HET0016对延迟性亚低温保护氧糖剥夺-复氧复糖离体神经元的强化效应
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摘要
目的探讨HET0016对延迟性亚低温保护氧糖剥夺-复氧复糖(oxygen-glucose deprivation and restoration,OGD/R)离体神经元的强化效应。方法新生24~48h的SD大鼠12只,随机分为对照组、氧糖剥夺(oxygen-glucose deprivation,OGD)组、OGD/R-亚低温(OGD/R-mild hypothermia,OGD/R-HM)组、OGD/R-HM-HET0016(OGD/R-HM-HET)组各3只,均分离海马神经元细胞培养7d,制备海马神经元OGD/R模型。对照组应用3mL含葡萄糖earle's平衡盐溶液正常条件下培养2h后更换正常培养液继续培养28h;OGD组应用3 mL无葡萄糖平衡盐,37℃、体积分数1%氧气培养箱中培养2h后更换正常培养液继续培养28h;OGD/R-HM组应用3mL无葡萄糖培养液、体积分数1%氧气培养箱培养2h后更换正常培养液,37℃、体积分数5%CO2培养箱中复氧4h后亚低温处理24h;OGD/R-HM-HET组于复氧前加入HET0016,调整终浓度1μmol/L,余处理同OGD/R-HM组。应用流式细胞仪检测4组细胞凋亡率。结果流式细胞仪检测结果显示,神经元细胞凋亡率在OGD组[(29.59±0.60)%]、OGD/R-HM组[(19.89±0.39)%]、OGD/R-HM-HET组[(14.17±0.25)%]均高于对照组[(1.63±0.42)%](P<0.05),且OGD组高于OGD/R-HM组和OGD/R-HM-HET组(P<0.05),OGD/R-HM组高于OGD/R-HM-HET组(P<0.05)。结论 HET0016对缺血缺氧4h后实施的亚低温保护离体OGD/R神经元具有一定的强化效应。
Objective To explore the strengthening effect of HET0016 on the protection of neurons in vitro by delayed mild hypothermia after oxygen-glucose deprivation and restoration(OGD/R)in rats.Methods Twelve neonatal SD rats aged 24 to 48 hold were randomly divided into control group,oxygen-glucose deprivation(OGD)group,OGD/R-mild hypothermia(OGD/R-MH)group,and OGD/R-MH-HET0016(OGD/R-MH-HET)group,with 3 rats in each group.The hippocampal neurons were isolated and cultured for 7 dto establish OGD/R models.Control group was cultured in3 mL earle's balanced salt solution containing glucose for 2 h,then the normal culture solution was replaced and the culture was continued for 28 h.OGD group was cultured in 3 mL glucose-free balanced salt solution at 37℃for 2 hin the incubator containing 1% oxygen,then the normal culture solution was replaced and the culture was continued for 28 h.OGD/R-MH group was cultured in 3 mL glucose-free solution for 2 hin incubator containing 1% oxygen,replaced with normal culture subjected to OGD for 2 h,restored of oxygen-glucose at 37 ℃in 5% CO2 incubator for 4 h,and treated with mild hypothermia for 24 h.OGD/R-MH-HET group was added HET0016 with the final concentration of 1μmol/L before restoration of oxygen-glucose,and other procedures were the same as OGD/R-MH group.The apoptosis rates were detected by flow cytometry in 4 groups.Results Flow cytometry result showed the apoptosis rate was the highest in OGD group((29.59±0.60)%),followed by OGD/R-MH group((19.89±0.39)%),OGD/R-MH-HET group((14.17±0.25)%)and control group((1.63±0.42)%)in turns(P<0.05).Conclusion HET0016 has a certain strengthening effect on the protection of neurons in vitro by delay mild hypothermia after hypoxia-ischemia for 4 hin rats.
Objective To explore the strengthening effect of HET0016 on the protection of neurons in vitro by delayed mild hypothermia after oxygen-glucose deprivation and restoration(OGD/R)in rats.Methods Twelve neonatal SD rats aged 24 to 48 hold were randomly divided into control group,oxygen-glucose deprivation(OGD)group,OGD/R-mild hypothermia(OGD/R-MH)group,and OGD/R-MH-HET0016(OGD/R-MH-HET)group,with 3 rats in each group.The hippocampal neurons were isolated and cultured for 7 dto establish OGD/R models.Control group was cultured in3 mL earle's balanced salt solution containing glucose for 2 h,then the normal culture solution was replaced and the culture was continued for 28 h.OGD group was cultured in 3 mL glucose-free balanced salt solution at 37℃for 2 hin the incubator containing 1% oxygen,then the normal culture solution was replaced and the culture was continued for 28 h.OGD/R-MH group was cultured in 3 mL glucose-free solution for 2 hin incubator containing 1% oxygen,replaced with normal culture subjected to OGD for 2 h,restored of oxygen-glucose at 37 ℃in 5% CO2 incubator for 4 h,and treated with mild hypothermia for 24 h.OGD/R-MH-HET group was added HET0016 with the final concentration of 1μmol/L before restoration of oxygen-glucose,and other procedures were the same as OGD/R-MH group.The apoptosis rates were detected by flow cytometry in 4 groups.Results Flow cytometry result showed the apoptosis rate was the highest in OGD group((29.59±0.60)%),followed by OGD/R-MH group((19.89±0.39)%),OGD/R-MH-HET group((14.17±0.25)%)and control group((1.63±0.42)%)in turns(P<0.05).Conclusion HET0016 has a certain strengthening effect on the protection of neurons in vitro by delay mild hypothermia after hypoxia-ischemia for 4 hin rats.
引文
[1]蔡清,薛辛东,富建华.新生儿缺氧缺血性脑病研究现状及进展[J].中国实用儿科杂志,2009,24(12):968-971.
[2]欧明明,任太亮,张燕,等.亚低温联合药物治疗新生儿缺氧缺血性脑病的研究进展[J].安徽医学,2015,36(12):1558-1561.
[3]ZHU J,WANG B,LEE J H,et al.Additive neuroprotection of a 20-HETE inhibitor with delayed therapeutic hypothermia after hypoxia-ischemia in neonatal piglets[J].Dev Neurosci,2015,37(4/5):376-389.
[4]YANG Z J,CARTER E L,KIBLER K K,et al.Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor[J].J Neurochem,2012,121(1):168-179.
[5]DRURY P P,BENNET L,GUNN A J.Mechanisms of hypothermic neuroprotection[J].Semin Fetal Neonatal Med,2010,15(5):287-292.
[6]斯妍娜,鲍红光,韩流,等.右美托咪定对大鼠海马神经细胞氧糖缺失/复氧复糖时OGG1mRNA表达的影响[J].中华麻醉学杂志,2013,33(8):1003-1006.
[7]秦彦昌,贾栋,于庆伟,等.RUNX1对氧糖剥夺诱导PC12细胞凋亡的保护作用研究[J].现代生物医学进展,2017,17(10):1838-1842.
[8]潘春留,闫雷,娜仁图娜拉,等.流式细胞术研究细胞凋亡的方法与技术[J].现代生物医学进展,2009,9(4):790-793,800.
[9]娜仁图娜拉,闫雷,赵瑞杰,等.流式细胞术及其在细胞凋亡检测中的应用[J].中国组织工程研究,2009,13(27):5353-5356.
[10]朱丝雨,郭明炎,曹铭辉.右美托咪啶对大鼠缺氧缺糖海马神经元凋亡的影响[J].岭南急诊医学杂志,2014,19(1):48-50.
[11]田青,叶文华,陶玉倩.体外培养的乳鼠皮层神经元氧糖剥夺后轴突损伤的实验观察[J].新医学,2016,47(11):730-734.
[12]朱俊超,彭振红,滕秀飞,等.HET0016对延迟性亚低温保护氧糖剥夺-复氧复糖大鼠神经元的改良作用:离体实验[J].中华麻醉学杂志,2016,36(4):484-487.
[13]朱俊超,白文娅,杨延超,等.HET0016联合亚低温对缺氧缺血性脑病新生猪仔脑神经元m-calpain蛋白表达的影响[J].中华麻醉学杂志,2016,36(4):505-507.
[14]YANG Z J,NI X,CARTER E L,et al.Neuroprotective effect of acid-sensing ion channel inhibitor psalmotoxin-1 after hypoxia-ischemia in newborn piglet striatum[J].Neurobiol Dis,2011,43(2):446-454.
[15]NI X,YANG Z J,WANG B,et al.Early antioxidant treatment and delayed hypothermia after hypoxia-ischemia have no additive neuroprotection in newborn pigs[J].Anesth Analg,2012,115(3):627-637.
[16]YANG Z J,WANG B,KWANSA H,et al.Adenosine A2A receptor contributes to ischemic brain damage in newborn piglet[J].J Cereb Blood Flow Metab,2013,33(10):1612-1620.
[17]杨尧,朱耀斌,范祥明,等.脑保护液联合尼莫地平对深低温停循环大鼠的脑保护作用[J].中华实用诊断与治疗杂志,2017,31(3):223-226.
[18]吴娜,范树信,宋达琳.缺血后处理对心肌缺血再灌注损伤过程中内质网应激的影响[J].中华实用诊断与治疗杂志,2016,30(11):1068-1070.
[2]欧明明,任太亮,张燕,等.亚低温联合药物治疗新生儿缺氧缺血性脑病的研究进展[J].安徽医学,2015,36(12):1558-1561.
[3]ZHU J,WANG B,LEE J H,et al.Additive neuroprotection of a 20-HETE inhibitor with delayed therapeutic hypothermia after hypoxia-ischemia in neonatal piglets[J].Dev Neurosci,2015,37(4/5):376-389.
[4]YANG Z J,CARTER E L,KIBLER K K,et al.Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor[J].J Neurochem,2012,121(1):168-179.
[5]DRURY P P,BENNET L,GUNN A J.Mechanisms of hypothermic neuroprotection[J].Semin Fetal Neonatal Med,2010,15(5):287-292.
[6]斯妍娜,鲍红光,韩流,等.右美托咪定对大鼠海马神经细胞氧糖缺失/复氧复糖时OGG1mRNA表达的影响[J].中华麻醉学杂志,2013,33(8):1003-1006.
[7]秦彦昌,贾栋,于庆伟,等.RUNX1对氧糖剥夺诱导PC12细胞凋亡的保护作用研究[J].现代生物医学进展,2017,17(10):1838-1842.
[8]潘春留,闫雷,娜仁图娜拉,等.流式细胞术研究细胞凋亡的方法与技术[J].现代生物医学进展,2009,9(4):790-793,800.
[9]娜仁图娜拉,闫雷,赵瑞杰,等.流式细胞术及其在细胞凋亡检测中的应用[J].中国组织工程研究,2009,13(27):5353-5356.
[10]朱丝雨,郭明炎,曹铭辉.右美托咪啶对大鼠缺氧缺糖海马神经元凋亡的影响[J].岭南急诊医学杂志,2014,19(1):48-50.
[11]田青,叶文华,陶玉倩.体外培养的乳鼠皮层神经元氧糖剥夺后轴突损伤的实验观察[J].新医学,2016,47(11):730-734.
[12]朱俊超,彭振红,滕秀飞,等.HET0016对延迟性亚低温保护氧糖剥夺-复氧复糖大鼠神经元的改良作用:离体实验[J].中华麻醉学杂志,2016,36(4):484-487.
[13]朱俊超,白文娅,杨延超,等.HET0016联合亚低温对缺氧缺血性脑病新生猪仔脑神经元m-calpain蛋白表达的影响[J].中华麻醉学杂志,2016,36(4):505-507.
[14]YANG Z J,NI X,CARTER E L,et al.Neuroprotective effect of acid-sensing ion channel inhibitor psalmotoxin-1 after hypoxia-ischemia in newborn piglet striatum[J].Neurobiol Dis,2011,43(2):446-454.
[15]NI X,YANG Z J,WANG B,et al.Early antioxidant treatment and delayed hypothermia after hypoxia-ischemia have no additive neuroprotection in newborn pigs[J].Anesth Analg,2012,115(3):627-637.
[16]YANG Z J,WANG B,KWANSA H,et al.Adenosine A2A receptor contributes to ischemic brain damage in newborn piglet[J].J Cereb Blood Flow Metab,2013,33(10):1612-1620.
[17]杨尧,朱耀斌,范祥明,等.脑保护液联合尼莫地平对深低温停循环大鼠的脑保护作用[J].中华实用诊断与治疗杂志,2017,31(3):223-226.
[18]吴娜,范树信,宋达琳.缺血后处理对心肌缺血再灌注损伤过程中内质网应激的影响[J].中华实用诊断与治疗杂志,2016,30(11):1068-1070.