5-LOX-CysLTs-CysLTsR表达规律及黄芩苷-栀子苷配伍对大鼠脑缺血抗炎作用机制研究
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摘要
目的动态观察脑卒中大鼠脑组织半胱氨酰白三烯(CysLTs)活性及其5-脂氧酶(5-LOX)、半胱氨酰白三烯受体1(CysLTs1)、半胱氨酰白三烯受体2(CysLTs2)表达水平变化规律,探讨黄芩苷-栀子苷(7:3)配伍抗脑缺血炎性损伤机制。方法线栓法制备大脑中动脉堵塞模拟脑缺血模型,模型成功后随机分为空白对照组(Control)、模型组(pMCAO)、黄芩苷-栀子苷(7:3)30mg/kg、45mg/kg、60mg/kg组、齐留通45mg/kg组、孟鲁司特0.5mg/kg组,持续35d给药治疗,每隔7d,神经功能评分观察神经保护作用,酶联免疫吸附实验(ELISA)法测脑组织CysLTs含量;蛋白质印迹(Western Blot)法检测脑组织5-LOX、CysLTs1、CysLTs2蛋白表达水平。结果与模型组相比,黄芩苷-栀子苷(7:3)30mg/kg、45mg/kg、60mg/kg组在脑缺血28d内可显著降低了CysLTs水平;Western Blot结果显示,5-LOX、CysLTs1和CysLTs2蛋白表达明显降低;至恢复期35d,各蛋白表达水平已无显著性差异。结论黄芩苷-栀子苷(7:3)配伍可减轻MCAO大鼠的神经功能障碍,其作用机制可能与抑制5-LOX活性、降低CysLTs1和CysLTs2蛋白表达有关。
Objective To dynamicly observe cysteinyl leukotriene(CysLTs) activity in brain tissue of stroke rats and its 5-lipoxygenase(5-LOX), cysteinyl leukotriene receptor 1(CysLTs1), cysteinyl The change of expression level of leukotriene receptor 2(CysLTs2), and to investigate the mechanism of anti-cerebral ischemic injury induced by baicalin-germangoside(7:3). Methods The model of middle cerebral artery occlusion simulated cerebral ischemia was prepared by suture embolism.The model was then randomly divided into blank control group(control), model group(pMCAO), baicalin-geniposide(7:3) 30 mg/kg, 45 mg/kg, 60 mg/kg groups, ziliutong 45 mg/kg group and montelukast 0.5 mg/kg group. The treatment lasted 35 days. Neuroprotective effect was observed by neurological function score every 7 days. The content of CysLTs in brain tissue was measured by ELISA, and the expression levels of 5-LOX, CysLTs1 and CysLTs2 in brain tissue were detected by Western Blot.Results Compared with the model group, baicalin-geniposide(7:3) 30 mg/kg, 45 mg/kg and 60 mg/kg groups could significantly reduce the level of CysLTs within 28 days after cerebral ischemia; Western Blot results showed that the expression of 5-LOX, CysLTs1 and CysLTs2 protein was significantly decreased; 35 days after recovery, there was no significant difference in the expression levels of each protein. Conclusion Baicalin-geniposide(7:3) combination can alleviate neurological dysfunction in MCAO rats, and its mechanism may be related to inhibiting 5-LOX activity, reducing the expression of CysLTs1 and CysLTs2 protein.
Objective To dynamicly observe cysteinyl leukotriene(CysLTs) activity in brain tissue of stroke rats and its 5-lipoxygenase(5-LOX), cysteinyl leukotriene receptor 1(CysLTs1), cysteinyl The change of expression level of leukotriene receptor 2(CysLTs2), and to investigate the mechanism of anti-cerebral ischemic injury induced by baicalin-germangoside(7:3). Methods The model of middle cerebral artery occlusion simulated cerebral ischemia was prepared by suture embolism.The model was then randomly divided into blank control group(control), model group(pMCAO), baicalin-geniposide(7:3) 30 mg/kg, 45 mg/kg, 60 mg/kg groups, ziliutong 45 mg/kg group and montelukast 0.5 mg/kg group. The treatment lasted 35 days. Neuroprotective effect was observed by neurological function score every 7 days. The content of CysLTs in brain tissue was measured by ELISA, and the expression levels of 5-LOX, CysLTs1 and CysLTs2 in brain tissue were detected by Western Blot.Results Compared with the model group, baicalin-geniposide(7:3) 30 mg/kg, 45 mg/kg and 60 mg/kg groups could significantly reduce the level of CysLTs within 28 days after cerebral ischemia; Western Blot results showed that the expression of 5-LOX, CysLTs1 and CysLTs2 protein was significantly decreased; 35 days after recovery, there was no significant difference in the expression levels of each protein. Conclusion Baicalin-geniposide(7:3) combination can alleviate neurological dysfunction in MCAO rats, and its mechanism may be related to inhibiting 5-LOX activity, reducing the expression of CysLTs1 and CysLTs2 protein.
引文
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[12]Borges T K,Alves E A,Vasconcelos H A,et al.Differences in the modulation of reactive species,lipid bodies,cyclooxygenase-2,5-lipoxygenase and PPAR-gamma in cerebral malaria-susceptible and resistant mice[J].Immunobiology,2017,222(4):604-619.
[13]石巧娟,郭红刚,楼琦,等.半胱氨酰白三烯受体拮抗剂通过下调自噬减轻长爪沙鼠的全脑缺血再灌注损伤[J].中国实验动物学报,2018,26(1):57-64.
[14]张丽慧,赵建波,王艳芳,等.半胱氨酰白三烯受体与脑损伤关系的研究进展[J].浙江大学学报 (医学版),2008,37(3):315-320.
[15]张壮,骆江云,黄静,等.白三烯D_4体外诱导小胶质细胞激活的作用[J].浙江大学学报 (医学版),2013,42(3):253-260.
[16]Sowndhararajan K,Deepa P,Kim M,et al.Neuroprotective and Cognitive Enhancement Potentials of Baicalin:A Review[J].Brain Sci,2018,8(6):1-24.
[17]Li F,Li W,Li X,et al.Geniposide attenuates inflammatory response by suppressing P2Y14 receptor and downstream ERK1/2 signaling pathway in oxygen and glucose deprivation-induced brain microvascular endothelial cells[J].J Ethnopharmacol,2016,185:77-86.
[2]张锦,张允岭,郭蓉娟,等.从“毒损脑络”到“毒损络脉”的理论探讨[J].北京中医药,2013,32(7):483-486.
[3]程发峰.精制清开灵注射液对脑缺血的治疗作用及对兴奋性毒性级联损伤的调控机制[D].北京:北京中医药大学,2011.
[4]曾明飞,潘林梅,朱华旭,等.黄连解毒汤中栀子苷在正常及脑缺血大鼠体内药动学研究[J].中草药,2010,41(4):617-620.
[5]李敏,王斌,唐志书,等.黄芩苷、栀子苷对大鼠脑缺血保护作用的机制[J].中药药理与临床,2012,28(3):34-36.
[6]刘阿萍,李敏,王斌,等.黄芩苷和栀子苷配伍对大鼠脑缺血再灌注损伤的作用研究[J].中药药理与临床,2016,32(1):46-48.
[7]李敏,王斌,唐志书,等.栀子苷和黄芩苷配伍对脑缺血损伤急性期和恢复早期大鼠海马中CysLT2R蛋白及mRNA表达的影响[J].中药药理与临床,2012,28(5):18-20.
[8]刘寒,张欢欢,康亚国,等.黄芩苷、栀子苷配伍对脑缺血大鼠恢复期炎症因子的影响[J].陕西中医药大学学报,2018,41 (3):75-81.
[9]Bederson J B,Pitts L H,Tsuji M,et al.Rat middle cerebral artery occlusion:evaluation of the model and development of a neurologic examination[J].Stroke,1986,17(3):472-476.
[10]朱晓磊.脑缺血损伤级联反应原理与胆酸、栀子甙配伍作用机制的研究[D].北京:北京中医药大学,2003.
[11]Zhou Y,Wei E Q,Fang S H,et al.Spatio-temporal properties of 5-lipoxygenase expression and activation in the brain after focal cerebral ischemia in rats[J].Life Sci,2006,79(17):1645-1656.
[12]Borges T K,Alves E A,Vasconcelos H A,et al.Differences in the modulation of reactive species,lipid bodies,cyclooxygenase-2,5-lipoxygenase and PPAR-gamma in cerebral malaria-susceptible and resistant mice[J].Immunobiology,2017,222(4):604-619.
[13]石巧娟,郭红刚,楼琦,等.半胱氨酰白三烯受体拮抗剂通过下调自噬减轻长爪沙鼠的全脑缺血再灌注损伤[J].中国实验动物学报,2018,26(1):57-64.
[14]张丽慧,赵建波,王艳芳,等.半胱氨酰白三烯受体与脑损伤关系的研究进展[J].浙江大学学报 (医学版),2008,37(3):315-320.
[15]张壮,骆江云,黄静,等.白三烯D_4体外诱导小胶质细胞激活的作用[J].浙江大学学报 (医学版),2013,42(3):253-260.
[16]Sowndhararajan K,Deepa P,Kim M,et al.Neuroprotective and Cognitive Enhancement Potentials of Baicalin:A Review[J].Brain Sci,2018,8(6):1-24.
[17]Li F,Li W,Li X,et al.Geniposide attenuates inflammatory response by suppressing P2Y14 receptor and downstream ERK1/2 signaling pathway in oxygen and glucose deprivation-induced brain microvascular endothelial cells[J].J Ethnopharmacol,2016,185:77-86.
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