麦冬皂苷D通过上调CYP2J3/EET减轻H2O2诱导的H9c2细胞氧化应激损伤

Ophiopogonin D upregulates CYP2J3/EETs to activate PI3K/AkteNOS pathway and rescue H2O2-induced H9c2 cell injury

目的探讨麦冬皂苷D(OP-D)对H2O2诱导的H9c2细胞氧化应激损伤的保护作用及机制。方法通过H2O2诱导建立H9c2细胞氧化损伤模型,细胞分为正常对照组(予以无血清培养基培养28 h)、H2O2损伤模型组(H2O2400μmol·L-1处理3 h)、OP-D 5,10和20μmol·L-1预处理组(OP-D预处理24 h后+H2O2400μmol·L-1处理3 h)、花生四烯酸CYP环氧酶活性抑制剂6-(2-炔丙基羟苯基)己酸(PPOH)干预组(OP-D20μmol·L-1+PPOH 10μmol·L-1,PPOH于OP-D处理前1 h加入细胞)。MTT法测定细胞活性,酶联免疫吸附法(ELISA)检测环氧-二十碳-三烯酸(11,12-DHET和14,15-DHET)含量,ELISA法检测细胞内丙二醛(MDA)、一氧化氮(NO)含量及乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性,流式细胞术(FCM)检测活性氧(ROS)水平及细胞凋亡水平、Western印迹法检测细胞色素P450 2J3(CYP2J3)、磷酸化Akt(p-Akt)蛋白和磷酸化内皮型一氧化氮合酶(p-e NOS)蛋白在细胞中的表达,利用PPOH进一步验证OP-D减轻细胞氧化应激的可能内在机制。结果 H2O2400μmol·L-1明显抑制H9c2细胞存活率(P<0.01),OP-D可明显增加H2O2损伤后细胞存活率(P<0.01);不同浓度OP-D增加11,12-DHET和14,15-DHET含量(P<0.05,P<0.01);OP-D增加H2O2损伤后细胞NO含量(P<0.05,P<0.01)、增强SOD活性(P<0.05)及降低MDA、LDH含量(P<0.05,P<0.01);OP-D显著降低H2O2损伤后细胞氧化应激及凋亡水平(P<0.05,P<0.01);OP-D预处理上调H2O2损伤后细胞CYP2J3蛋白和m RNA表达(P<0.05,P<0.01)、激活PI3K/Akt-e NOS通路的磷酸化水平(P<0.05,P<0.01);提前给予PPOH后,OP-D保护效应被抑制(P<0.05,P<0.01)。结论 OP-D能减轻H2O2所致的H9c2细胞损伤,可能是通过诱导CYP2J3表达及增加11,12-DHET和14,15-DHET含量,激活PI3K通路促进其下游因子Akt和e NOS磷酸化发挥作用。

OBJECTIVE To explore the protective effect and mechanism of ophiopogonin D(OP-D)on oxidative stress injury of H9c2 cells induced by H2O2.METHODS An oxidative damage model of H9c2 cells was established by H2O2 induction.The cells were divided into control group(cultured in serum-free medium for 28 h),H2O2 injury model group(treated with H2O2400μmol·L-1 for 3 h),OP-D 5,10 and 20μmol·L-1 pretreatment groups(treated with H2O2400μmol·L-1 for 3 h after OP-D pretreatment for 24 h),and an inhibitor of CYP2J3,6-(2-proparglyloxyphenyl)hexanoic acid(PPOH)group(OP-D 20μmol·L-1+PPOH 10μmol·L-1,PPOH was added to the cells 1 h before OP-D treatment).Cell activity was measured by MTT method,levels of dihydroxyeicosatrienoic acid(11,12-DHET and 14,15-DHET respectively)were detected by enzyme-linked immunosorbent assay(ELISA),while levels of malondialdehyde(MDA),nitric oxide(NO)and activity of lactate dehydrogenase(LDH),superoxide dismutase(SOD)were detected by assay kits.Flow cytometry(FCM)was used to detect reactive oxygen species(ROS)and apoptosis.Western blotting was used to detect the expressions of CYP2J3,Akt phosphorylation(p-Akt)protein and endothelial nitric oxide synthase phosphorylation(p-eNOS)protein in cells,and the possible mechanism by which OP-D reduces oxidative stress was further verified with PPOH.RESULTS H2O2400μmol·L-1 significantly inhibited H9c2 cell viability(P<0.01),and OP-D significantly increased the cell survival rate after H2O2 injury(P<0.01).Different concentrations of OP-D increased the level of 11,12-DHET and 14,15-DHET(P<0.05,P<0.01).OP-D increased the level of NO in cells after H2O2-induced injury(P<0.05,P<0.01),enhanced the activity of SOD(P<0.05),and decreased the level of MDA and LDH(P<0.05,P<0.01).OP-D significantly reduced oxidative stress and apoptosis after H2O2 injury(P<0.05,P<0.01).OP-D pretreatment increased the protein and mRNA expression of CYP2J3(P<0.05,P<0.01)and the phosphorylation of PI3K/Akt-eNOS pathway after H2O2 injury(P<0.05,P<0.01).After PPOH was given in advance,the protective effect of OP-D was inhibited(P<0.05,P<0.01).CONCLUSION OP-D can reduce H2O2-induced H9c2 cell damage,which may be related to the activation of PI3K pathway and the phosphorylation of its downstream factors Akt and eNOS by inducing CYP2J3 expression and increasing the contents of 11,12-DHET and 14,15-DHET.