【作者】 张书刚；

【导师】 张颖冬；

【作者基本信息】 南京医科大学 ， 神经病学， 2009， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease, AD)是一种进行性神经变性疾病,是老年期痴呆中最常见的类型。全世界约有2400万AD患者,其中我国约有400万左右。随着疾病进展,患者出现记忆、思维、行为等方面的症状,严重影响日常工作及社会生活,给家庭和社会带来沉重的负担,已经成为一个严重的社会和医疗问题。但到目前为止,AD的发病机制尚不完全清楚,临床缺乏理想的治疗药物。因此,研究AD的发病机制及研发有效的防治药物显得尤为重要。一些对AD患者脑内新病理现象的认识,为阐明AD的神经元死亡机制提供了新的线索,也为AD的治疗带来新的希望。越来越多的证据显示神经元凋亡的出现伴随细胞周期蛋白的激活。在各种神经应激情况下,完成有丝分裂的成熟神经元重新进入细胞周期,但是终极分化的神经元由于自身有丝分裂能力的限制,无法协调的经过细胞周期的各个阶段,最终导致凋亡性细胞死亡。因此异常出现的细胞周期依赖性蛋白激酶(cyclin-dependent kinases, Cdks)在AD的细胞死亡中起关键作用。据此推论,应用药物干预Cdks的活性,可能为AD的治疗提供新的有效的药物。但是Cdks抑制剂作为神经保护剂的研究刚刚起步,并且多在细胞水平和急性脑缺血模型中进行。靛玉红(indirubin)是中国医学科学院在传统中成药当归芦荟丸中发现的抗白血病有效成分。目前已经清楚它是一种选择性的Cdks抑制剂,可以在纳摩尔水平抑制Cdk1、Cdk2、Cdk5的活性,但其水溶性和脂溶性都很差,且有一定的肝毒性。3’单肟靛玉红(Indirubin-3’-monoxime, IMX),为其衍生物,不仅是强有力的Cdks抑制剂,而且毒性低、分子量小、有较好的膜通透性,IMX可以与ATP竞争Cdks激酶的催化位点。本文工作旨在研究Cdks抑制剂IMX对β淀粉样蛋白25-35(β-amyloid protein 25-35, Aβ25-35)所致SH-SY5Y细胞凋亡的保护作用,并在此基础上研究其对tau蛋白磷酸化的影响及其可能机制。本文包括以下两部分工作:第一部分IMX对Aβ25-35所致SH-SY5Y细胞毒性的保护作用目的:探讨Cdks抑制剂IMX对Aβ25-35所致SH-SY5Y细胞凋亡的保护作用。方法:采用细胞培养法,以神经母细胞瘤SH-SY5Y细胞系为材料制备Aβ25-35损伤的离体细胞模型。通过Cell Counting Kit 8(CCK-8)分析不同剂量IMX和(或)Aβ25-35在不同时间点对细胞存活率的影响;采用相差显微镜观察细胞形态并配以Hoechst染色检测细胞核固缩;用流式细胞仪检测细胞凋亡。结果:(1) IMX浓度高至3.0μM以上时可使细胞活性逐渐降低,与对照组相比差异有显著性意义(在3.0μM和6.0μM组,P<0.01;在12μM组,P<0.001);(2)10-50μM Aβ25-35可使细胞活性逐渐降低,与对照组相比差异有显著性意义(在10-30μM组,P < 0.01;在40-50μM组,P < 0.001);(3)同时给予0.25、0.5、1.0μM的IMX可使细胞活性明显升高,与损伤组相比差异有显著性意义(与损伤组比较,在0.25μM组,P<0.05;在0.5、1.0μM组,P<0.01);(4)Hoechst 33258染色结果显示,Aβ25-35处理组凋亡细胞明显增多,出现核固缩、凋亡小体,凋亡细胞升高至42.4±3.86% (P <0.001),而0.5和1.0μM IMX给药后凋亡细胞明显减少,阳性细胞分别减少至27.4±3.09%和19.2±2.02% (P< 0.001);(5)流式细胞仪实验证实,与正常对照组相比,20μM Aβ25-35处理组细胞凋亡率显著升高,可达20.33±2.02%(P<0.01),1.0μM IMX与Aβ25-35同时孵育后,细胞凋亡率降低至12.4±1.82% (与模型组比较,P<0.01 )。在40μM处理组,Aβ25-35处理组凋亡细胞百分率可从对照组的8.1±1.42%增加至50.2±4.77%,而0.5和1.0μM IMX给药后凋亡细胞百分率分别恢复至33.9±3.09%和24.5±3.47%(P<0.001)。结论:IMX能显著减少Aβ25-35所致的SH-SY5Y细胞凋亡,从而抑制其神经毒性作用。第二部分IMX减轻Aβ25-35所致SH-SY5Y细胞tau蛋白磷酸化的作用及其机制研究目的:探讨IMX减轻Aβ25-35诱导的SH-SY5Y细胞tau蛋白过度磷酸化的作用及其可能机制。方法:(1)SH-SY5Y细胞分别与0、0.5和1.0μM IMX预孵育24 hr后,加入40μM老化的Aβ25–35,继续孵育48 hr,应用caspase-3活性检测试剂盒观察各组细胞caspase-3活性变化(。2)用0.5μM和1.0μM IMX预处理SH-SY5Y细胞1 hr后,加入终浓度为20μM凝聚态Aβ25-35,再孵育6 hr,应用蛋白免疫印迹的方法,观察tau蛋白在pS396、pS199、pT205位点的磷酸化水平,并检测磷酸化糖原合酶激酶3beta(p-glycogen synthase kinase 3β, p-GSK3β)(Ser9)的表达;(3)同样的方法处理细胞后24 hr和2 hr,应用蛋白免疫印迹的方法,观察c-jun N末端激酶(c-jun N-terminal kinase, JNK)和细胞外信号调节激酶(extracellular signal regulated protein kinases, ERKs)的活化情况,以探讨这些蛋白在tau蛋白磷酸化中的作用。结果:(1)caspase-3蛋白激酶活性在Aβ25–35诱导的AD细胞模型中明显增高,IMX可以降低其激酶活性;(2)空白对照组tau蛋白在pS396、pS199、pT205位点的磷酸化水平较低,Aβ25–35损伤后tau蛋白磷酸化明显增加,IMX给药可以减少损伤组中tau蛋白磷酸化水平;(3)Aβ25–35损伤后p-GSK3β(Ser9)的表达较空白对照组减少,意味着GSK3β的磷酸化增加,而IMX给药可以增加损伤组中p-GSK3β(Ser9)的表达水平,从而抑制GSK3β的磷酸化;(4)空白对照组p-JNK和p-ERK的表达水平较低,Aβ25–35损伤后其表达明显增加,但IMX给药后蛋白表达无明显变化。结论:(1)IMX可通过抑制caspase-3活化,在一定程度减轻凝聚态的Aβ25-35所致的SH-SY5Y细胞凋亡;(2)IMX可以通过抑制tau蛋白在pS396、pS199、pT205位点的磷酸化水平,从而减轻Aβ25–35的细胞毒性;(3)GSK3β参与了Aβ25–35诱导细胞tau蛋白磷酸化的作用,IMX可通过诱导失活形式的p-GSK3β(Ser9)表达增加而抑制GSK3β的活性,进而抑制Aβ诱导的细胞tau蛋白磷酸化;(4)p-JNK和p-ERK没有参与IMX抑制Aβ诱导的细胞tau蛋白磷酸化的作用。更多还原

【Abstract】 Alzheimer’s disease is a progressive neurodegenerative disease, which is the most common form of dementia. There are as many as 24 million patients in the world and about 4 million people living with Alzheimer’s disease in China. With the progress of the disease, it can cause problems with memory, thinking and behavior severe enough to affect work, daily or social life. AD has become a heaven burden for our society and many families. Up to now, the exact pathogenesis of AD is not clear. It is urgent to elucidate the mechanism of AD and search for new treatments for this debilitating disease.Some new recognitions of pathologic feature of AD patients have provided new important clues about the mechanisms of neuronal apoptosis which make us to see new desire of AD therapy. Accumulating data have shown that neuronal death is accompanied by the induction of cell cycle proteins in a variety of experimental neuronal models. It is indicated that post-mitotic neurons attempt to reenter the cell cycle in respond to various neuronal stressors. But due to their post-mitotic state, these neurons enter into an adverse condition which would create a conflict of signals and result in cell death by apoptosis.These results suggest that aberrant cell cycle reentry may be an important mechanism of neuron loss in neurological disorders. Not surprisingly, treatment with pharmacological inhibitors of Cdks may therefore potentiate the role for treatment of AD. However, the development of highly selective clinical therapies targeting Cdks activity is still in its infancy. Some relative reports used in vitro and acute cerebral ischemia models. At present, it is destitute to develop effective, hypotoxic, selective Cdks inhibitors which can pass the blood brain barrier. Indirubin was identified as the active component of Danggui Luhui Wan, a mixture pill of 11 herbal medicines traditionally utilized against certain types of leukemias by the Chinese Academy of Medicine. It is a selective Cdks inhibitor, which can efficiently inhibit the activities of Cdk1, Cdk2 and Cdk5. But the water-solubility and liposolubility are poor. Indirubin-3’-monoxime (IMX), a synthetic derivative of indirubin, is a potent inhibitor of Cdks with low molecular weight, with better solubility characteristics than indirubins. It has hypotoxicity and membrane permeability and acts by competition with ATP at the catalytic site of Cdks. The aim of the present study is to evaluate the neuroprotective effect of IMX against Aβ-induced apoptosis in cultured neuroblastoma SH-SY5Y cells. In addition, we have explored the effect on tau protein hyperphosphorylation and its possible mechanisms.The study included the following two parts:PartⅠThe neuroprotective effect of IMX against Aβ25-35-induced apoptosis in cultured neuroblastoma SH-SY5Y cellsAim: To investigate the neuroprotective effect of IMX against Aβ25-35-induced apoptosis in cultured neuroblastoma SH-SY5Y cells. Methods: Alzheimer’s disease cellular model was established by cultured neuroblastoma SH-SY5Y cells with Aβ25-35-treatment. Cell Counting Kit 8(CCK-8) assay was used to measure the cell viability of Aβ25-35 and(or) IMX at different time and concentrations.The morphology of SH-SY5Y cell apoptosis of different groups was observed by phase-contrast microscopy and Hoechst 33258 staining. In addition, neuronal apoptosis was measured by a flow cytometric assay. Results: (1) Treatment with a dose of more than 3.0μM IMX resulted in a significant reduction in cell viability, which is compared with the control group (P<0.01 at 3.0 and 6.0μM; P<0.001 at 12.0μM).(2)Treatment with a dose of 10-50μM of aged Aβ25–35 for 48 hr and 72 hr or 30-50μM Aβ25–35 for 24 hr resulted in a significant reduction in cell viability, which is compared with that in the control group (P<0.01 at 10-30μM; P<0.001 at 40-50μM).(3) Pretreatment with IMX (0.25–1.0μM) concentration-dependently reversed Aβ25–35-induced SH-SY5Y cell death (P<0.05 at 0.25μM; P<0.01 at 0.5 and 1.0μM).(4) Aβ25–35-treated cells exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. After 48 hr exposure of 40μM of aged Aβ25–35, the number of apoptotic cells was markedly increased to 42.4±3.86% (P< 0.001), which, however, was prevented by the addition of 0.5 and 1.0μM IMX. The percentage of apoptotic cells was 27.4±3.09% and 19.2±2.02%, respectively (n=5, P<0.001). (5) Apoptotic cells of Aβ25–35-treated-alone group were markedly increased to 20.33±2.02% (P<0.01) with 20μM Aβ25-35 treatment. This increase, however, was prevented by the addition of 1.0μM IMX. The percentage of apoptotic cells was 12.4±1.82% (P<0.01). Basal apoptotic cell death was 8.1±1.42% and rose to 50.2±4.77% in cells treated for 48 hr with 40μM of aged Aβ25–35 (n=5, P <0.001). However, pretreatment with 0.5 and 1.0μM of IMX concentration-dependently suppressed the apoptosis induced by 40μM of aged Aβ25–35 for 48 hr, and the percentage of apoptotic cells was 33.9±3.09% and 24.5±3.47%, respectively (n=5, P<0.001). Conclusion: IMX markedly reversed Aβ25–35-induced neurotoxicity, indicating the neuroprotective activity of IMX. PartⅡThe effect and its possible mechanisms of IMX on Aβ25-35-induced tau phosphorylation in SH-SY5Y cellsAim: To investigate the effect and its possible mechanisms of IMX on Aβ25-35-induced tau phosphorylation in SH-SY5Y cells. Methods: (1) SH-SY5Y was incubated with 0, 0.5 and 1.0μM IMX. Following 24 hr treatment, 40μM of aged Aβ25–35 was added. 48 hr later, caspase-3 activity was measured with the caspase-3 assay kit. (2) 0.5μM and 1.0μM IMX were added in the serum-deprived media of SH-SY5Y cells for 1 hr prior to the 6 hr Aβ25-35 (20μM) exposure. Western blotting analysis was performed to detect tau phosphorylation at the site of pS396, pS199 and pT205 and p-GSK3β(Ser9)expression. (3) The cultures were treated as described above, 24 hr and 2 hr later, western blotting analysis was performed to detect p-JNK and p-ERK expression. Results: (1) Caspase-3 activity was significantly increased in the Aβ25-35 treated group when compared to the control group (P<0.01). These changes were partially prevented by pretreatment with IMX (P<0.05). (2)In the Aβ25-35-treated group, protein levels of phosphorylated tau at the site of pS396, pS199 and pT205 detected by western blotting were significantly increased when compared to the control group (P<0.05). However, these changes were partially or completely prevented by pretreatment with IMX (P<0.05). (3) After Aβ25–35 treatment, the expression of p-GSK3β(Ser9) was decreased, which meaned that GSK3βphosphorylation was increased. However, the expression of p-GSK3β(Ser9) was significantly increased when pretreatment with IMX (P<0.05). (4) The expression of p-JNK and p-ERK were significantly increased in the Aβ25-35-treated group compared to the control group (P<0.05). But there were no significant changes after IMX treatment. Conclusion: (1) IMX can inhibit caspase-3 activity efficiently to ameliorate Aβ25–35-induced neurotoxicity. (2) IMX attenuates Aβ25–35-induced neurotoxicity possibly by inhibiting tau phosphorylation at the site of pS396, pS199 and pT205. (3) IMX decreases tau phosphorylation by inhibiting GSK3βphosphorylation. (4) The expression of p-JNK and p-ERK were not involved the effect of IMX.更多还原