酪氨酸激酶抑制剂AG490抗过氧化氢对血管内皮细胞的保护作用及其机制

Protective effects and mechanism of AG490 against H2 02-induced vascular endothelial cells injury

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、400μmol/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（400μmol/L）建立HUVECs损伤模型，实验分4组：对照组、H：O：组、H：02＋AG490（20p,mol／L）组、AG490（20μmol/L）组。各组细胞处理4h，MTr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；Westernblot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H，O：（100、200、400μmol/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H202组：0．637±0．030、H202＋AG20μmoL/I。组：0．847±0．030；凋亡率H202组：59．1044-1．572、H202＋AG20μmol/L组：36．454±1．403，P〈0．01）；经AG490和H202共处理的内皮细胞与H：O：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bel-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。

Objective To investigate the protective effects and mechanism of AG490, a specific in hibitor of the Janus kinase 2 （JAK2） kinase, against H2 02-induced vascular endothelial cells injury. Meth ods The cultured human umbilical vein endothelial cells （HUVECs） were treated with H202 （ 100,200 and 400 μmol/L） for 2,4 and 6 h. Methyl thiazol tetrazolium （MTY） method was used to detect the survival rate of cells. HUVECs treated with H202 （400 μmoL/L） for 4 h were selected for further experiments. Trials are divided into four groups： control, H202, H202 ＋ AG490 （20 μmol/L）, AG490 （20 μmol/L） groups. MT＇F 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 phosphoryla ted-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 H202 （ 100, 200, 400 p.mol/L） for 2, 4 and 6 h, the survival rate of HUVECs was decreased significantly. The survival rate was significantly increased by AG490 treat ment, and the apoptosis rate was decreased significantly （ P 〈 0. 01 ） [ survival ratio, H2 02 group ： O. 637 ± 0. 030, H202 ± AG 20 μmol/L group： 0. 847 ±0. 030; apoptotic rate, H202 group： 59. 104 ± 1. 572, H202 ± AG 20 p, mol/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 H2 O2 -induced injury on cultured HUVECs via inhibiting the JAK2/ STAT3 and anti-apoptotie signaling pathways.