降钙素基因相关肽对高糖下新生大鼠心肌细胞缺氧复氧损伤的影响

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

目的 评价降钙素基因相关肽（CGRP）对高糖下新生大鼠心肌细胞缺氧复氧（A/R）损伤的影响.方法 取原代培养的出生1 ～3 d SD大鼠心肌细胞,在含有10％胎牛血清培养基中培养,细胞接种于6孔细胞培养板,接种密度10×10^5/ml,3 ml/孔,采用随机数字表法,将其分为5组（n=9）,正常糖浓度对照组（NG组）用正常糖浓度（葡萄糖5.5 mmol/L）培养基孵育细胞72 h;高糖对照组（HG组）用高糖（葡萄糖25.0 mmol/L）培养基孵育细胞72 h;HG＋ A/R组用高糖培养基孵育细胞72 h后,行缺氧3h,复氧2h;HG＋ A/R＋ CGRP组用高糖培养基孵育细胞72 h后加入CGRP（终浓度为10^-8 mol/L）,1h后行缺氧3h,复氧2h;HG＋ MR＋ CGRP＋ CGRP8-37组用高糖培养基孵育细胞72 h后加入CGRP（终浓度为10^-8 mol/L）和CGRP8-37（CGRP受体拮抗剂,终浓度10^-7 mol/L）,1h后行缺氧3h,复氧2h.处理后收集细胞,采用TUNEL法检测心肌细胞凋亡情况,计算凋亡指数（AI）.于缺氧3h和复氧2h时分别收集细胞培养液,测定乳酸脱氢酶（LDH）活性.结果 与NG组比较,HG组AI、细胞培养液LDH活性升高（P＜0.01）;与HG组比较,HG＋ A/R组AI、细胞培养液LDH活性升高（P＜0.01）;与HG＋ A/R组比较,HG＋ A/R＋ CGRP组AI、细胞培养液LDH活性降低（P＜0.01）,HG＋ A/R＋ CGRP＋CGRP8-37组上述指标比较差异无统计学意义（P＞0.05）;与HG＋ A/R＋ CGRP组比较,HG＋ A/R＋ CGRP＋ CGRP8-37组AI、细胞培养液LDH活性升高（P＜0.01）.结论 CGRP可通过与CGRP受体结合减轻高糖下新生大鼠心肌细胞缺氧复氧损伤.

Objective To evaluate the effect of calcitonin gene-related peptide （CGRP） on anoxia- reoxygenation （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 × 10^5/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 ceils 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 tbr 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-s 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 tool/L） and CGRP8-37 （CGRP receptor antagonist, final concentration 10^-7 mol/L） was then added to the culture nmdia 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 wer 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 LI）H 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.