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

 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Ca²⁺-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);Ca²⁺-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。

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

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 Ca2+-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 Ca2+ 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.