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目的研究重组人促红素(EPO)对高糖诱导乳鼠心肌细胞凋亡的影响,以及内质网应激(ERS)在其中的作用。方法分离新生SD大鼠心肌细胞,培养72 h后用高糖诱导心肌细胞凋亡,以甘露醇作为高渗对照,并用不同浓度EPO(5、10、20、40 U·m L-1)预处理心肌细胞48 h,流式细胞仪检测各组细胞凋亡率,选取EPO最佳干预浓度;将实验分成五组即正常糖(NG)组、EPO+NG组、高糖(HG)组、EPO+HG组和ERS抑制剂4-苯基丁酸(PBA)+HG组,流式细胞仪测各组凋亡率,激光共聚焦显微镜检测各组钙离子的平均荧光强度,Real-time PCR和Western blot分别检测各组促红素受体(EPOR)、ERS标志物GRP78、钙内流蛋白Serca2a的m RNA和蛋白表达。结果高糖诱导后心肌细胞凋亡率(41.36±10.49)%,显著高于正常心肌细胞凋亡率(16.08±3.97)%(P<0.05),甘露醇组对正常心肌细胞凋亡无显著影响(P>0.05);20 U·m L-1 EPO预处理高糖诱导的心肌细胞后凋亡率为(20.97±10.55)%,为最佳干预浓度。与NG组比较,EPO+NG组各指标均无显著差异(P>0.05),HG组凋亡率和钙离子荧光强度显著增加,EPOR、Serca2a的m RNA及蛋白表达降低,GRP78 m RNA及蛋白表达升高(均P<0.05);与HG组相比,PBA+HG组和EPO+HG组凋亡率和钙离子荧光强度降低,EPOR、Serca2a的m RNA及蛋白表达升高,GRP78 m RNA及蛋白表达降低(均P<0.05);PBA+HG组和EPO+HG组各指标均无显著差异(P>0.05)。结论高糖可通过介导ERS并引发钙稳态失衡诱导心肌细胞凋亡;EPO能够抑制高糖诱导心肌细胞的凋亡,该作用可能与抑制ERS,增强钙内流蛋白表达、维持细胞内钙离子平衡有关。
Objective To investigate the effect of recombinant human erythropoietin (EPO) on cardiomyocyte apoptosis induced by high glucose and the role of endoplasmic reticulum stress (ERS) in neonatal rat cardiomyocytes. Methods Cardiomyocytes from neonatal SD rats were isolated and cultured for 72 h. Cardiomyocytes were induced to apoptosis by high glucose. Mannitol was used as hypertonic control and pretreated with different concentrations of EPO (5, 10, 20 and 40 U · m L -1) The cardiomyocytes were treated for 48 h, the apoptosis rate of each group was detected by flow cytometry and the best concentration of EPO was selected. The experiment was divided into five groups: normal glucose (NG) group, EPO + NG group, high glucose group EPO + HG group and ERS inhibitor 4-phenylbutyric acid (PBA) + HG group. The apoptosis rate of each group was measured by flow cytometry. The average fluorescence intensity of calcium in each group was detected by confocal laser scanning microscopy. Real-time PCR Western blot were used to detect the expression of m RNA and protein of erythroid receptor (EPOR), ERS marker GRP78 and Ca Ca protein Serca2a. Results The apoptosis rate of cardiomyocytes induced by high glucose was (41.36 ± 10.49)%, which was significantly higher than that of normal cardiomyocytes (16.08 ± 3.97)% (P <0.05). The apoptosis of normal cardiomyocytes was not affected by mannitol (P> 0.05). The apoptotic rate of 20 U · m L-1 EPO pretreated with high glucose was (20.97 ± 10.55)%, which was the best intervention concentration. Compared with NG group, the indexes of EPO + NG group showed no significant difference (P> 0.05), the apoptosis rate and Ca2 + fluorescence intensity of HG group increased significantly, while the expression of m RNA and protein of EPOR and Serca2a decreased. GRP78 mRNA and protein (P <0.05). Compared with HG group, the apoptotic rate and Ca2 + fluorescence intensity of PBA + HG group and EPO + HG group were decreased, while m RNA and protein expression of EPOR and Serca2a increased. GRP78 mRNA (P <0.05). There was no significant difference between PBA + HG group and EPO + HG group (P> 0.05). Conclusion High glucose can induce cardiomyocyte apoptosis by mediating ERS and inducing imbalance of calcium homeostasis. EPO can inhibit the apoptosis of cardiomyocytes induced by high glucose, which may be related to the inhibition of ERS, enhancement of calcium influx protein expression and maintenance of intracellular calcium Ion balance.