利拉鲁肽对缺氧和高糖所致心肌细胞钙超载和细胞凋亡的影响

Effects and possible mechanism of liraglutide on hypoxia and high glucose-induced calcium overload and apoptosis in neonatal rat cardiomyocytes in vitro

目的研究利拉鲁肽对缺氧和高糖所致心肌细胞钙超载和细胞凋亡的影响。方法采用原代培养的乳鼠心肌细胞,建立缺氧和高糖模型。实验分为:正常对照组、利拉鲁肽对照组、缺氧和高糖模型组、利拉鲁肽处理组、胰高血糖素样肽-1(glucagon like peptide-1,GLP-1)受体抑制剂组、高渗对照组6组。采用荧光分光光度法检测心肌细胞内钙离子变化,化学荧光法检测活性氧族(reactive oxygen species,ROS)水平、利用生化方法检测Ca^(2+)-ATP和Na^+-K^+-ATP酶活性、RT-PCR技术检测钙敏感的钙蛋白酶(μ-calpain)基因表达、流式细胞仪测定细胞凋亡率、ELISA法检测细胞caspase-3活性。结果与正常对照组相比,缺氧高糖模型组细胞内ROS水平、μ-calpain mRNA表达量、caspase-3活性均显著升高(P<0.01);Ca^(2+)-ATP和Na^+-K^+-ATP酶活性显著降低(P<0.01);游离钙离子浓度由(117.19±15.04)nmol/L增加至(508.53±26.11)nmol/L,细胞凋亡率由(3.95±0.12)%增加至(31.03±4.30)%(P<0.01)。利拉鲁肽处理组细胞较缺氧高糖模型组上述参数均明显改善,游离钙离子浓度和细胞凋亡率显著降低(P<0.01);GLP-1R抑制剂exendin(9-39)可拮抗利拉鲁肽的上述作用(P<0.05)。结论利拉鲁肽可明显抑制缺氧和高糖所致心肌细胞钙超载及μ-calpain基因的上调,降低caspase-3水平,减少心肌细胞凋亡,对心肌细胞具有保护作用。

Objective To investigate the effect and possible mechanism of liraglutide on hypoxia and high glucose-induced calcium overload and apoptosis in neonatal rat cardiomyocytes in vitro. Methods A model of hypoxia and high glucose was established using primarily cultured neonatal rat cardiomyocytes. Then,cells were divided into six groups： normal control group,liraglutide control group,hypoxia and high glucose model group,liraglutide treatment group,GLP-1R antagonist group,and high osmolality control group. The concentration of intracellular dissociative calcium in myocardial cells was determined with fluorospectrophotometry. The levels of Ca^（2 ＋）-ATPase,Na~＋-K~＋-ATPase and ROS were measured with biochemical approaches. The apoptosis rate was observed by a flow cytometer. The level of μ-calpain mRNA was examined with RT-PCR method. The level of caspase-3 was measured with ELISA.Results Compared with normal control group,the levels of expression of ROS,caspase-3,and μ-calpain mRNA were significantly increased in hypoxia and high glucose model group（ P〈0. 01）,the activity of Ca2 ＋-ATPase and Na＋-K＋-ATPase decreased（ P〈0. 01）,the concentration of free Ca^（2 ＋） increased from 117. 19 ± 15. 04 nmol/L to 508. 53 ± 26. 11 nmol/L,and the apoptosis rate increased from（ 3. 95 ± 0. 12） % to（ 31. 03 ± 4. 30） %. Liraglutide improved the parameters mentioned above（ P〈0. 01）. However,exendin（ 9-39）, an antagonist of GLP-1R, attenuated the protective effect of liraglutide under hypoxia and high glucose.Conclusions Liraglutide can obviously inhibit calcium influx of myocardial cells and μ-calpain up-regulation,and decrease the level of caspase-3 and cell apoptosis.