| Objective:To investigate the effect of CGRP on H/R induced injury in cardiomyocytes incubated in low/high glucose medium. The mechanisms of CGRP on H/R induced injury were investigated. Methods:1.Cardiomyocytes culture, identification and induction of H/R injury. The hearts were collected from the neonates of SD rats, within 72 h of birth. Then the tissues of the ventricles were minced and digested by collagenase Type Ⅱ. The cardiomyocytes were centrifuged and collected in six-well cell culture clusters. Identification of cardiomyocytes was achieved using immunohistochemical assay(SABC method) with antibodies against sarcometric α-actin. After 72 h of incubation, the cardiomyocytes were treated with the hypoxia for 3 h followed by the reoxygenation for 2 h. Apoptosis was examined by TUNEL.2.The effect of CGRP on H/R induced injury. Cells were cultured in low glucose medium for 72 h. Twelve wells of the cultured cells were randomly assigned to four groups(n=3):(1) The control group, the cells were kept for 5h without any treatments or drugs.(2) The H/R group, without any other treatments, the cells were treated with the hypoxia for 3 h followed by the reoxygenation for 2h.(3) The CGRP postconditioning group(H/R+CGRP), cells were treated with the hypoxia/reoxygenation and with CGRP, given at 10-8 mol/L immediately before the start of the reoxygenation.(4) The CGRP8-37 antagonizing group(H/R+ CGRP8-37+CGRP), CGRP8-37(10-7mol/L) was administrated 1 h before the induction of cell-hypoxia, then the treatments of hypoxia/reoxygenation and CGRP(10-8 mol/L) were followed as scheduled. Apoptosis was detected by TUNEL. Culture medium was collected from each well. The changes of LDH and caspase-3 were determined by ELISA.3.The mechanisms of CGRP in attenuation of cardiomyocyte injury induced by H/R. Groups and treatments were according to method 2. The JC-1 assay was performed to analysis the mitochondrial membrane potential(MMP). Fluo-3 AM and Rhod-2 AM were performed to analysis cytosolic and mitochondrial calcium. Cytosolic reactive oxygen species(ROS) were evaluated by DCFH-DA.4. The effect of CGRP on H/R induced injury in high glucose medium. Cardiomyocytes were cultured in low and high glucose DMEM respectively. Fifteen wells of cultured cardiomyocytes were randomly assigned to five groups(n=3):(1) LG group, cells were cultured in low glucose DMEM, without any treatment with any test agent.(2) HG group, high glucose group, cells were cultured in high glucose medium, without any treatment with any test agent.(3) HG+H/R group, cells were cultured in high glucose medium and exposed to hypoxia/reoxygenation.(4) HG+H/R+CGRP group, cells were cultured in high glucose medium and exposed to hypoxia/reoxygenation. Cells were treated with CGRP(10-8 mol/L) at the beginning of reoxygenation.(5) HG+H/R+CGRP+CGRP8-37 group, cells were cultured in high glucose medium. CGRP8-37(10-7mol/L) was administrated 1 h before the induction of cell-hypoxia, then the treatments of hypoxia/reoxygenation and CGRP(10-8 mol/L) were followed as scheduled. Apoptosis was detected by TUNEL. Culture medium was collected from each well. The changes of LDH and caspase-3 were determined by ELISA.5.The mechanisms of CGRP in attenuation of cardiomyocytes incubated in high glucose medium injury induced by hypoxia/reoxygenation. Groups and treatments were according to method 4. Cytosolic and mitochondrial calcium was analyzed by Fluo-3 AM and Rhod-2 AM. Cytosolic reactive oxygen species(ROS) were evaluated by DCFH-DA. Results:1.The model of neonatal SD rats cardiomyocytes was successfully established, presenting more than 90% of the cultured cells were cardiomyocytes with microscope.2.The model of H/R was established. Compared with the control group, the myocardial apoptosis was significantly greater in H/R group(P<0.05).3.The apoptosis rate, medium concentrations of LDH and caspase-3, concentrations of the cytosolic and mitochondrial calcium, concentration of cytosolic ROS was significantly greater(P<0.05), MMP was significantly lower(P<0.05), compared to control group. CGRP significantly attenuated the changes of the cardiomyocyte apoptosis rate, medium concentrations of LDH and caspase-3, concentrations of the cytosolic and mitochondrial calcium, concentration of cytosolic ROS, MMP induced by H/R. CGRP8-37 reversed the effect.4.The apoptosis rate, medium concentrations of LDH and caspase-3, concentrations of the cytosolic and mitochondrial calcium, concentration of cytosolic ROS in HG group was significantly greater than LG group(P < 0.05). The apoptosis rate, medium concentrations of LDH and caspase-3, concentrations of the cytosolic and mitochondrial calcium, concentration of cytosolic ROS in HG+H/R group was significantly greater than HG group(P < 0.05). The effect of HG+H/R was attenuated by CGRP. CGRP8-37 reversed the effect. Conclusion:1. The cardiomyocytes were injured by H/R, CGRP can attenuate the effect of H/R, CGRP8-37 reversed the effect of CGRP.2. The cardiomyocytes were injured by high glucose medium, H/R induces severer injury in cardiomyocytes, CGRP can attenuate the effect of HG+H/R, CGRP8-37 reversed the effect of CGRP.3. The findings in this study suggest that CGRP may prevent cardiomyocyte injury induced by hypoxia/reoxygenation via secure homeostasis of cytosolic and mitochondrial calcium, cytosolic ROS and MMP. |
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