麦冬皂苷D通过上调CYP2J2/EETs抗Ang Ⅱ诱导的内皮细胞凋亡
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摘要
探讨麦冬皂苷D(OP-D)对血管紧张素Ⅱ(Ang Ⅱ)诱导内皮细胞凋亡的影响及其机制,为中药的安全性及有效性提供参考依据。MTT法检测Ang Ⅱ对细胞存活率的影响;检测OP-D对Ang Ⅱ诱导的细胞总蛋白含量的影响;酶标仪测定OP-D对Ang Ⅱ诱导的细胞乳酸脱氢酶(LDH)释放率的影响;Western blot和RT-PCR法检测OP-D及外源性11,12-EET对Ang Ⅱ诱导的细胞中JNK及其下游靶分子c-Jun的磷酸化水平表达;流式法检测细胞凋亡率;最后进一步借助CYP450酶抑制剂PPOH及JNK特异性抑制剂SP600125,探究OP-D保护内皮细胞的可能内在机制。结果显示,不同剂量的Ang Ⅱ(1×10~(-8)~1×10~(-6)mol·L~(-1))对细胞存活率没有显著的影响,1×10~(-6)mol·L~(-1)的Ang Ⅱ处理24 h可诱导细胞总蛋白含量的增加(P<0.05,P<0.01),刺激LDH释放率的增多(P<0.001),JNK和c-Jun的磷酸化水平明显增加(P<0.01,P<0.001),caspase-3的蛋白表达上调(P<0.01),细胞凋亡率增高(P<0.001)。提前给予SP600125显著抑制了JNK和c-Jun的磷酸化水平。OPD预处理后,能明显降低Ang Ⅱ诱导的细胞总蛋白含量(P<0.01)及LDH的释放(P<0.01,P<0.01),减少JNK和c-Jun的磷酸化水平,降低caspase-3的表达及细胞凋亡率,提前给予PPOH后,OP-D保护效应被抑制。11,12-EET预处理降低JNK和c-Jun的磷酸化水平(P<0.05)。结果表明Ang Ⅱ通过JNK/c-Jun途径诱导细胞凋亡;OP-D通过诱导CYP2 J2表达及增加11,12-EET含量抗Ang Ⅱ激活JNK/c-Jun通路所诱导的细胞损伤及凋亡。
This study aimed to investigate the effect and mechanism of ophiopogonin D(OP-D) on Ang Ⅱ-induced HUVECs apoptosis,in order to provide a reliable basis for the safety and efficacy of traditional Chinese medicines. The effect of Ang Ⅱ on survival and total proteins content of HUVECs were measured by MTT and Western blotting. The effect of OP-D on Ang Ⅱ-induced lactate dehydrogenase( LDH) release rate in HUVECs was measured by enzyme standard instrument. The effects of OP-D and 11,12-EET on phosphorylation of JNK/c-Jun induced by Ang Ⅱ were measured by Western blot and RT-PCR with the help of JNK specific inhibitor SP600125 and CYP450 isozymes selective inhibitor 6-( 2-propargyloxyphenyl) hexanoic acid( PPOH). The cell apoptosis was assayed by flow cytometry. According to the results,different doses of Ang Ⅱ had no significant effect on cell survival; treatment with Ang Ⅱat 1 × 10~(-6) mol·L~(-1) could increase the release of LDH( P < 0. 001),improve the JNK and c-Jun phosphorylation levels( P < 0. 01,P < 0. 001),increase the expression of caspase-3( P < 0. 01),and promote the apoptosis of HUVECs( P < 0. 001). The phosphorylation of JNK and c-Jun could be inhibited by the pre-treatment with SP600125,11,12-EET and OP-D. Pre-treatment with OP-D could significantly reduce the release of LDH induced by Ang Ⅱ stimulation,decrease the expression of caspase-3,and diminish the apoptosis of cells. The protective effect of OP-D was suppressed,when being pretreated with PPOH. The experimental results showed that the apoptosis of HUVECs induced by Ang Ⅱ may be associated with JNK/c-Jun signaling pathway. OP-D-mediated CYP2 J2 expression increased 11,12-EET levels,and could remarkably resist Ang Ⅱ-induced injury and apoptosis of cells,which is associated with the maintenance of endothelium homeostasis.
This study aimed to investigate the effect and mechanism of ophiopogonin D(OP-D) on Ang Ⅱ-induced HUVECs apoptosis,in order to provide a reliable basis for the safety and efficacy of traditional Chinese medicines. The effect of Ang Ⅱ on survival and total proteins content of HUVECs were measured by MTT and Western blotting. The effect of OP-D on Ang Ⅱ-induced lactate dehydrogenase( LDH) release rate in HUVECs was measured by enzyme standard instrument. The effects of OP-D and 11,12-EET on phosphorylation of JNK/c-Jun induced by Ang Ⅱ were measured by Western blot and RT-PCR with the help of JNK specific inhibitor SP600125 and CYP450 isozymes selective inhibitor 6-( 2-propargyloxyphenyl) hexanoic acid( PPOH). The cell apoptosis was assayed by flow cytometry. According to the results,different doses of Ang Ⅱ had no significant effect on cell survival; treatment with Ang Ⅱat 1 × 10~(-6) mol·L~(-1) could increase the release of LDH( P < 0. 001),improve the JNK and c-Jun phosphorylation levels( P < 0. 01,P < 0. 001),increase the expression of caspase-3( P < 0. 01),and promote the apoptosis of HUVECs( P < 0. 001). The phosphorylation of JNK and c-Jun could be inhibited by the pre-treatment with SP600125,11,12-EET and OP-D. Pre-treatment with OP-D could significantly reduce the release of LDH induced by Ang Ⅱ stimulation,decrease the expression of caspase-3,and diminish the apoptosis of cells. The protective effect of OP-D was suppressed,when being pretreated with PPOH. The experimental results showed that the apoptosis of HUVECs induced by Ang Ⅱ may be associated with JNK/c-Jun signaling pathway. OP-D-mediated CYP2 J2 expression increased 11,12-EET levels,and could remarkably resist Ang Ⅱ-induced injury and apoptosis of cells,which is associated with the maintenance of endothelium homeostasis.
引文
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[15]侯炳旭,冯丽英.JNK信号通路介导的凋亡在疾病中的作用[J].世界华人消化杂志,2011,19(17):1819.
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[2]You W T,Tao Z,Ma Z C,et al.Ophiopogonin D maintains Ca2+homeostasis in rat cardiomyocytes in vitro by up regulating CYP2 J3/EETs and suppressing ER stress[J].Acta Pharmacol Sin,2016,37(3):368.
[3]Askari A,Thomson S J,Edin M L,et al.Roles of the epoxygenase CYP2 J2 in the endothelium[J].Prostaglandins Other Lipid Mediat,2013,107(12):56.
[4]Chen W,Zheng G,Yang S,et al.CYP2J2 and EETs Protect against oxidative stress and apoptosis in vivo and in vitro following lung ischemia/reperfusion[J].Cell Physiol Biochem,2014,33(6):1663.
[5]Huang X,Wang Y,Zhang Z,et al.Ophiopogonin D and EETs ameliorate AngⅡ-induced inflammatory responses via activating PPARαin HUVECs[J].Biochem Biophys Res Commun,2017,490:123.
[6]郝颖,周京敏,王克强,等.通心络对血管紧张素Ⅱ致大鼠内皮损伤的保护作用[J].中华中医药杂志,2009,24(7):855.
[7]任娇,孙惜时,田憬若,等.大豆异黄酮对内皮细胞损伤保护作用的研究进展[J].大豆科学,2014,33(1):135.
[8]Kopf P G,Gauthier K M,Zhang D X,et al.AngiotensinⅡregulates adrenal vascular tone through zona glomerulosa cell-derived eets and dhets[J].Hypertension,2011,57(2):323.
[9]Menden H,Welak S,Cossette S,et al.Lipopolysaccharide(LPS)-mediated angiopoietin-2-dependent autocrine angiogenesis is regulated by NADPH oxidase 2(Nox2)in human pulmonary microvascular endothelial cells.[J].J Biol Chem,2015,290(9):5449.
[10]Phillips M I,Kagiyama S.AngiotensinⅡas a pro-inflammatory mediator[J].Curr Opin Investig Drugs,2002,3(4):569.
[11]Pueyo M E,Gonzalez W,Nicoletti A,et al.AngiotensinⅡstimulates endothelial vascular cell adhesion molecule-1 via nuclear factor-kappa B activation induced by intracellular oxidative stress[J].Arterioscler Thromb Vasc Biol,2000,20(3):645.
[12]孟晨,袁彩华,张晨晨,等.麦冬皂苷D通过减轻内质网应激对阿霉素所致心肌损伤产生保护作用[J].药学学报,2014(8):1117.
[13]张赢予,张国辉,孟晨,等.麦冬皂苷D缓解多柔比星诱导的心肌细胞损伤[C].昆明:第十六届中国科协年会——分3环境污染及职业暴露与人类癌症学术研讨会,2014.
[14]蒋凤荣,张旭,洪艳丽.麦冬皂苷D对过氧化氢造模的HUVEC保护作用机制研究[J].现代生物医学进展,2008,8(9):1646.
[15]侯炳旭,冯丽英.JNK信号通路介导的凋亡在疾病中的作用[J].世界华人消化杂志,2011,19(17):1819.
[16]蔡艳,蒋伟,周爱玲,等.苦参素通过p38/JNK信号途径对海马神经元凋亡的影响[J].中国中药杂志,2017,42(4):731.