【作者】 王燕；

【导师】 秦正红；

【作者基本信息】 苏州大学 ， 药理学， 2010， 博士

【摘要】 目的:研究核因子-κB (Nuclear Factor-kappa B, NF-κB)/p53信号通路在谷氨酸受体介导的神经兴奋性毒性中的作用及机制。方法:采用脑立体定位纹状体内给药法,建立谷氨酸受体介导的大鼠神经兴奋性毒性的体内模型,RT-PCR和Western Blot检测喹啉酸(Quinolinic acid, QA)引起的凋亡相关蛋白——p53、PUMA、Bax、Bcl-2和自噬相关蛋白——DRAM (damage-regulated autophagy modulator)、LC3 (microtubule-associated protein 1 light chain 3)、Beclin1的mRNA和蛋白表达的变化。DNA ladder及尼氏染色法观察NF-κB抑制剂SN50和p53抑制剂PFT-α(Pifithrin-alpha)对模型大鼠纹状体神经元的保护作用;RT-PCR和Western Blot检测SN50和PFT-α对模型大鼠纹状体内凋亡相关蛋白p53、PUMA (p53 up-regulate modulator of apoptosis)、Bax、Bcl-2和自噬相关蛋白DRAM、LC3、Beclin1的mRNA和蛋白表达的影响。尼氏染色法观察Cathepsin L抑制剂Z-FY-DMK对模型大鼠纹状体神经元的保护作用;免疫组织化学法检测Z-FY-DMK对模型大鼠纹状体内NF-κB激活的影响;Western Blot检测Z-FY-DMK对模型大鼠纹状体内IκBα(inhibitor of NF-kB, alpha isoform)、p-IκBα(phospho-IκB alpha)、自噬相关蛋白DRAM、Beclin1、P62蛋白表达的影响。采用谷氨酸处理原代培养的大鼠胎鼠纹状体神经元,建立谷氨酸受体介导的神经兴奋性毒性的体外模型,LDH法检测SN50、PFT-α、Z-FY-DMK对神经元存活率的影响;免疫细胞化学法检测SN50和PFT-α对谷氨酸引起的线粒体膜电位的影响;Western Blot检测Z-FY-DMK对兴奋性毒性细胞模型中自噬相关蛋白Beclin1和P62蛋白表达的影响。结果:本实验成功建立了大鼠神经兴奋性毒性的体内模型。RT-PCR和Western Blot结果显示,模型大鼠纹状体内p53、PUMA、Bax表达增加,Bcl-2表达降低和DRAM、LC3、Beclin1表达上调。SN50、PFT-α给药组与模型组相比,DNA ladder凋亡条带明显减弱,损伤面积显著减小(p<0.01);RT-PCR和Western Blot结果显示SN50和PFT-α可对抗QA模型大鼠纹状体中p53、PUMA、Bax上调、Bcl-2下调、DRAM、LC3、Beclin1上调。Z-FY-DMK给药组与模型组相比,损伤面积显著减小(p<0.01);免疫组化结果显示Z-FY-DMK可抑制QA模型大鼠纹状体内NF-κB的核转位;Western Blot结果显示Z-FY-DMK可对抗QA模型大鼠纹状体中IκBα降解和磷酸化、以及DRAM、Beclin1上调和P62的下调。本实验成功建立了大鼠胎鼠的原代纹状体神经元兴奋性毒性的体外模型。LDH法结果显示SN50、PFT-α、Z-FY-DMK可减少谷氨酸引起的神经元死亡;免疫细胞化学法结果显示SN50和PFT-α可抑制谷氨酸受体介导的线粒体膜电位下降;Western Blot结果显示Z-FY-DMK可对抗谷氨酸引起的Beclin1和P62蛋白表达的变化。结论:谷氨酸受体介导的神经兴奋性毒性中,NF-κB抑制剂和p53抑制剂通过影响NF-κB/p53信号通路,抑制神经元自噬和凋亡的发生;Cathepsin L抑制剂Z-FY-DMK通过抑制谷氨酸受体介导的IκBα降解和磷酸化、NF-κB核转位、自噬激活,最终阻断NF-κB/p53信号通路,从而对谷氨酸受体激活引起的纹状体神经元损伤起保护作用。更多还原

【Abstract】 Aim: To study the roles of Nuclear Factor-kappa B (NF-κB)/p53 pathway in autophagy and apoptosis mediated by glutamate receptors.Methods: The in vivo excitotoxic model was produced with stereotaxic administration of Quinolinic acid (QA) into unilateral striatum. The changes in the expression of p53 and its target genes involved in apoptosis and autophagy including PUMA, Bax, Bcl-2, damage-regulated autophagy modulator (DRAM), and other autophagic proteins including microtubule-associated protein 1 light chain 3 (LC3), and beclin1 were assessed with RT-PCR and Western blot analysis. The neuroprotective effects of the NF-κB inhibitor SN50 and the p53 inhibitor Pifithrin-alpha (PFT-α) were assessed with internucleosomal DNA fragmentation and Cresyl violet staining. Effects of SN50 or PFT-αon QA-induced increases in the level of p53, PUMA, Bax, decreases in the level of Bcl-2, and increases in the level of DRAM, LC3, Beclin1 were detected with RT-PCR and Western blot analysis. The neuroprotective effects of the Cathepsin L inhibitor Z-FY-DMK were assessed with Cresyl violet staining. Effects of Z-FY-DMK on QA-induced NF-κB activation were detected with immunohistochemical analysis. Effects of Z-FY-DMK on QA-induced IκBαdegradation and phosphorylation, changes in the levels of DRAM, Beclin1, P62 were detected with Western blot analysis. The in vitro excitotoxic model was produced with glutamate treatment of primary striatum neurons. The neuroprotective effects of SN50, PFT-αand Z-FY-DMK were assessed with LDH. Effects of SN50 and PFT-αon glutamate-induced decrease of mitochondrial transmembrane potential were assessed with immunofluorescence analysis. Effects of Z-FY-DMK on glutamate-induced changes of protein levels of Beclin1 and P62 were observed using Western blot analysis.Results: We have successfully established in vivo model of excitotoxicity by QA. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl-2. QA also upregulated DRAM, the ratio of LC3-II/LC3-I and the levels of Beclin1. QA- induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50 or PFT-α(p<0.01). SN50 and PFT-αreversed QA-induced increases in the expression of p53, PUMA, Bax, decrease in Bcl-2, and up-regulation of DRAM, the ratio of LC3-II/LC3-I and the levels Beclin1 in the striatum. QA-induced loss of striatal neurons were robustly inhibited by Z-FY-DMK (p<0.01). Z-FY-DMK reversed QA-induced activation of NF-κB. Z-FY-DMK inhibited QA-induced IκBαdegradation and phosphorylation, up-regulation of DRAM, Beclin1, and down-ragulation of P62. We have successfully established the in vitro excitotoxic model with glutamate in cultured primary striatal neurons. LDH tests showed that the damage of neurons induced by excitotoxicity was significantly depressed by the treatment with SN50, PFT-αand Z-FY-DMK. Immunofluorecsence analysis showed that glutamate-induced depolarization of mitochondrial membrane was attenuated by SN50 and PFT-α. Western blot analysis showed that glutamate-induced Beclin1 upragulation and P62 downregulation were attenuated by Z-FY-DMK.Conclusions: These results suggest that over-stimulation of glutamate receptors can induce NF-κB-dependent expression of p53. NF-κB/p53 pathway participates in excitotoxic neuronal death through both apoptotic and autophagic mechanisms. Cathepsin L inhibitor has neuroprotective effects by inhibiting glutamate receptors-induced IκBαdegradation and phosphorylation, NF-κB nuclear translocation and activation of autophagy.更多还原