异氟醚后处理对局灶性脑缺血-再灌注大鼠海马神经损伤的影响
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摘要
目的观察异氟醚后处理是否通过Wnt/β-catenin信号通路减轻局灶性脑缺血-再灌注损伤(cerebral ischemia-reperfusion injury,CIRI)大鼠海马神经元损伤。方法 60只雄性SD大鼠随机分为五组,每组12只。采用大脑中动脉栓塞法(middle cerebral artery occlusion,MCAO)缺血90min、再灌注24h制备大鼠局灶性CIRI模型:假手术组(S组)、模型组(M组)、异氟醚后处理+模型组(MI组)、Wnt/β-catenin拮抗剂Dicckkopf-1(DKK-1)+异氟醚后处理+模型组(MDI组)、DKK-1+模型组(MD组)。S组仅分离暴露一侧颈内动脉,不插入线栓。MI和MDI组于再灌注即刻行1.5%异氟醚后处理60min。MDI组和MD组于建立模型前30min行侧脑室注射DKK-1(5μg/kg)。所有模型组大鼠在再灌注24h后,采用Longa评分法进行神经行为学评分,HE染色观察神经细胞形态学变化,免疫组化检测大鼠海马CA1区β-catenin的表达,Tunel荧光检测大鼠海马CA1区神经细胞凋亡情况,Western Blot检测Bcl-2、Bax、β-catenin及GSK-3β蛋白含量,并计算Bcl-2/Bax比值。结果与S组比较,M组大鼠神经行为学评分、神经细胞损伤及凋亡数目、Bax以及GSK-3β蛋白含量均明显增加(P<0.05),而β-catenin蛋白含量及Bcl-2/Bax比值明显减少(P<0.05)。与M组比较,MI组大鼠神经行为学评分、神经细胞损伤及凋亡数目以及Bax蛋白含量明显减少(P<0.05),Bcl-2和β-catenin蛋白含量以及Bcl-2/Bax比值明显增加(P<0.05)。与MI组比较,MDI组大鼠神经行为学评分、神经细胞损伤及凋亡数目及Bax、GSK-3β蛋白含量明显增加(P<0.05),而Bcl-2、β-catenin蛋白含量及Bcl-2/Bax比值明显减小(P<0.05)。结论异氟醚后处理可能是通过影响Wnt/β-catenin信号通路,调控Bcl-2及Bax的表达,从而减轻局灶性CIRI大鼠海马神经元损伤。
Objective To investigate whether Wnt/β-catenin signaling pathway mediating the neuroprotection of isoflurane post-conditioning in hippocampal neurons damage induced by ischemia/reperfusion injury in rats.Methods According to the randomized principle,60 male Sprague-Dawley rats were randomly divided into five groups(12 rats in each group):sham group(group S),model group(group M),ISO+model group(group MI),ISO+model+DKK-1 group(group MDI)and model+DKK-1 group(group MD).A rat model of middle cerebral artery occlusion(MCAO)was established with 90 min ischemia followed by 24 hreperfusion.Group S was only exposed to one side of the internal carotid artery without fishing line.Isoflurane post-conditioning groups(group MI,MDI)were immediately treated with 1.5%isoflurane for 60 min at the onset of reperfusion.DKK-1(5μg/kg)was injected intracerebroventricularly 30 min before the model established in group MDI and group MD.After reperfusion for 24 h,Longa score method was used for neurological deficit score.HE staining and Tunel fluorescence was employed to observe the morphological changes of neurons.Immunohistochemistry and Western Blot were applied to detect the expression of target protein in CA1 region.Results Compared with group S,the neurobehavioral score,the number of apoptosis and the expression of Bax and GSK-3βprotein in group M all increased(P<0.05),while the expression ofβ-catenin and Bcl-2/Bax ratio decreased(P<0.05);Compared with group M,the neurobehavioral score,the number of apoptosis and the expression of Bax protein were significantly decreased(P<0.05),while the expression of Bcl-2,β-catenin protein and the Bcl-2/Bax ratio were significantly increased(P<0.05)in group MI.Compared with group MI,the neurobehavioral score,the number of apoptosis,Bax and GSK-3βprotein in group MDI were significantly increased(P<0.05),while the Bcl-2,β-catenin protein expression,and Bcl-2/Bax ratio were significantly decreased(P<0.05).Conclusion Isoflurane post-conditioning may protect the hippocampus neurons against cerebral ischemic reperfusion-induced damage via the way that the Wnt/β-catenin signaling pathway regulates the expression levels of Bcl-2 and Bax proteins in rats.
Objective To investigate whether Wnt/β-catenin signaling pathway mediating the neuroprotection of isoflurane post-conditioning in hippocampal neurons damage induced by ischemia/reperfusion injury in rats.Methods According to the randomized principle,60 male Sprague-Dawley rats were randomly divided into five groups(12 rats in each group):sham group(group S),model group(group M),ISO+model group(group MI),ISO+model+DKK-1 group(group MDI)and model+DKK-1 group(group MD).A rat model of middle cerebral artery occlusion(MCAO)was established with 90 min ischemia followed by 24 hreperfusion.Group S was only exposed to one side of the internal carotid artery without fishing line.Isoflurane post-conditioning groups(group MI,MDI)were immediately treated with 1.5%isoflurane for 60 min at the onset of reperfusion.DKK-1(5μg/kg)was injected intracerebroventricularly 30 min before the model established in group MDI and group MD.After reperfusion for 24 h,Longa score method was used for neurological deficit score.HE staining and Tunel fluorescence was employed to observe the morphological changes of neurons.Immunohistochemistry and Western Blot were applied to detect the expression of target protein in CA1 region.Results Compared with group S,the neurobehavioral score,the number of apoptosis and the expression of Bax and GSK-3βprotein in group M all increased(P<0.05),while the expression ofβ-catenin and Bcl-2/Bax ratio decreased(P<0.05);Compared with group M,the neurobehavioral score,the number of apoptosis and the expression of Bax protein were significantly decreased(P<0.05),while the expression of Bcl-2,β-catenin protein and the Bcl-2/Bax ratio were significantly increased(P<0.05)in group MI.Compared with group MI,the neurobehavioral score,the number of apoptosis,Bax and GSK-3βprotein in group MDI were significantly increased(P<0.05),while the Bcl-2,β-catenin protein expression,and Bcl-2/Bax ratio were significantly decreased(P<0.05).Conclusion Isoflurane post-conditioning may protect the hippocampus neurons against cerebral ischemic reperfusion-induced damage via the way that the Wnt/β-catenin signaling pathway regulates the expression levels of Bcl-2 and Bax proteins in rats.
引文
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[17]Zhang BQ,Zheng GY,Han Y,et al.Ilexonin a promotes neuronal proliferation and regeneration via activation of the canonical wnt signaling pathway after cerebral ischemia reperfusion in rats.Evid Based Complement Alternat Med,2016,2016:9753189.
[18]Huang C,Fu XH,Zhou D,et al.The role of wnt/β-catenin signaling pathway in disrupted hippocampal neurogenesis of temporal lobe epilepsy:apotential therapeutic target?Neurochem Res,2015,40(7):1319-1332.
[2]Cao L,Feng C,Li L,et al.Contribution of microrna-203to the isoflurane preconditioning-induced neuroprotection.Brain Res Bull,2012,88(5):525-528.
[3]Yuan M,Ge M,Yin J,et al.Isoflurane post-conditioning down-regulates expression of aquaporin 4in rats with cerebral ischemia/reperfusion injury and is possibly related to bone morphogenetic protein 4/smad1/5/8signaling pathway.Biomed Pharmacother,2018,97:428-438.
[4]Wang S,Yin J,Ge M,et al.Transforming growth-beta 1contributes to isoflurane postconditioning against cerebral ischemia-reperfusion injury by regulating the c-jun n-terminal kinase signaling pathway.Biomed Pharmacother,2016,78:280-290.
[5]Zu G,Guo J,Che N,et al.Protective effects of ginsenoside rg1on intestinal ischemia/reperfusion injury-induced oxidative stress and apoptosis via activation of the wnt/β-catenin pathway.Sci Rep,2016,6:(38480).
[6]Khazipov R,Zaynutdinova D,Ogievetsky E,et al.Atlas of the postnatal rat brain in stereotaxic coordinates.Front Neuroanat,2015,9(12):161.
[7]Longa EZ,Weinstein PR,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke,1989,20(1):84-91.
[8]Bak IJ,Misgeld U,Weiler M,et al.The preservation of nerve cells in rat neostriatal slices maintained in vitro:a morphological study.Brain Res,1980,197(2):341-353.
[9]Abe K,Aoki M,Kawagoe J,et al.Ischemic delayed neuronal death.A mitochondrial hypothesis.Stroke,1995,26(8):1478-1489.
[10]Bielen H,Houart C.The wnt cries many:wnt regulation of neurogenesis through tissue patterning,proliferation,and asymmetric cell division.Dev Neurobiol,2014,74(8):772-780.
[11]Yang Y,Li Z,Chen G,et al.Gsk3βregulates ameloblast differentiation via wnt and tgf‐βpathways.J Cell Physiol,2018,233(7):5322-5333.
[12]Zheng W,Lin P,Ma Y,et al.Psoralen promotes the expression of cyclin d1in chondrocytes via the wnt/β-catenin signaling pathway.Int J Mol Med,2017,40(5):1377-1384.
[13]Tao J,Abudoukelimu M,Ma YT,et al.Secreted frizzled related protein 1protects h9c2cells from hypoxia/re-oxygenation injury by blocking the wnt signaling pathway.Lipids Health,2016,15:72.
[14]García de la Cadena S,Massieu L.Caspases and their role in inflammation and ischemic neuronal death.Focus on caspase-12.Apoptosis,2016,21(7):763-777.
[15]Wang Q,Zhang L,Yuan X,et al.The relationship between the bcl-2/bax proteins and the mitochondria-mediated apoptosis pathway in the differentiation of adipose-derived stromal cells into neurons.Plos One,2016,11(10):e0163327.
[16]Chuffa LG,Lupi Júnior LA,Maia Lima AF.Sex steroid receptors and apoptosis-related proteins are differentially expressed in polycystic ovaries of adult dogs.Tissue Cell,2016,48(1):10-17.
[17]Zhang BQ,Zheng GY,Han Y,et al.Ilexonin a promotes neuronal proliferation and regeneration via activation of the canonical wnt signaling pathway after cerebral ischemia reperfusion in rats.Evid Based Complement Alternat Med,2016,2016:9753189.
[18]Huang C,Fu XH,Zhou D,et al.The role of wnt/β-catenin signaling pathway in disrupted hippocampal neurogenesis of temporal lobe epilepsy:apotential therapeutic target?Neurochem Res,2015,40(7):1319-1332.