultured for 72 h and 18-hole cells were selected and randomly divided into 3 groups（n=6）:（1）5-HD+CGRP+A/R group:5-HD(5×10^-4mol/L)was given 30 minutes before anoxia,and CGRP was given after 10minutes later,and then the same as A/R group.（2）Glibenclamide+CGRP+A/R group:Glibenclamide(10^-5mol/L)was given 30 minutes before anoxia,and CGRP was given after 10 minutes later,and then the same as A/R group.（3）HMR-1098+CGRP+A/R group:HMR-1098(10^-5mol/L)was given 30 minutes before anoxia,CGRP was given after 10 minutes,and then the same as A/R group.After reoxygenation,the activity of Caspase-3 and Caspase-9 were detected by spectrophotometry.6.PKA/PKC pathway in CGRP activated mitoK_ATP channels in the role.The cultured cells were cultured for 72h and 12-hole cells were selected and randomly divided into 2 groups（n=6）:（1）H-89+CGRP+A/R group:H-89(3×10^-6mol/L)was given 30minutes before anoxia,CGRP was given after 10 minutes,and then the same as A/R group;（2）chelerythine+CGRP+A/R group:chelerythine(2×10^-6mol/L),and then the same A/R group.After reoxygenation,the activity of Caspase-3 and Caspase-9 were detected by spectrophotometry.Results:1.Rat neonatal rat cardiomyocytes model was established successfully.After testing,cardiomyocytes accounted for more than 90%of the total number of cultured cells.2.Neonatal rat cardiomyocytes anoxia/reoxygenation model was established successfully,anoxia/reoxygenation apoptosis rate of cardiomyocytes increased significantly.3.A large number of CGRP receptors exist in normal neonatal rat cardiomyocytes,and in the nucleus and cytoplasm are distributed.4.Compared with the blank control group,the activities of Caspase-3 and Caspase-9in A/R group were significantly increased（P<0.05）.Compared with A/R group,the activities of Caspase-3 and Caspase-9 in CGRP+A/R group were significantly decreased（P<0.05）,but this effects can be reversed by CGRP_8-37（P<0.05）.5.Compared with A/R group,the activities of Caspase-3 and Caspase-9 in DZ+A/R group were significantly decreased（P<0.05）.6.Compared with CGRP+A/R group,the activities of Caspase-3 and Caspase-9 in Glibenclamide+CGRP+A/R group were significantly increased（P<0.05）.The activity of Caspase-3 and Caspase-9 in HMR-1098+CGRP+A/R group did not increase significantly,which was not significantly different from CGRP+A/R group.In addition,compared with CGRP+A/R group,the activity of Caspase-3 and Caspase-9 in 5-HD+CGRP+A/R group was significantly increased（P<0.05）.7.Compared with CGRP+A/R group,the activity of Caspase-3 and Caspase-9 in H-89+CGRP+A/R group had an increasing trend,but there was no statistical significance.The activities of Caspase-3 and Caspase-9 in chelerythine+CGRP+A/R group were significantly increased（P<0.05）.Conclusions:1.Neonatal rat cardiomyocytes undergo anoxia/reoxygenation injury,which can induce the significant increase of the activity of caspase-3 and Caspase-9 in cardiomyocytes.Pretreatment with CGRP before anoxia could significantly decrease the activity of Caspase-3 and Caspase-9 in cardiomyocytes and alleviate cardiomyocyte injury.2.Activation of mitoK_ATP channel opening can reduce neonatal cardiomyocytes anoxia/reoxygenation injury,play a role in myocardial protection.3.CGRP has a protective effect on anoxia/reoxygenated cardiomyocytes,which may be related to the activation of protein kinase C and the opening of mitoK_ATP channel.