CAJ | 학술논문

目的：探讨心肌细胞缺氧复氧（H／R）时是否存在异常的ROS／JNK／Egr－1信号通路。方法将培养的H9c2心肌细胞随机分为以下组别：对照（ control）组、control＋ROS激活剂黄嘌呤氧化酶／次黄嘌呤（ con＋XO／HX）组、H／R组、H／R＋ROS清除剂依达拉奉（ EDA）组、H／R＋ROS清除剂N－乙酰基－L－半胱氨酸（ NAC）组、H／R＋JNK抑制剂SP600125组。建立心肌细胞H／R模型，采用不同浓度的EDA（2×10－6、2×10－5、2×10－4 M）、NAC（5×10－4、2×10－3 M、8×10－3 M）、XO／HX（1mU／mL／1．2×10－4 M ，3mU／mL／3．6×10－4 M，5mU／mL／6．0×10－4 M）、SP600125（2×10－5 M）处理细胞。流式细胞仪法检测 H9c2心肌细胞内 ROS 水平。Western blot方法检测心肌细胞Egr－1、p－JNK、JNK蛋白表达的变化。结果 H／R组ROS水平及Egr－1蛋白表达均显著高于control组（ P＜0．05）；2种中、高浓度的ROS清除剂EDA和NAC均能明显降低H／R所致的ROS的高水平和Egr－1蛋白的高表达（均P＜0．05）。而2种低剂量的ROS清除剂对H／R所致的Egr－1蛋白表达以及ROS水平的影响差异无统计学意义。 ROS水平与Egr－1蛋白表达呈高度正相关（ r＝0．91，P＜0．01）。 XO／HX各浓度组JNK激活程度显著高于对照组，且随着XO／HX浓度的增加，JNK激活程度越明显（均P＜0．05）。 H／R组JNK激活水平显著高于control组，使用ROS清除剂EDA与NAC和JNK抑制剂后，JNK活性明显降低（ P＜0．05）。 H／R组Egr－1蛋白表达水平显著高于control组，而JNK抑制剂可以显著抑制H／R所致Egr－1蛋白的高表达（ P＜0．05）。结论 H／R可导致H9c2心肌细胞ROS／Egr－1信号通路激活，JNK的活化于此通路中起了重要的介导作用。
목적：탐토심기세포결양복양（H／R）시시부존재이상적ROS／JNK／Egr－1신호통로。방법장배양적H9c2심기세포수궤분위이하조별：대조（ control）조、control＋ROS격활제황표령양화매／차황표령（ con＋XO／HX）조、H／R조、H／R＋ROS청제제의체랍봉（ EDA）조、H／R＋ROS청제제N－을선기－L－반광안산（ NAC）조、H／R＋JNK억제제SP600125조。건립심기세포H／R모형，채용불동농도적EDA（2×10－6、2×10－5、2×10－4 M）、NAC（5×10－4、2×10－3 M、8×10－3 M）、XO／HX（1mU／mL／1．2×10－4 M ，3mU／mL／3．6×10－4 M，5mU／mL／6．0×10－4 M）、SP600125（2×10－5 M）처리세포。류식세포의법검측 H9c2심기세포내 ROS 수평。Western blot방법검측심기세포Egr－1、p－JNK、JNK단백표체적변화。결과 H／R조ROS수평급Egr－1단백표체균현저고우control조（ P＜0．05）；2충중、고농도적ROS청제제EDA화NAC균능명현강저H／R소치적ROS적고수평화Egr－1단백적고표체（균P＜0．05）。이2충저제량적ROS청제제대H／R소치적Egr－1단백표체이급ROS수평적영향차이무통계학의의。 ROS수평여Egr－1단백표체정고도정상관（ r＝0．91，P＜0．01）。 XO／HX각농도조JNK격활정도현저고우대조조，차수착XO／HX농도적증가，JNK격활정도월명현（균P＜0．05）。 H／R조JNK격활수평현저고우control조，사용ROS청제제EDA여NAC화JNK억제제후，JNK활성명현강저（ P＜0．05）。 H／R조Egr－1단백표체수평현저고우control조，이JNK억제제가이현저억제H／R소치Egr－1단백적고표체（ P＜0．05）。결론 H／R가도치H9c2심기세포ROS／Egr－1신호통로격활，JNK적활화우차통로중기료중요적개도작용。
Objective To investigate whether ROS/JNK/Egr-1 signaling pathway was activated in cardiomyocytes after hypoxia/reoxygenation ( H/R).Methods H9c2 cardiomyocytes were grouped randomly as follows: control group, H/R group, control +the ROS donor xanthine oxidase /hypoxanthine (XO/HX) group, H/R +the ROS scavenger edaravones (EDA) group, H/R +the ROS scavenger N-acetyl-L-cysteine (NAC) group, H/R +JNK inhibitor SP60012 group.To establish H9c2 H/R models and the myocardial cells were treated with different concentrations of EDA (2 × 10 -6,2 ×10 -5,2 ×10 -4 M), NAC (5 ×10 -4,2 ×10 -3,8 ×10 -3 M), XO/HX (1mU/mL/1.2 ×10 -4 M , 3mU/mL/3.6 ×10 -4 M, 5mU/mL/6.0 × 10 -4 M) and SP600125 (2 ×10 -5 M).ROS level was measured by flow cytometry, and Egr-1, p-JNK and total JNK protein levels were detected by Western blot.ResuIts ROS levels and Egr-1 protein levels in H/R group were significantly higher than control group (P<0.05).The moderate and high concentrations EDA and NAC of ROS scavenger significantly decreased the high levels of ROS and Egr-1 protein ( P<0.05 ) , but there were no significant differences of low concentration.There was a significant positive correlation between ROS levels and Egr-1 protein (r=0.91,P<0.01).JNK activation levels in each concentrations of XO/HX were significantly higher than control group, and JNK activation increased with the increasing of XO/HX concentrations (P<0.05).JNK activation level in H/R group was higher than control group, after treated by EDA and NAC of ROS scavenger and JNK inhibitor, JNA activation reduced (P<0.05).Egr-1 protein levels in H/R group was higher than that in control group, and JNK inhibitor reduced the expression of Egr-1 protein induced by H/R.ConcIusion H/R activates ROS/Egr-1 signaling pathway in H9c2 cardiomyocytes, and JNK activation plays an important role in this pathway.