Ero1α在缺氧复氧致H9C2细胞内质网应激时的表达变化及意义

Change and significance of Ero1α expression on endoplasmic reticulum stress induced by hypoxia/reoxygenation in rat cardiomycytes

目的:观察内质网氧化还原酶(Endoplasmic oxidoreductin-1-like protein alpha,Ero1α)表达与内质网应激(endoplasmic reticulum stress,ERS)、细胞凋亡的关系。方法:将培养的H9C2心肌细胞随机分组:对照组(Control组)、缺氧/复氧1、3、6、12 h组(H/R1组,H/R3组,H/R6组,H/R12组)、4-苯基丁酸钠预处理+缺氧复氧6 h组(4PBA+H/R6组)。对照组心肌细胞正常培养至实验结束,H/R组行缺氧3 h后复氧1、3、6、12 h,4PBA+H/R6组分别于缺氧前2 h给4PBA再行缺氧/复氧6 h过程。Hoechst33258染色荧光显微镜下观察细胞凋亡情况。Western blot测定Ero1α,内质网应激蛋白葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白(CHOP)、半胱氨酸门冬氨酸特异性蛋白酶12(caspase-12)的表达。结果:与对照组比较,Ero1α蛋白表达水平随着复氧时间的延长Ero1α表达量逐渐升高(P<0.01),在复氧3 h明显升高,6 h达到高点。Grp78在缺氧复氧后即明显升高(P<0.01),在复氧3 h达到高峰。细胞凋亡率也显著升高,其中复氧6 h最为显著。以缺氧3 h复氧6 h作为观测点,H/R6组Ero1α、GRP78、CHOP、Caspase-12蛋白表达较对照组明显增加(P<0.01);给予4PBA干预后,Ero1α、GRP78、CHOP、caspase-12蛋白表达较H/R6组明显下降(P<0.05,P<0.01)。结论:缺氧复氧能够诱导心肌细胞凋亡及内质网应激,缺氧复氧诱导大鼠心肌细胞内质网应激时Ero1α的表达可出现相应的变化并与细胞凋亡相关。

AIM： To explore the relationship between Ero1α expression and ERS and apoptosis in rat hypoxia/reoxygenation cardiomyocyte model. METHODS： The H9C2 cardiomyocytes were randomly divided into groups： control group, hypoxia / reoxygenation 1 h, 3 h, 6 h, 12 h group （H/R1 group, H/R3 group, H/R6 group, H/R12 group）, 4-PBA ＋ H/R6 group. H9C2 cardiomyocytes of con- trol group were cultured under normal condition till the end of experiment. Cardiomyocytes of H/t（ group underwent hypoxia for 3 hours followed by reoxygenation for 1 h, 3 h, 6 h, 12 h. 4PBA were given 2 hours before hypoxia in the 4PBA ＋ H/R6 group. Hoechst 33258 staining was used to observe the apoptosis of cells under fluorescence microscope. The protein expression of Erolct, glucose-regulated proteins 78 （ Grp78 ）, C/EBP homologous protein （CHOP）, easpase-12 were detected by Western blot. RESULTS：Compared with the control group, the expression level of Erolct protein increased with the time of reoxygenation. The expression of Erolct increased significantly at 3 h after reoxygenation and reached high point at 6 h （ compared with the control group, P 〈 0.01 ）. Grp78 increased significantly af- ter hypoxia and reoxygenation （compared with the control group, P 〈0.01 ）, reached a peak at 3 h after reoxygenation. The apoptosis rate was also signif- icantly increased, of which the most significant was at reoxygenation 6 h. Take hypoxia 3 h reoxygen- ation 6 h as the observation point, the expression of Ero1α GRP78, CHOP and Caspase-12 in H/R6 group was significantly higher than that in control group（ P 〈 0.01 ）. After administration of 4PBA, Ero1α, GRP78, CHOP, Caspase-12 protein expression was significantly lower than that in H/R6 group （P 〈 0.05, P 〈 0.01 ）. CONCLUSION ： Hypoxi- a/reoxygenation can induce cardiomyocyte apoptosis and endoplasmic reticulum stress. The expression of Ero1α in cardiomyocytes induced by hypoxia/reoxy- genation can be changed. The expression of Ero1α is related to apoptosis.