松弛素保护心肌微血管内皮细胞缺氧复氧损伤的机制

Relaxin protects myocardial microvascular endothelial cells from hypoxia-reoxygenation injury

背景:研究报道松弛素可显著改善病理因素导致的心肾功能障碍,抑制心肌肥厚、抗纤维化以及改善缺血再灌注损伤等,但其对内皮细胞缺氧复氧损伤的保护机制尚不明确。目的:探讨松弛素保护心肌微血管内皮细胞缺氧复氧损伤的机制。方法:取生长状态良好的小鼠心肌微血管内皮细胞系(H5V),分3组处理:对照组常规培养33 h,模型组常规培养24 h后给予6 h缺氧+3 h复氧模拟心肌缺氧再灌注损伤,松弛素组常规培养(加180 ng/mL松弛素)24 h后给予6 h缺氧+3 h复氧(加180 ng/mL松弛素)模拟心肌缺氧再灌注损伤。检测细胞通透性与Caspase-3的表达,细胞上清中肿瘤坏死因子α、白细胞介素1β和白细胞介素6水平,RT-PCR检测细胞中钙黏蛋白、Akt1、GSK-3βmRNA表达;Western blot检测细胞中钙黏蛋白、Akt、GSK-3β蛋白表达。结果与结论:(1)与对照组比较,模型组细胞通透性增强(P<0.05),细胞内Caspase-3表达升高(P<0.05);与模型组比较,松弛素组细胞通透性降低(P<0.05),细胞内Caspase-3表达降低(P<0.05);(2)与对照组比较,模型组上清中肿瘤坏死因子α、白细胞介素1β和白细胞介素6水平升高(P<0.05);与模型组比较,松弛素组3种炎症因子水平降低(P<0.05);(3)3组间细胞中钙黏蛋白、Akt1、GSK-3βmRNA表达比较差异无显著性意义(P>0.05);(4)3组间细胞中钙黏蛋白、Akt、GSK-3β总蛋白表达比较差异无显著性意义(P>0.05);与对照组比较,模型组磷酸化钙黏蛋白、p-Akt、p-GSK-3β蛋白表达降低(P<0.05);与模型组比较,松弛素组磷酸化钙黏蛋白、p-Akt、p-GSK-3β蛋白表达升高(P<0.05);(5)结果表明,松弛素治疗通过增强钙黏蛋白表达,减少缺氧复氧引起的小鼠心肌微血管内皮细胞损伤,抑制炎症因子的释放,减轻细胞凋亡,该作用可能与激活Akt/GSK-3β信号通路有关。

BACKGROUND:Relaxin can significantly improve cardiac and renal dysfuction caused by pathological factors,inhibit myocardial hypertrophy,have an anti-fibrosis effect,and improve ischemia-reperfusion injury.H owever,its protective mechanism against hypoxia-reoxygenation inju ry of endothelial cells remains unclear.OBJECTIVE:To investigate the protective mechanism of relaxin against hypoxia-reoxygenation injury of myocardial microvascular endothelial cells.METHODS:Mouse myocardial microvascular endothelial cell line(H5V cells)was used for the experiment.Cells were treated by three different interventions:in control group,cells we re normally cultured for 33 hours;in model group,cells we re treated by 6-hour hypoxia followed by 3-hour reoxygenation;and in relaxin group,24 hours of routine culture(180 ng/mL relaxin),6 hours of hypoxia and 3 hours of reoxygenation(180 ng/mL relaxin)were given to simulate myocardial hypoxia-reperfusion injury.Cell permeability and Caspase-3 activity we re then detected.Levels of tumor necrosis factor-α,interleukin-1βand interleukin-6 in cell supernatants were detected by ELISA.Expressions of VE-cadherin,Akt,and GSK-3β at mRNA and protein levels were detected by RT-PCR and western blot,respectively.RESULTS AND CONCLUSION:Compared with the control group,the cell permeability and expression of caspase-3 increased significantly in the model group(P<0.05).Compared with the model group,the cell permeability and expression of caspase-3 decreased in the relaxin group(P<0.05).Moreover,the levels of tumor necrosis factor-α,interleukin-1βand inte rleukin-6 were elevated in the model group,while the levels were significantly decreased after relaxin treatment(all P<0.05).There were no significantly changes in the mRNA and protein expressions of VE-cadherin,Akt1,and GSK-3β mRNA among three groups(all P>0.05).Compared with the control group,the expression of phosphorylated VE-cadherin,Akt1 and GSK-3β were decreased in the model group(P<0.05),and relaxin treatment reve rsed these changes to the control levels(P<0.05).To conclude,relaxin treatment could enhance VE-cadherin expression,reduce hypoxiareoxygenation-induced microvascular endothelial cell damage,inhibit inflammatory cytokine release,and reduce cell apoptosis,which may be related to the activation of the Akt/GSK-3β signaling pathway.