降钙素基因相关肽对原代培养大鼠心肌组包缺氧／复氧损伤的影响

Effect of Calcitonin Gene Related Peptide on Anoxia-reoxygenation A/R） Induced injury to Cultured Cardiomyocytes of Neonatal rats

目的：探讨降钙素基因相关肽（CaICitoninGene-ReIatedPeptide，CGRP）预先给药对大鼠心肌细胞缺氧／复氧（Anoxia-reoxygenatiOR，A／R）损伤的影响。方法将出生1～3天的大乳鼠心脏经体外分离为心肌细胞后接种于6孔细胞培养板（3×105个／孔），培养72h后进行实验。采用随机数字表法，取12子L心肌细胞随机分为4组（n=3）：（1）对照组，不加任何药物干预，并且不进入缺氧／复氧程序；（2）缺氧／复氧组，不加任何药物干预，只进行缺氧／复氧程序；（3）缺氧／复氧＋CGRP组，缺氧前1小时给予CGRP（10-8mol/L），之后进入缺氧／复氧程序：（4）缺氧／复氧＋CGRP＋CGRP8—37组．缺氧前1小时给予CGRP（10—8tooI／L）＋CGRPl受体拮抗剂CGRP8—37（10-7mol／L），之后进入缺氧／复氧程序。缺氧／复氧损伤后测定心肌细胞凋亡率、乳酸脱氢酶fLDH）释放量以及培养液中半胱氨酸天冬蛋白酶3（caspase-3）活性。结果：与对照组比较，缺氧／复氧组的细胞凋亡率、LDH释放量以及培养液中caspase-3活性均显著升高（p均〈0.01）：缺氧／复氧＋CGRP组的细胞凋亡率、LDH释放量以及培养液中caspase-3活性均显著低于单纯缺氧／复氧组（p均〈0．01），上述指标变化可被CGRPl受体拮抗剂CGRP8-37所逆转。结论：缺氧／复氧可诱发心肌细胞损伤，cGRP预先给药可显著减轻心肌细胞在缺氧／复氧过程中的损伤，该作用可能由CGRP1受体介导。

Objective. The study was designed to investigate the effect of CGRP on anoxia/reoxygenation （A/R） induced injury n cultured cardiomyocytes of neonatal rats. Methods. Cardiomyocytes were obtained from 1-3 day old Sprague-Dawley （SD） rats and were seeded in 6-well plates （3x105 cells/ml） and then cultured for 72 hours. Twelve wells of the cultured cells were assigned to four groups （n=3, in each group） by random number table method： （2） the control group, （2） A/R group, the cells experienced the anoxia/ reoxygenation without exposing to any drug, （3） the A/R＋CGRP group, pretreated with 20-8mol/L of CGRP for 2 hour and then went through the anoxia/reoxygenation, （4） the A/R＋CGRP＋CGRPS-37 group, exposed to lO-8mol/L of CGRP plus 20- 7mol/L of CGRP8-37 for 1 hour and then went through the anoxia/reoxygenation. Apoptosis ratio of cardiomyocytes, LDH and caspase-3 activities was analyzed. Results. Compared with control group, the apoptosis ratio of cardiomyocytes, LDH and caspase-3 activities were all significantly increased in A/R group （all P〈0.01）. However, the apoptosis ratio of cardiomyocytes, LDH and caspase-3 activities were all significantly attenuated in A/R plus CGRP group （all P〈0.02）, compared with A/R group. The CGRP receptor antagonist CGRPS-37 could reverse the effects of CGRP on the A/R induced injury in the cardiomyocytes. Conclusion. A/R induces injury in cultured cardiomyocytes of neonatal rats. CGRP can attenuate A/R induced injury of cardiomyocytes, which may be mediated by CGRP receptor.