CAJ | 학술논문

目的探讨酪氨酸激酶(JAK)抑制剂AG490对血管内皮细胞抗过氧化氢(H2O2)损伤的保护作用及其机制.方法分别用不同浓度H2O2(100、200、400 μmol/L)处理体外培养的人脐静脉内皮细胞(HUVECs)2、4、6h,噻唑蓝(MTT)法测定内皮细胞活力;H2O2(400 μmol/L)建立HUVECs损伤模型,实验分4组:对照组、H2O2组、H2O2+ AG490(20μmol/L)组、AG490(20μmol/L)组.各组细胞处理4h,MTT法检测各组细胞存活率;原位末端转移酶标记(TUNEL)法测定各组细胞凋亡率;Western blot法检测各组细胞中磷酸化酪氨酸蛋白激酶2(p-JAK2)、磷酸化信号传导与转录激活因子-3(p-STAT3)、B细胞淋巴瘤/白血病-2相关X蛋白(bax)和B细胞淋巴瘤/白血病-2(bcl-2)的表达.结果经不同浓度的H2O2(100、200、400 μmol/L)处理内皮细胞2、4、6h后,内皮细胞的存活率明显降低(P ＜0.01);AG490能明显提高H2O2损伤的内皮细胞存活率,降低细胞凋亡率(存活率H2O2组:0.637 ±0.030、H2O2 +AG 20 μmol/L组:0.847±0.030;凋亡率H2O2组:59.104±1.572、H2O2+ AG 20μmol/L组:36.454±1.403,P＜0.01);经AG490和H2O2共处理的内皮细胞与H2O2损伤组比较,p-JAK2和p-STAT3的表达显著降低(P＜0.01);凋亡相关蛋白bax的表达显著降低,但是抗凋亡蛋白bcl-2的表达显著升高(P＜0.01).结论 AG490对内皮细胞抗过氧化氢损伤有保护作用,其抗氧化能力可能和抑制JAK2/STAT3信号通路及抗凋亡有关.
목적 탐토락안산격매(JAK)억제제AG490대혈관내피세포항과양화경(H2O2)손상적보호작용급기궤제.방법 분별용불동농도H2O2(100、200、400 μmol/L)처리체외배양적인제정맥내피세포(HUVECs)2、4、6h,새서람(MTT)법측정내피세포활력;H2O2(400 μmol/L)건립HUVECs손상모형,실험분4조:대조조、H2O2조、H2O2+ AG490(20μmol/L)조、AG490(20μmol/L)조.각조세포처리4h,MTT법검측각조세포존활솔;원위말단전이매표기(TUNEL)법측정각조세포조망솔;Western blot법검측각조세포중린산화락안산단백격매2(p-JAK2)、린산화신호전도여전록격활인자-3(p-STAT3)、B세포림파류/백혈병-2상관X단백(bax)화B세포림파류/백혈병-2(bcl-2)적표체.결과 경불동농도적H2O2(100、200、400 μmol/L)처리내피세포2、4、6h후,내피세포적존활솔명현강저(P ＜0.01);AG490능명현제고H2O2손상적내피세포존활솔,강저세포조망솔(존활솔H2O2조:0.637 ±0.030、H2O2 +AG 20 μmol/L조:0.847±0.030;조망솔H2O2조:59.104±1.572、H2O2+ AG 20μmol/L조:36.454±1.403,P＜0.01);경AG490화H2O2공처리적내피세포여H2O2손상조비교,p-JAK2화p-STAT3적표체현저강저(P＜0.01);조망상관단백bax적표체현저강저,단시항조망단백bcl-2적표체현저승고(P＜0.01).결론 AG490대내피세포항과양화경손상유보호작용,기항양화능력가능화억제JAK2/STAT3신호통로급항조망유관.
Objective To investigate the protective effects and mechanism of AG490,a specific inhibitor of the Janus kinase 2 (JAK2) kinase,against H2O2-induced vascular endothelial cells injury.Methods The cultured human umbilical vein endothelial cells (HUVECs) were treated with H2O2 (100,200 and 400 μmol/L) for 2,4 and 6 h.Methyl thiazol tetrazolium (MTT) method was used to detect the survival rate of cells.HUVECs treated with H2O2 (400 μmol/L) for 4 h were selected for further experiments.Trials are divided into four groups:control,H2O2,H2O2 + AG490 (20 μmol/L),AG490 (20 μmol/L) groups.MTT method was used to detect the survival rate.TdT-mediated dUTP nick end labeling (TUNEL) was used to detect the apoptosis rate of cells.Western boltting was used to detect the expression of phosphorylated-Janus kinase 2 (p-JAK2),phosphorylated-signal transducer and activator of transcription 3 (p-STAT3),B cell lymphoma/leukemia-2 associated X protein (bax) and B cell lymphoma/leukemia-2 (bcl-2) proteins.Results After treatment with H2O2 (100,200,400 μmol/L) for 2,4 and 6 h,the survival rate of HUVECs was decreased significantly.The survival rate was significantly increased by AG490 treatment,and the apoptosis rate was decreased significantly (P ＜ 0.01) [survival ratio,H2O2 group:0.637 ±0.030,H2O2 + AG 20 μmoL/L group:0.847 ± 0.030; apoptotic rate,H2O2 group:59.104 ± 1.572,H2O2 + AG 20 μmol/L group:36.454 ± 1.403].In addition,the expression of p-JAK2,p-STAT3 and bax was decreased significantly,and that of bcl-2 was increased significantly (P ＜ 0.01).Conclusion AG490 treatment can fight against H2O2-induced injury on cultured HUVECs via inhibiting the JAK2/STAT3 and anti-apoptotic signaling pathways.