【作者】 洪荔枝；

【导师】 张慧灵；

【作者基本信息】 苏州大学 ， 药理学， 2010， 硕士

【摘要】 目的:研究苦参碱(Matrine)对缺血性神经元损伤的保护作用及其抑制NF-κB信号的机制。方法:采用大鼠永久性大脑中动脉阻塞(permanent middle cerebral artery occlusion, pMCAO)方法,建立脑缺血的大鼠模型。采用原代培养大脑皮层神经元氧糖剥夺(oxygen-glucose deprivation,OGD)方法,建立脑缺血的细胞模型。免疫荧光法和LDH法分别检测OGD诱导的神经元MAP2阳性细胞数和乳酸脱氢酶漏出率;Hoechst33258染色观察Matrine对缺血神经元凋亡的影响。Western Blot方法检测Matrine对缺血区NF-κB信号通路相关蛋白IκBα,NF-κB p65表达的影响。免疫荧光双标记法分析MAP2阳性神经元NF-κB的核转位。Real Time PCR测定OGD诱导的神经元p53和c-Myc mRNA的表达。结果:LDH结果显示Matrine (50-200μM)能剂量依赖性地降低神经元OGD12h后LDH的漏出率(P<0.01)。免疫荧光法结果显示OGD 12 h模型组MAP2阳性细胞数较正常组明显减少(P<0.01),Matrine可明显增加MAP2阳性细胞数(P< 0.05, P< 0.01)。Hoechst33258染色结果显示OGD12h凋亡细胞明显增多(P<0.01),Matrine可明显减少凋亡细胞的数量(P< 0.05, P< 0.01)。Western Blot结果显示,与假手术组相比,在大脑中动脉阻塞3h,6h,12h,24h后,缺血区IκBα的表达水平在3h开始下降,12h达到最低,24h又开始上升;而细胞核NF-κB p65的表达水平在3h开始上升,12h达到峰值,24h又开始下降。静脉注射Matrine后,Matrine (12.5-50 mg/kg)组明显升高了缺血区IκBα的表达水平,而降低了NF-κB p65的表达水平,并呈现一定的剂量依赖性。Western Blot结果显示,神经元在OGD0h, 1h,3h,6h,12h后,IκBα的表达水平随OGD时间延长逐渐降低,在12h达到最低;而神经元细胞核NF-κB p65的表达水平在1h开始上升,12h达到峰值。给与Matrine后,Matrine (50-200μM)能剂量依赖性地升高神经元IκBα的表达水平,降低神经元细胞核NF-κB p65的表达水平。免疫荧光结果进一步显示: OGD 3h神经元NF-κB的核转位显著增加,6-12h达高峰(P<0.01);Matrine可显著抑制OGD 12h诱导的神经元NF-κB p65核转位(P< 0.05, P< 0.01)。Real Time PCR结果显示:与NF-κB p65核转位的高峰时间相一致,OGD 12h明显诱导神经元NF-κB的靶基因p53和c-MycmRNA表达(P<0.01),而Matrine可显著下调p53和c-Myc mRNA的表达(P< 0.05, P< 0.01)。结论:1.证实了Matrine对缺血性神经元损伤具有直接保护作用,并能抑制神经元细胞凋亡。2. Matrine的神经保护作用可能与抑制神经元NF-κB信号通路激活及其促凋亡靶基因p53和c-Myc mRNA的表达有关。更多还原

【Abstract】 Aim: To investigate the protection of matrine on ischemic neuronal injury and its mechanism of inhibition of NF-κB signaling.Methods: In this study, we established two cerebral ischemia-hypoxia models in in vivo and in vitro. Permanent middle cerebral artery occlusion (pMCAO) model was induced by using intraluminal filament technique in rats. Primary neurons was exposed to a paradigm of ischemic insult by using an oxygen-glucose deprivation (OGD) device. The injury of neurons induced by OGD was detected with LDH test, MAP2 immunofluorescence and Hoechst33258 staining. The signaling pathway of NF-κB related protein NF-κB p65 and IκBαwere assessed with Western Blot analysis. Double immunofluorescence was employed to determine the nuclear translocation of NF-κB in MAP2 positive cell. Real Time PCR was used to measure the expression of p53 mRNA and c-Myc mRNA.Results: LDH showed that Matrine (50-200μM) reduced LDH leakage in a dose-dependent manner 12h after OGD (P< 0.01). Immunofluorescence revealed that matrine markedly increased the number of MAP2 positive cells 12h after OGD (P< 0.05, P< 0.01). Hoechst33258 staining showed that a significant increase in the number of neuronal apoptosis was observed 12h after OGD(P< 0.01), and matrine significantly reduced the number of neuronal apoptosis (P< 0.05, P< 0.01).Western Blot analysis revealed that expression of IκBαwas decreased following pMCAO and peaked at 12h (P<0.01); and the level of nuclear NF-κB p65 was increased and peaked at 12h (P<0.01). Matrine(12.5-50 mg/kg) markedly increased the level of IκBαand decreased the level of nuclear NF-κB p65 in ischemic region in a dose-dependent manner (P< 0.05). Western Blot analysis demonstrated that the expression of IκBαwas declined following OGD-induced neuron injury and peaked at 12h (P<0.01); and the level of nuclear NF-κB p65 was up-regulated and peaked at 12h (P<0.01). Matrine markedly increased the level of IκBαand reduced the level of nuclear NF-κB p65 in neurons (P< 0.05). Consistent with the Western Blot results, immunofluorescence further revealed that a significant increase in the nuclear translocation of NF-κB was observed as early as 3h (P< 0.01) after OGD, and peaked at 6-12 h (P< 0.01). Matrine strongly inhibited NF-κB nuclear translocation 12h after OGD (P< 0.05, P< 0.01). Furthermore, consistent with the peak time of NF-κB nuclear translocation, the expression of NF-κB target gene p53 mRNA and c-Myc mRNA was up-regulated 12h after OGD(P< 0.01), and matrine dramatically down-regulated the expression of p53 mRNA and c-Myc mRNA (P< 0.05, P< 0.01).Conclusion: The current findings provide the first evidences that matrine has a direct neuroprotective activity on cerebral ischemic neuronal injury and suppress the neuronal apoptosis, and this effect may be associated with its blocking the activation of NF-κB and inhibiting proapoptotic target gene of NF-κB p53 and c-Myc in neurons.更多还原