GSK3β抑制在异氟醚后处理减轻SH-SY5Y细胞缺血再灌注损伤中的作用
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摘要
【目的】研究糖原合成酶激酶3β(GSK3β)抑制在异氟醚后处理减轻SH-SY5Y细胞缺血再灌注损伤的神经保护中所起的作用。【方法】将SH-SY5Y细胞进行分成3组(n=24):Control组、OGD组及OGD+Isoflurane组。Control组细胞常规培养;OGD组进行1 h氧糖剥夺(OGD)及20 h模拟再灌注;OGD+Isoflurane组进行1 h的OGD及20 h模拟再灌注,OGD开始时立即暴露于2%异氟醚1 h。检测各组模拟再灌注1 h后LDH释放水平及总GSK3β和磷酸化GSK3β表达水平。将高选择性的GSK3β抑制剂chir 98014或chir 99021分别加入OGD组或OGD+Isoflurane组,检测各组模拟再灌注1 h后LDH释放水平。【结果】OGD组LDH释放水平增高,与Control组相比差异有统计学意义(P<0.05)。OGD+Isoflurane组LDH释放减少,与OGD组相比差异有统计学意义(P<0.05)。与Control组相比,OGD组与OGD+Isoflurane组GSK3β表达差异无统计学意义(P>0.05),p-GSK3β表达增加,差异有统计学意义(P<0.05);与OGD组相比,OGD+Isoflurane组p-GSK3β表达增加,差异有统计学意义(P<0.05)。GSK3β抑制剂复合2%异氟醚组与单独使用GSK3β抑制剂组相比,LDH释放减少,差异有统计学意义(P<0.05),与单独使用异氟醚组相比,LDH释放差异无统计学意义(P>0.05)。【结论】异氟醚后处理对SH-SY5Y细胞缺血再灌注损伤具有神经保护效应。异氟醚后处理保护效应部分通过抑制GSK3β发挥作用。
【Objective】 To study the effect of GSK3β inhibition on isoflurane post-conditioning-induced neuroprotection of SHSY5 Y cells against ischemia / reperfusion. 【Method】 SH-SY5 Y cells were divided into three groups(n = 24): Control group, OGD group, OGD + Isoflurane group. Control group: the cells were cultured regularly; OGD group: the cells were subjected to a 1-h OGD(oxygen and glucose deprivation) and a 20-h simulated reperfusion; OGD + Isoflurane group: 2% isoflurane was applied for 1 h during the early phase of simulated reperfusion. Cell injury was quantified by lactate dehydrogenase(LDH) release. Phospho-GSK3βat Ser 9 and total GSK3β were quantified at 1 h after the OGD with western blotting. Two kinds of high selective GSK3β inhibitors were added and LDH was detected. 【Results】(1) OGD increased LDH release(P < 0.05), while isoflurane post-treatment reduced LDH release(P < 0.05).(2)Isoflurane post-treatment also significantly increased the phosphorylation of GSK3β at Ser 9 at 1 h after the OGD(P < 0.05).(3)GSK3β inhibitors reduced OGD and simulated reperfusion-induced LDH release(P < 0.05). The combination of GSK3β inhibitors and isoflurane post-conditioning induced a greater protection than GSK3β inhibitors alone(P < 0.05), but it did not induce a greater protection than isoflurane post-conditioning alone(P < 0.05). 【Conclusion】 Isoflurane post-conditioning can provide neuroprotection in SH-SY5 Y cells and it may be partly mediated by GSK3β inhibition.
【Objective】 To study the effect of GSK3β inhibition on isoflurane post-conditioning-induced neuroprotection of SHSY5 Y cells against ischemia / reperfusion. 【Method】 SH-SY5 Y cells were divided into three groups(n = 24): Control group, OGD group, OGD + Isoflurane group. Control group: the cells were cultured regularly; OGD group: the cells were subjected to a 1-h OGD(oxygen and glucose deprivation) and a 20-h simulated reperfusion; OGD + Isoflurane group: 2% isoflurane was applied for 1 h during the early phase of simulated reperfusion. Cell injury was quantified by lactate dehydrogenase(LDH) release. Phospho-GSK3βat Ser 9 and total GSK3β were quantified at 1 h after the OGD with western blotting. Two kinds of high selective GSK3β inhibitors were added and LDH was detected. 【Results】(1) OGD increased LDH release(P < 0.05), while isoflurane post-treatment reduced LDH release(P < 0.05).(2)Isoflurane post-treatment also significantly increased the phosphorylation of GSK3β at Ser 9 at 1 h after the OGD(P < 0.05).(3)GSK3β inhibitors reduced OGD and simulated reperfusion-induced LDH release(P < 0.05). The combination of GSK3β inhibitors and isoflurane post-conditioning induced a greater protection than GSK3β inhibitors alone(P < 0.05), but it did not induce a greater protection than isoflurane post-conditioning alone(P < 0.05). 【Conclusion】 Isoflurane post-conditioning can provide neuroprotection in SH-SY5 Y cells and it may be partly mediated by GSK3β inhibition.
引文
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[9]Gomez L,Paillard M,Thibault H,et al.Inhibition of GSK3 beta by postconditioning is required to prevent opening of the mitochondrial permeability transition pore during reperfusion[J].Circulation,2008,117(21):2761-2768.
[10]Feng J,Lucchinetti E,Ahuja P,et al.Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3beta[J].Anesthesiology,2005,103(5):987-995.
[11]Petit-paitel A,Brau F,Cazareth J.Involvement of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP/MPPtreated neurons[J].PLos One,2009,4(5):5491-5500.
[12]Bhat RV,Shanley J,Correll MP,et al.Regulation and localization of tyrosine 216 phosphorylation of glycogen synthase kinase-3beta in cellular and animal models of neuronal degeneration[J].Proc Natl Acad Sci USA,2000,97(20):11074-11079.
[13]Gao X,Zhang H,Takahashi T,et al.The Akt signaling pathway contributes to post-conditioning’s protection against stroke:the protection is associated with the MAPK and PKC pathways[J].Neurochemistry,2008,105(3):943-955.
[14]Xie HR,Hu Ls,Li GY.SH-SY5Y human neuroblastoma cell line:in vitro cell model of dopaminergic neurons in Parkinson’s disease[J].Chin Med J:Engl,2010,123(8):1086-1092.
[15]Ismail N,Ismail M,Fathy SF,et al.Neuroprotective effects of gerralnated brown rice against hydrogen peroxide induced cell death in human SH-SY5Y Cells[J].Int J Mol Sci,2012,13(8):9692-9708.
[2]Zhou Y,Lekic T,Fathali N,et al.Isoflurane posttreatment reduces neonatal hypoxic-ischemic brain injury in rats by the sphingosine-1-phosphate/phosphatidylinositol-3-kinase/Akt pathway[J].Stroke,2010,41(7):1521-1527.
[3]Zhao H.Ischemic post-conditioning as a novel avenue to protect against brain injury after stroke[J].Cereb Blood Flow Metab,2009,29(5):873-885.
[4]李国福,符加红,赵阳.挥发性麻醉剂后处理对SHSY5Y神经细胞缺血再灌注损伤的影响[J].中国现代医学杂志,2011,21(35):4367-4370.LI GF,Fu JH,Zhao Y.Effects of volatile anesthetic postconditioning on ischemia reperfusion induced SHSY5Y nerve cell injury[J].China J Modern Med,2011,21(35):4367-4370.
[5]Cohen MV,Yang XM,Downey JM.The pH hypothesis of post-conditioning:staccato reperfusion reintroduces oxygen and perpetuates myocardial acidosis[J].Circulation,2007,115(14):1895-1903.
[6]Gross GJ,Auchampach JA.Reperfusion injury:does it exist?[J].Mol Cell Cardiol,2007,42(1):12-18.
[7]Matsui T,Rosenzweig A.Convergent signal transduction pathways controlling cardiomyocyte survival and function:the role of PI 3-kinase and Akt[J].Mol Cell Cardiol,2005,38(1):63-71.
[8]Gosens R,Dueck G,Rector E,et al.Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation[J].Physiol Lung Cell Mol Physiol,2007,293(5):L1348-L1358.
[9]Gomez L,Paillard M,Thibault H,et al.Inhibition of GSK3 beta by postconditioning is required to prevent opening of the mitochondrial permeability transition pore during reperfusion[J].Circulation,2008,117(21):2761-2768.
[10]Feng J,Lucchinetti E,Ahuja P,et al.Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3beta[J].Anesthesiology,2005,103(5):987-995.
[11]Petit-paitel A,Brau F,Cazareth J.Involvement of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP/MPPtreated neurons[J].PLos One,2009,4(5):5491-5500.
[12]Bhat RV,Shanley J,Correll MP,et al.Regulation and localization of tyrosine 216 phosphorylation of glycogen synthase kinase-3beta in cellular and animal models of neuronal degeneration[J].Proc Natl Acad Sci USA,2000,97(20):11074-11079.
[13]Gao X,Zhang H,Takahashi T,et al.The Akt signaling pathway contributes to post-conditioning’s protection against stroke:the protection is associated with the MAPK and PKC pathways[J].Neurochemistry,2008,105(3):943-955.
[14]Xie HR,Hu Ls,Li GY.SH-SY5Y human neuroblastoma cell line:in vitro cell model of dopaminergic neurons in Parkinson’s disease[J].Chin Med J:Engl,2010,123(8):1086-1092.
[15]Ismail N,Ismail M,Fathy SF,et al.Neuroprotective effects of gerralnated brown rice against hydrogen peroxide induced cell death in human SH-SY5Y Cells[J].Int J Mol Sci,2012,13(8):9692-9708.