3,4-二羟基苯乙酮调控JNK/Nrf2信号通路减轻ox-LDL诱导的内皮细胞损伤的作用研究

Effect of 3,4⁃dihydroxyacetophenone-regulated JNK/Nrf2 signaling path⁃way on ox-LDL-induced endothelial cell injury

目的:观察3,4‐二羟基苯乙酮(3,4-DHAP)减轻氧化型低密度脂蛋白(ox-LDL)诱导的内皮细胞损伤的作用,并从c-Jun氨基末端激酶(JNK)/核因子E2相关因子2(Nrf2)信号通路来探讨其可能的作用机制。方法:将常规培养的EA.hy926细胞随机分为空白对照(CON)组、3,4‐DHAP(0.1 mmol/L)对照(3,4-DHAP-C)组、ox-LDL(100 mg/L)组,ox-LDL+低剂量(0.01 mmol/L)3,4‐DHAP(3,4-DHAP-L)组及ox-LDL+高剂量(0.1 mmol/L)3,4-DHAP(3,4‐DHAP-H)组。通过免疫荧光法观察细胞活性氧(ROS)的生成,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活力,硝酸还原酶法检测一氧化氮(NO)水平;酶联免疫吸附法(ELISA)检测内皮素1(ET-1)水平,CCK-8法检测细胞活力,TUNEL法检测细胞凋亡情况;RT-qPCR法检测Nrf2和血红素加氧酶1(HO-1)的mRNA表达;Western blot法检测JNK、磷酸化JNK(p-JNK)、Nrf2和HO-1蛋白水平。结果:与ox-LDL组比较,ox-LDL+3,4‐DHAP-H组ROS生成显著减少(P<0.05),SOD活性显著升高(P˂0.05),NO水平显著升高(P<0.05),而ET-1水平降低(P˂0.05),细胞凋亡显著减少,活力显著增强(P˂0.05),Nrf2和HO-1 mRNA及蛋白表达显著升高(P˂0.05),p-JNK/JNK比值显著增大(P˂0.05),Nrf2核内表达显著增多(P˂0.05)。结论:3,4‐DHAP能够有效地减轻ox-LDL诱导的EA.hy926细胞损伤,其机制可能与激活JNK/Nrf2信号通路,抑制氧化应激反应有关。

AIM:To study the effect the effect of 3,4dihydroxyacetophenone(3,4-DHAP)on endothelial cells induced by oxidized low-density lipoprotein(ox-LDL),and to explore the possible mechanism.METHODS:The routinely cultured EA.hy926 cells were randomly divided into blank control(CON)group,3,4-DHAP(0.1 mmol/L)control(3,4-DHAP-C)group,ox-LDL(100 mg/L)group,ox-LDL+high-dose(0.1 mmol/L)3,4-DHAP(3,4-DHAP-H)group and ox-LDL+low-dose(0.01 mmol/L)3,4-DHAP(3,4-DHAP-L)group.The production of reactive oxygen species(ROS)was observed by immunofluorescence method,the superoxide dismutase(SOD)activity was measured indirectly by xanthine oxidase method,the nitric oxide(NO)level was detected by nitrate reductase method,and the endothelin-1(ET-1)level was detected by enzyme-linked immunosorbent assay(ELISA).Cell viability was detected by CCK-8 assay,and apoptosis was detected by TUNEL method.The mRNA expression of nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)was detected by RT-qPCR.The protein levels of c-Jun N-terminal kinase(JNK),phosphorylated JNK(p-JNK),Nrf2 and HO-1 were detected by Western blot.RESULTS:Compared with ox-LDL group,ROS production was significantly reduced(P˂0.05),SOD activity significantly increased(P˂0.05),NO level significantly increased(P˂0.05),ET-1 level decreased(P˂0.05),apoptosis significantly reduced,and the cell viability was significantly enhanced(P˂0.05)in 3,4-DHAP-H group.The mRNA and protein expression of Nrf2,HO-1 and the ratio of p-JNK/JNK were also significantly increased in 3,4-DHAP-H group(P˂0.05).CONCLUSION:Treatment with 3,4-DHAP effectively reduces the injury of EA.hy926 cells induced by ox-LDL.The mechanism may be related to the activation of JNK/Nrf2 signaling pathway and the inhibition of oxidative stress.