丁苯酞对S-亚硝基化谷胱甘肽诱导脑微血管内皮细胞损伤的保护作用及机制研究
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摘要
目的:构建S-亚硝基化谷胱甘肽(S-nitrosoglutathione,GSNO)诱导脑微血管内皮细胞的损伤模型,探讨丁苯酞(Butylphthalide,dL-NBP)对脑微血管内皮细胞的保护作用与可能的分子调控机制。方法:建立损伤bEnd.3细胞模型,以MTT法检测细胞存活率。DCFH染色检测细胞活性氧水平。Western blot法检测p-ERK,ERK,cleaved caspase 9,pro-caspase 9,cleaved caspase 3,pro-caspase 3蛋白表达。RT-PCR法检测糖皮质激素受体(GR)、SGK、MKP-1基因表达水平。结果:dL-NBP(5,10,20μmol/L)可减少GSNO引起的脑微血管内皮细胞损伤,提高细胞存活率,减少ROS发生。此外,dL-NBP可激活SGK和MKP-1转录水平的增加,上调ERK磷酸化,抑制cleaved caspase 9,cleaved caspase 3蛋白上调。结论:dL-NBP对GSNO诱导的脑微血管内皮细胞损伤具有保护作用,其作用机制与激活PR活性下游基因SGK和MKP-1,上调ERK磷酸化水平,抑制caspase级联反应密切相关。
AIM: To investigate the protection of Butylphthalide(dL-NBP) on oxidative damage in brain microvascular endothelial cells, and to further find the potential mechanisms of dL-NBP effects. METHODS: The brain microvascular endothelial cells were injured by GSNO. The cell viability was measured by MTT assay. ROS was observed by DCFH staining. ERK, p-ERK,cleaved caspase 9,pro-caspase 9,cleaved caspase 3 and pro-caspase 3 were detected by Western blot. The gene expressions of GR, SGK and MKP-1 were detected by RT-PCR assay. RESULTS:dL-NBP(10,20 μmol/L) protected endothelial cells against GSNO-induced injury and ROS release. dL-NBP increased SGK and MKP-1 gene expression. dL-NBP up-regulated ERK phosphorylation. dL-NBP inhibited cleaved caspase 9 and cleaved caspase 3. CONCLUSION: dL-NBP attunates bEnd.3 cells GSNO-induced injury, and its mechanism is related with activating GR relative SGK and MKP-1. dL-NBP increases ERK phosphorylation and down-regulates caspase decades.
AIM: To investigate the protection of Butylphthalide(dL-NBP) on oxidative damage in brain microvascular endothelial cells, and to further find the potential mechanisms of dL-NBP effects. METHODS: The brain microvascular endothelial cells were injured by GSNO. The cell viability was measured by MTT assay. ROS was observed by DCFH staining. ERK, p-ERK,cleaved caspase 9,pro-caspase 9,cleaved caspase 3 and pro-caspase 3 were detected by Western blot. The gene expressions of GR, SGK and MKP-1 were detected by RT-PCR assay. RESULTS:dL-NBP(10,20 μmol/L) protected endothelial cells against GSNO-induced injury and ROS release. dL-NBP increased SGK and MKP-1 gene expression. dL-NBP up-regulated ERK phosphorylation. dL-NBP inhibited cleaved caspase 9 and cleaved caspase 3. CONCLUSION: dL-NBP attunates bEnd.3 cells GSNO-induced injury, and its mechanism is related with activating GR relative SGK and MKP-1. dL-NBP increases ERK phosphorylation and down-regulates caspase decades.
引文
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[15] Xiong Z,Lu W,Zhu L,et al.Dl-3-n-Butylphthalide treatment enhances hemodynamics and ameliorates memory deficits in rats with chronic cerebral hypoperfusion[J].Front Aging Neurosci,2017,9:238.
[16] Wang HM,Zhang T,Huang JK,et al.3-N-butylphthalide(NBP) attenuates the amyloid- -induced inflammatory responses in cultured astrocytes via the nuclear factor-κB signaling pathway[J].Cell Physiol Biochem,2013,32(1):235-242.
[17] Yan JP,Liu QB,Dou Y,et al.Activating glucocorticoid receptor-ERK signaling pathway contributes to ginsenoside Rg1 protection against β-amyloid peptide-induced human endothelial cells apoptosis[J].J Ethnopharmacol,2013,147:456-466.
[18] 张洁,陈志武.山红花总黄酮对大鼠神经元及EA.hy926内皮细胞超极化作用及机制[J].中国临床药理学与治疗学,2017,22(6):606-610.
[19] 韩晨阳,张晓玲,官俏兵,等.丁苯酞对于糖氧剥夺条件下人脑微血管内皮细胞能量代谢的影响及保护作用[J].中国临床药理学与治疗学,2018,23(12):1329-1334.
[20] 胡琴,李鸣,肖家平,等.丁苯酞序贯治疗急性脑梗死50例[J].医药导报,2017,36(4):409-412.
[21] Yan JP,Huang X,Zhu DY,et al.Enhanced aerobic glycolysis by S-nitrosoglutathione via HIF-1α associated GLUT1/Aldolase A axis in human endothelial cells[J].J Cell Biochem,2017,118:2443-2453.
[2] Feng L,Sharma A,Niu F,et al.TiO2-Nanowired delivery of DL-3-n-butylphthalide(DL-NBP) attenuates blood-Brain barrier disruption,brain edema formation,and neuronal damages following concussive head injury[J].Mol Neurobiol,2018,55(1):350-358.
[3] Li L,Zhang B,Tao Y,et al.DL-3-n-butylphthalide protects endothelial cells against oxidative/nitrosative stress,mitochondrial damage and subsequent cell death after oxygen glucose deprivation in vitro[J].Brain Res,2009,1290:91-101.
[4] Zhang P,Xu RX,Guo Y,et al.DL-3-n-butylphthalide promotes dendrite development in cortical neurons subjected to oxygen-glucose deprivation/reperfusion[J].Cell Biol Int,2018,42(8):1041-1049.
[5] Liu CY,Zhao ZH,Chen ZT,et al.DL-3-n-butylphthalide protects endothelial cells against advanced glycation end product-induced injury by attenuating oxidative stress and inflammation responses[J].Exp Ther Med,2017,14(3):2241-2248.
[6] Zhang T,Jia W,Sun X.3-n-Butylphthalide(DL-NBP) reduces apoptosis and enhances vascular endothelial growth factor(VEGF) up-regulation in diabetic rats[J].Neurol Res,2010,32(4):390-396.
[7] Chang Q,Wang XL.Effects of chiral 3-n-butylphthalide on apoptosis induced by transient focal cerebral ischemia in rats[J].Acta Pharmacol Sin,2003,24(8):796-804.
[8] Liao DH,Xiang DX,Dang RL,et al.Neuroprotective effects of dl-3-n-butylphthalide against doxorubicin-induced neuroinflammation,oxidative stress,endoplasmic reticulum stress,and behavioral changes[J].Oxid Med Cell Longev,2018(2018):9125601.
[9] Ma S,Xu S,Liu B,et al.Long-term treatment of l-3-n-butylphthalide attenuated neurodegenerative changes in aged rats[J].Naunyn Schmiedebergs Arch Pharmacol,2009,379(6):565-574.
[10] Huang JZ,Chen YZ,Su M,et al.dl-3-n-Butylphthalide prevents oxidative damage and reduces mitochondrial dysfunction in an MPP(+)-induced cellular model of Parkinson's disease[J].Neurosci Lett,2010,475(2):89-94.
[11] Zhang C,Zhao S,Zang Y,et al.The efficacy and safety of Dl-3n-butylphthalide on progressive cerebral infarction:A randomized controlled STROBE study[J].Medicine,2017(30):7257.
[12] Jia J,Wei C,Liang J,et al.The effects of DL-3-n-butylphthalide in patients with vascular cognitive impairment without dementia caused by subcortical ischemic small vessel disease:A multicentre,randomized,double-blind,placebo-controlled trial[J].Alzheimers Dement,2016,12(2):89-99.
[13] Cui LY,Zhu YC,Gao S,et al.Ninety-day administration of dl-3-n-butylphthalide for acute ischemic stroke:A randomized,double-blind trial[J].Chin Med J(Engl),2013,126(18):3405-3410.
[14] Liu Z,Wang H,Shi X,et al.DL-3-n-Butylphthalide (NBP) provides neuroprotection in the mice models after traumatic brain injury via Nrf2-ARE signaling pathway[J].Neurochem Res,2017,42(5):1375-1386.
[15] Xiong Z,Lu W,Zhu L,et al.Dl-3-n-Butylphthalide treatment enhances hemodynamics and ameliorates memory deficits in rats with chronic cerebral hypoperfusion[J].Front Aging Neurosci,2017,9:238.
[16] Wang HM,Zhang T,Huang JK,et al.3-N-butylphthalide(NBP) attenuates the amyloid- -induced inflammatory responses in cultured astrocytes via the nuclear factor-κB signaling pathway[J].Cell Physiol Biochem,2013,32(1):235-242.
[17] Yan JP,Liu QB,Dou Y,et al.Activating glucocorticoid receptor-ERK signaling pathway contributes to ginsenoside Rg1 protection against β-amyloid peptide-induced human endothelial cells apoptosis[J].J Ethnopharmacol,2013,147:456-466.
[18] 张洁,陈志武.山红花总黄酮对大鼠神经元及EA.hy926内皮细胞超极化作用及机制[J].中国临床药理学与治疗学,2017,22(6):606-610.
[19] 韩晨阳,张晓玲,官俏兵,等.丁苯酞对于糖氧剥夺条件下人脑微血管内皮细胞能量代谢的影响及保护作用[J].中国临床药理学与治疗学,2018,23(12):1329-1334.
[20] 胡琴,李鸣,肖家平,等.丁苯酞序贯治疗急性脑梗死50例[J].医药导报,2017,36(4):409-412.
[21] Yan JP,Huang X,Zhu DY,et al.Enhanced aerobic glycolysis by S-nitrosoglutathione via HIF-1α associated GLUT1/Aldolase A axis in human endothelial cells[J].J Cell Biochem,2017,118:2443-2453.