【作者】 薛宏宇；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 本研究旨在探讨桃叶珊瑚苷在糖尿病脑病大鼠模型和过氧化氢（H₂O₂）诱导PC12细胞凋亡模型中的作用及机制。将Wistar大鼠一次性腹腔注射链脲佐菌素（STZ;60mg/kg）,建立糖尿病模型。糖尿病大鼠在正常饲养条件下,检测不同时期的大鼠血糖、体重,认知能力,海马神经元细胞的凋亡情况,确定糖尿病脑病发病时间。研究表明,STZ诱导形成的糖尿病大鼠饲养65d后表现出学习、记忆能力下降,海马CA1区神经元细胞大量死亡,因此糖尿病大鼠引发脑病时间确定为糖尿病发病后第65d。给患糖尿病脑病的大鼠腹腔注射不同剂量的桃叶珊瑚苷,确定桃叶珊瑚苷的药物剂量和药用时间,通过Y迷宫检测大鼠的认知能力,HE染色检测大鼠海马神经元细胞的凋亡情况。结果表明,1mg/kg桃叶珊瑚苷组与糖尿病脑病组相比具有一定的保护作用,大鼠的学习、记忆功能得到一定的改善。随着药物剂量的增加,其保护作用也增强。而5mg/kg和10mg/kg桃叶珊瑚苷组,海马CA1区存活神经元细胞数量与脑病组相比明显增多,大鼠学习、记忆功能得到很大程度的改善,但5mg/kg和10mg/kg两个剂量组之间无显著性差异。因此,以5mg/kg剂量对不同给药时间进行了检测,结果表明15d和30d两个剂量组之间无论在体重、血糖,还是在认知功能上均无显著性差异,故本实验确定桃叶珊瑚苷的给药剂量为5mg/kg,给药时间为15d。进一步运用电子显微镜、原位细胞死亡检测技术、免疫组化技术、Western blotting检测技术和紫外分光光度仪探讨桃叶珊瑚苷的神经保护作用机制。结果表明,糖尿病脑病大鼠的海马内GSH-PX、SOD和CAT酶活性显著降低,NOS酶和MDA活性明显增强,Bcl-2蛋白的表达减少,Bax蛋白表达和凋亡细胞数量均明显增加;而桃叶珊瑚苷治疗组海马内GSH-PX、SOD和CAT酶活力明显提高,Bcl-2蛋白表达量增加,抑制了NOS酶和MDA活性和Bax蛋白表达,大大降低了海马神经元细胞的凋亡数量。这些结果表明,桃叶珊瑚苷能够通过调控海马内抗氧化酶活性,清除海马内过多的自由基,通过调节NOS酶的活性,减少自由基的生成,降低体内自由基含量,并通过有效调节线粒体中Bcl-2和Bax蛋白的表达,从而抑制了海马神经元的细胞凋亡,最终实现对海马神经元细胞的保护。利用H₂O₂（0.25mM）诱导PC12细胞建立体外细胞凋亡模型之后,用桃叶珊瑚苷进行处理观察其对PC12细胞的作用。结果表明,桃叶珊瑚苷能够抑制H₂O₂对PC12细胞的毒性。荧光染色检测发现桃叶珊瑚苷治疗组与H₂O₂组相比细胞存活数量明显增多,凋亡数量明显减少;透射电镜观察H₂O₂处理组细胞呈现核固缩、胞质空泡化、但胞膜仍保持完整;桃叶珊瑚苷与H₂O₂共培养,细胞的形态只发生轻微变化;流式细胞仪分析细胞凋亡率,H₂O₂组为35.1±3.0%,桃叶珊瑚苷治疗组为23.1±1.1%;与H₂O₂组相比桃叶珊瑚苷治疗组的Bcl-2蛋白的表达明显增加,Bax蛋白的表达明显降低;Westernblotting检测结果与流式细胞仪分析结果相似;桃叶珊瑚苷治疗组的半胱氨酸酶-3活性明显降低;分光光度法检测LDH释放率的结果表明H₂O₂使得LDH的释放率明显增高,桃叶珊瑚苷有效缓解了LDH的释放。上述结果说明,桃叶珊瑚苷抑制H₂O₂诱导PC12细胞凋亡的机制,体现在保护细胞膜的功能,抑制Bcl-2蛋白表达的下降和Bax蛋白表达的升高,拮抗Caspase-3的激活。更多还原

【Abstract】 The purpose of this study mainly aims at observation the effects of aucubin and its action mechanism in diabetic encephalopathy rat model and H₂O₂-induced PC12 cells.In this study, we determined the effect and therapeutic mechanism of aucubin on diabetic encephalopathy. With the exception of a control group, all rats were received intraperitoneal injections of streptozotocin （STZ; 60 mg/kg） to induce Type 1 DM. From d 65 after induction of diabetes, rats were treated with various doses and different injective time of aucubin. Cognition function was assayed by Y maze and morphological changes in hippocampal neurons were observed. In diabetic rats, the function of learning and memory was impaired and the neuron density was remarkably decreased. Various dose of aucubin significantly reduced the working errors during behavioral testing, and attenuated neuron loss in hippocampal region in diabetic encephalopathy rats. Aucubin showed a significant neuroprotective effect and improved learning in diabetic encephalopathy rats.Neuroprotection was estimated by the indexes of behavior and histology. Behavioral testing was performed in a Y-maze and the surviving neurons in CA1 to SC of the hippocampus were counted under a microscope. In addition, the apoptotic neurons in the CA1 of the hippocampus were also examined by using the TUNEL staining. In order to clarify the mechanism of aucubin’s neuroprotection, the activities of endogenous antioxidants and nitric oxide synthase （NOS） together with the content of lipid peroxide in the hippocampus were assayed. The results proved that aucubin significantly reduced the content of lipid peroxide, regulated the activities of antioxidant enzymatic and decreased the activity of NOS and MDA. All these effects indicated that aucubin was a potential neuroprotective agent and its neuroprotective effects were achieved, at least in part by promoting endogenous antioxidant enzymatic activities. Hippocampi were excised for visual （light and transmission electron microscopic） and immunochemical （Western blot; immunohistochemical） assessments of the CA1 subfield for apoptosis and expression of regulatory proteins Bcl-2 and Bax. Treatment responses to all the parameters examined （body weight, plasma glucose, Y-maze error rates, pyramidal cell ultrastructure, proportions of apoptotic cells, levels of expression of Bcl-2 and Bax, and survivability of neuronal cells） were identical: These findings confirm the association of apoptosis with the encephalopathic effects of diabetes mellitus, and suggest a major role of the expression levels of Bcl-2 and Bax in the regulation of apoptotic cell death. All of the results suggest that aucubin could effectively inhibit apoptosis by modulating the expressions of Bcl-2 and Bax genes.In this study, the effect of aucubin on H₂O₂-induced apoptosis （0.25 mM） was studied by using a rat pheochromocytoma （PC12） cell line. Hoechst 33258 assays revealed the appearance of a collection of multiple chromatin and fragmented apoptotic nuclei upon H₂O₂ treatment. however, the incidence of apoptotic nuclei was significantly reduced when cells were treated with aucubin. Transmission electron microscopy was found preferable to alternatives for distinguishing between nuclei and organelles. Obvious apoptotic morphological changes were observed in the H₂O₂ induced cells. Addition of aucubin to the H₂O₂ clearly enlarged the apoptotic nuclei, which were also smoother and resembled those of the control cells. When the apoptotic cells were analyzed quantified using flow cytometry, when PC12 cells were concurrently treated with aucubin, the percentage of apoptotic cells decreased from 35.1±3.0 to 23.1±1.1%. Bcl-2 protein increased and Bax protein declined in aucubin treatment. Western blot analysis confirmed the changes in anti-apoptotic and pro-apoptotic proteins in PC12 cells, as did Bcl-2 and Bax levels, which coincided with the flow cytometry results. H₂O₂ treatment caused a time-dependent increase in caspase-3 proteolytic activity, when aucubin was added caspase-3 activity declined. On the basis of these findings, we speculate that the neuroprotective effects of aucubin may be achieved by the regulation of Bcl-2 family members, as well as modulating the caspases cascade activation.更多还原