Effect of calcitonin gene-related peptide on anoxia-reoxygenation induced injury to neonatal rat cardiomyocytes incubated in high glucose medium

Abstract

Objective To evaluate the effect of calcitonin gene-related peptide (CGRP) on anoxiareoxygenation (A/R)-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium.Methods Cardiomyocytes were obtained from 1-3-day old Sprague-Dawley rats and cultured in the culture medium containing 15％ bovine calf serum and then seeded onto 6-well plates at a density of 10 × 105/ml (3 ml/well).The cells were randomly divided into 5 groups (n =9 each):normal glucose medium control group (NG group),high glucose medium group (HG group),high glucose medium + A/R group (HG+ A/R group),high glucose medium + A/R + CGRP group (HG + A/R + CGRP group),and high glucose medium + A/R + CGRP+ CGRP8-37 group (HG + A/R + CGRP + CGRP8-37 group).The cells were incubated in normal glucose (5.5 mmol/L) medium for 72 h in NG group.In HG group,the cells were incubated in high glucose (25.0 mmol/L) medium for 72 h.In HG + A/ R group,the cells were incubated in high glucose medium for 72 h and then exposed to 3 h of anoxia followed by 2 h of reoxygenation.In group HG + A/R + CGRP,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10-8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10 8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP8-37 (final concentration 10-8 mol/L) and CGRP8-37 (CGRP receptor antagonist,final concentration 10-7 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.Apoptosis in cardiomyocytes was detected using TUNEL and apoptosis index (AI) was calculated.The lactate dehydrogenase (LDH) activity in the culture medium was analyzed.Results AI and LDH activity were significantly higher in HG group than in NG group,and in HG + A/R group than in HG group.Compared with HG + A/R group,AI and LDH activity were significantly decreased in HG + A/R + CGRP group,while no significant changes were found in HG + A/R + CGRP + CGRP8-37 group.Compared with HG + A/R + CGRP group,AI and LDH activity were significantly increased in HG + A/R + CGRP + CGRP8-37 group.Conclusion CGRP attenuates A/R-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium via combing with CGRP receptor. Key words: Calcitonin gene-related peptide; Diabetes mellitus; Cell hypoxia; Myocytes, cardiac ; Infant, newborn