【作者】 张梅；

【导师】 冯鉴强；

【作者基本信息】 中山大学 ， 生理学， 2008， 博士

【摘要】 研究背景随着人口老龄化程度的提高,神经系统退行性疾病成为威胁人类健康的重要敌人。如何防治这一类疾病也成为当前医学研究的重点之一。氧化应激引起的神经元凋亡和坏死是导致神经系统退行性疾病的重要原因。目前对这类疾病的治疗限于使用抗氧化剂减轻氧化应激所致的细胞损伤和凋亡,属于被动治疗的范畴。如何通过外源性刺激调动机体内在的保护机制来主动的对抗氧化损伤,将是防治这一类疾病新的方向。适应性细胞保护是指预先给予弱损害因素,可阻止或减轻随后强损害因素引起的细胞损伤,即通过某些“亚伤害性”应激预处理调动机体内在的保护代偿机制,使机体适应或减轻后续的更严重损害。适应性细胞保护作为机体维持稳态的天然机制,特点在于不改变攻击因子性质的情况下,通过外源性的适宜刺激（预处理）调动机体自身的保护机制,加强细胞对损伤的防护能力。所以适应性细胞保护作用的存在为我们主动防治神经系统退行性疾病提供了可能性。本实验室利用肾上腺嗜铬细胞瘤（PC12）细胞建立了H₂O₂预处理诱导的适应性细胞保护模型。前期研究表明H₂O₂预处理通过降低细胞内活性氧水平、抑制线粒体膜电位降低、增强bcl-2和Survivin蛋白的表达发挥细胞保护作用。并且诱导性一氧化氮合酶（iNOS）、环氧化物酶（COX-2）的表达增高和JAK-STAT信号通路的激活也参与H₂O₂预处理诱导的抗凋亡作用。结果提示H₂O₂预处理诱导的适应性细胞保护作用机制复杂,可能涉及到多条信号通路的激活。NF-κB和PI3K/Akt作为与氧化应激后细胞命运（存活或死亡）密切相关的信号通路,受到越来越多的关注。1、NF-κB信号通路与氧化应激NF-κB是被报道的第一个对由ROS或H₂O₂诱导的氧化应激产生反应的真核转录因子。大量的研究证实NF-κB可以介导细胞存活或死亡两条通路。在氧化损伤情况下构成性的NF-κB活性为神经细胞存活所必须,而使用NF-κB的抑制剂可降低不同应激情况下神经元的存活。但是相反的证据表明阻止NF-κB的核转位可以减轻氧化应激情况下的神经细胞死亡。2、PI3K/Akt信号通路与氧化应激当细胞面临氧化损伤时,PI3K/Akt通路的活化对细胞存活非常重要。PI3K/Akt通路的活化在中枢和外周神经元均发挥抗氧化作用。活化的Akt可以保护神经细胞免受氧化性毒物诱导的细胞凋亡。但是不同研究指出,某些诱导细胞凋亡的刺激可导致Akt的表达下调,提示PI3K/Akt通路不参与某些应激情况下的细胞存活。所以面对氧化损伤,NF-κB和PI3K/Akt信号通路对细胞功能影响复杂。NF-κB和PI3K/Akt信号通路是否在H₂O₂预处理诱导的适应性细胞保护中发挥作用?本课题将对这一问题开展研究。第一部分NF-κB信号通路在H₂O₂预处理适应性细胞保护中的作用研究目的揭示NF-κB信号通路是否参与H₂O₂预处理诱导的细胞保护作用。方法1.细胞损伤程度的判断:MTT法测定PC12细胞的存活率,比色法测定乳酸脱氢酶的活性。2.细胞凋亡的判断:Hoechst33258染色检测细胞形态学改变,流式细胞术检测DNA含量的变化。3.细胞免疫化学染色显示NF-κB的核转位。4.电泳迁移实验（EMSA）测定NF-κB的DNA结合活性。5.比色法测定caspase-3的活性。6. Western blot分析相关蛋白质表达水平的变化。7.用SPSS 14.0 for Windows统计软件进行数据分析。数据用均数±标准差（±S）表示,用单因素方差分析（One-way ANOVA）进行均数间的多重比较,检验水准α=0.05。结果1. H₂O₂预处理诱导NF-κB的表达Western blotting结果显示,氧化应激和H₂O₂预处理可以诱导PC12细胞NF-кB的表达。并且NF-κB抑制剂TPCK减少H₂O₂预处理对NF-κB的表达上调。2. H₂O₂预处理引起NF-κB的核转位细胞免疫化学染色结果显示,H₂O₂预处理PC12细胞能明显地促进NF-κB发生核转移。3. H₂O₂预处理增强了NF-κB的DNA结合活性EMSA电泳结果显示H₂O₂预处理使核蛋白DNA复合体的数量明显增多,提示H₂O₂预处理增加NF-κB的DNA结合活性。4. NF-κB介导H₂O₂预处理适应性细胞保护作用①NF-κB抑制剂TPCK拮抗H₂O₂预处理减轻细胞损伤的作用:MTT检测结果显示,H₂O₂预处理可以使PC12细胞对抗随后高浓度H₂O₂引起的细胞存活率下降,而在预处理前使用TPCK,细胞的成活率明显降低。细胞上清LDH测定结果显示,TPCK对抗了H₂O₂预处理对LDH释放的抑制作用,使PC12细胞释放LDH明显增多。②TPCK拮抗了H₂O₂预处理减轻细胞凋亡的作用:Hoechst 33258染色结果显示,预处理明显减少凋亡细胞的数量。但在预处理前使用TPCK则可使凋亡细胞的数量增加。流式细胞仪检测结果表明,H₂O₂预处理PC12细胞可使损伤浓度H₂O₂引起的细胞凋亡率显著下降,而预处理前使用TPCK明显增加细胞的凋亡率。5. NF-κB以caspase依赖的方式影响凋亡H₂O₂预处理明显降低损伤浓度的H₂O₂引起caspases-3活性升高,预处理前使用TPCK明显增加了caspase-3的活性。6. H₂O₂预处理通过NF-κB途径诱导了HSP70和HSP90的表达H₂O₂预处理上调300μM H₂O₂损伤时HSP 70和HSP 90的表达,TPCK抑制了H₂O₂预处理诱导的HSP 70和HSP 90表达上调。结论1、H₂O₂预处理可以上调PC12细胞NF-κB的表达,并增强其核转移和转录活性。2、NF-κB的表达和激活参与了H₂O₂预处理诱导的适应性细胞保护作用（抗细胞损伤和细胞凋亡）。其抗细胞凋亡作用涉及caspase依赖的信号通路。3、HSP90和HSP70作为NF-κB激活转录后的效应蛋白,可能是NF-κB介导H₂O₂预处理的细胞保护作用机制之一。第二部分PI3K/Akt信号通路在H₂O₂预处理适应性细胞保护中的作用研究目的揭示PI3K/Akt信号通路是否参与H₂O₂预处理诱导的细胞保护作用。方法1.细胞损伤程度的判断:MTT法测定细胞的存活率,比色法测定乳酸脱氢酶的活性。2.细胞凋亡的判断:Hoechst33258染色检测细胞形态学改变,流式细胞术检测DNA含量的变化。3.比色法测定caspase-3的活性。4. Western blot分析相关蛋白质表达水平的变化。5.用SPSS 14.0 for Windows统计软件进行数据分析。数据用均数±标准差（±S）表示,用单因素方差分析（One-way ANOVA）进行均数间的多重比较,检验水准α=0.05。结果1. H₂O₂预处理以PI3K依赖的方式诱导p-akt的表达Western blot结果显示, H₂O₂预处理可以诱导PC12细胞p-akt的表达。PI3K抑制剂ly294002拮抗了H₂O₂预处理引起的p-akt表达。2. PI3K/Akt通路介导H₂O₂预处理适应性细胞保护作用①PI3K抑制剂ly294002拮抗H₂O₂预处理减轻细胞损伤的作用:MTT检测结果显示,H₂O₂预处理可以使PC12细胞对抗随后高浓度H₂O₂引起的细胞存活率下降,而在预处理前使用ly294002,细胞的成活率明显降低。细胞上清LDH测定结果显示,ly294002使PC12细胞释放LDH明显增多,对抗了H₂O₂预处理抑制的LDH释放增加。②Ly294002拮抗H₂O₂预处理减轻细胞凋亡的作用:Hoechst 33258染色结果显示,预处理明显减少凋亡细胞的数量。但在预处理前使用ly294002则可使凋亡细胞的数量增加。流式细胞仪检测结果表明,H₂O₂预处理PC12细胞可使损伤浓度H₂O₂引起的细胞凋亡率显著下降,而预处理前使用TPCK明显增加细胞的凋亡率。3. Ly294002以caspase依赖的方式影响凋亡H₂O₂预处理明显降低损伤浓度的H₂O₂引起caspases-3活性升高,预处理前使用ly294002明显增加了caspase-3的活性。4. H₂O₂预处理通过PI3K/Akt途径上调了HSP70和HSP90的表达H₂O₂预处理上调300μM H₂O₂损伤时HSP 70和HSP 90的表达,ly294002抑制了H₂O₂预处理诱导的HSP 70和HSP 90表达上调。5. H₂O₂预处理通过PI3K/Akt途径激活NF-κB信号通路研究表明,PI3K的阻断剂ly294002可以抑制H₂O₂对NF-κB的激活,说明在H₂O₂预处理引起的适应性细胞保护作用中,PI3K/Akt信号通路位于NF-κB信号通路的上游。结论1、H₂O₂预处理通过PI3K依赖途径引起Akt的的活化。2、PI3K/Akt信号通路的激活参与了H₂O₂预处理诱导的适应性细胞保护作用（抗细胞损伤和细胞凋亡）。其抗细胞凋亡作用涉及caspase依赖的信号通路。3、HSP90和HSP70作为PI3K/Akt信号通路激活后的效应蛋白,可能是PI3K/Akt介导H₂O₂预处理的细胞保护作用机制之一。4、在H₂O₂预处理引起的适应性细胞保护作用中,PI3K/Akt信号通路位于NF-κB信号通路的上游。总结本研究表明:在PC12细胞PI3K/Akt和NF-κB信号通路均介导了H₂O₂预处理诱导的适应性细胞保护作用。H₂O₂预处理通过PI3K依赖的方式激活akt,活化后的akt可以引起NF-κB通路的激活,通过抑制caspase-3活性来对抗凋亡,同时促进某些应激基因的快速表达,合成对细胞具有保护作用的蛋白质HSP90和HSP70,来使细胞耐受后续的严重损伤。更多还原

【Abstract】 BackgroundWith the progress of aging, neurodegenerative diseases became a menace to human’s health. Therefore the pivot of medical research turned to how to prevent this kind of disease. Necrosis and apoptosis of neuron induced by oxidative stress is the main reason of neurodegenerative diseases. At present, prevention and treatment to this kind of diseases was limited to reducing the injury and apoptosis resulting from oxidative damages by using anti-oxidant, which belong to a passive process. How to resist oxidative injury actively will be a new direction for prevention of neurodegenerative diseases through enhancing ability of endogenous defensive system stimulated by extrinsic substance.Adaptive cytoprotection means treated cells with mild insults before strong insults can relieve cell injury induced by strong damages, and the mechanism of which is due to the sublethal stress （mild insults） triger endogenous defense system which make organism adapt to subsequent strong insults. Serve as a common mechanism on maintaining steady state, adaptive cytoprotection have a characteristic of enhancing ability of endogenous defensive system through extrinsic stimuli without changing quality of insults. Therefore adaptive cytoprotection provides possibility for prevention of neurodegenerative diseases actively.Cytoprotection model of H₂O₂ precondition had been established in PC12 cells in our laboratory, and previous study indicated that H₂O₂ precondition induced cytoprotection via reducing intracellular reactive oxygen species （ROS） level, preventing the reduction of mitochondrial membrane potential （MMP）, and enhancing expression of Bcl-2 and Survivin. Moreover increasing expression of inducible nitric oxide synthase （iNOS）, cyclooxygenase-2 （COX-2） and activation of JAK-STAT signal pathway participated in anti-apoptotic induced by H₂O₂ precondition. The results suggest that the mechanism of H₂O₂ precondition-induced cytoprotection is complicated, which may involve in activation of multi-pathway.NF-κB and PI3K/Akt signal pathway gathered more attentions because of its closed relation with cell fate （survival and death） under oxidative stress circumstance.1. NF-κB signal pathway and oxidative stressNF-κB was the first eukaryotic transcription factor reported to response to oxidative stress such as ROS and H₂O₂. A number of study demonstrated that NF-κB mediates cell survival and death. When Confronted with oxidative injury constitutive NF-kB activity was necessary for neuron survival, and inhibition of NF-κB reduce neuron survival under different oxidative stress. On contrary, some evidences indicated blocking of NF-κB transition into nuclei can reduce neuron death induced by oxidative stress.2. PI3K/Akt signal pathway and oxidative stressActivation of PI3K/Akt pathway is very important for cell survival to oxidative stress. Moreover activation of PI3K/Akt pathway played an anti-oxidant role in central and peripheral neuron and can protect neuron against apoptosis induced by oxidative toxicant. But different study showed that some apoptosis–induced stimuli down-regulate expression of Akt, which suggest PI3K/Akt pathway may not participate in cell survival under some stress.Therefore the effect of NF-κB and PI3K/Akt signal pathway on cell fate is complicated when confronted with oxidative injury. Whether NF-κB and PI3K/Akt signal pathway play a role in H₂O₂ precondition-induced cytoprotection is still not clarified. This study will investigate this question. Part one Role of NF-κB signal pathway in H₂O₂ preconditioning-induced adaptive cytoprotectionObjectiveTo discover whether NF-κB signal pathway participate in H₂O₂ precondition induced- cytoprotection.Methods1. Estimation of damages on cells: MTT was used to detect the viability of PC12 cell, and the cytotoxicity was quantitatively assessed by measuring the amount of lactate dehydrogenase （LDH） released from damaged cells into the cell culture medium.2. Assessment of cell apoptosis: morphological changes were determined by using Hoechst 33258 staining, and flow cytometry （FCM） analysis was used to measure the rate of apoptosis.3. Immunocytochemical staining was adopted to detect the nuclear translocation of NF-κB.4. Electrophoretic mobility shift assay （EMSA） was used to estimate the ability of NF-κB binding to DNA.5. Activity of caspase-3 was measured by colorimetry.6. Western blot analysis was used to measure the expression of protein.7. All data are expressed as mean + SD. The differences between groups were evaluated by one-way analyses of variance （ANOVA） using SPSS 14.0. Unless indicated, all experiments were performed at least three times with similar results. Differences were considered significant at P<0.05.Results1. H₂O₂ precondition causes expression of NF-κBAnalysis of western blot indicated oxidative stress and H₂O₂ precondition induced expression of NF-κB in PC 12 cells. Moreover inhibitor of NF-κB, N-tosyl-L-phenylalanine chloromethyl ketone （TPCK） reduced the expression of NF-κB induced by H₂O₂ precondition.2. H₂O₂ precondition results in nuclear translocation of NF-κBImmunocytochemical staining showed that H₂O₂ precondition significantly promote nuclear translocation of NF-κB.3. H₂O₂ precondition enhances NF-κB DNA binding activityAnalysis of EMSA showed that H₂O₂ precondition enhance the amount of nuclear protein and DNA complex, which indicated that H₂O₂ precondition caused increasing in NF-κB DNA binding activity in PC12 cells.4. NF-κB mediates adaptive cytoprotection induced by H₂O₂ precondition①TPCK antagonize relieving in cell damages induced by H₂O₂ precondition: measurement of MTT showed that H₂O₂ precondition resist reducing in viability induced by high dose H₂O₂ in PC 12 cells, and TPCK administered before H₂O₂ precondition significantly decreased the viability of cell. Assessment of LDH in cell culture medium showed that H₂O₂ precondition decrease release of LDH induced by high dose H₂O₂ in PC 12 cells, and TPCK administered before H₂O₂ precondition significantly increased the release of LDH in PC 12 cell.②TPCK antagonize relieving in cell apoptosis induced by H₂O₂ precondition: Hoechst 33258 staining showed that H₂O₂ precondition reduce numbers of apoptic cell, but TPCK administered before H₂O₂ precondition significantly increase numbers of apoptic cell. Analysis of FCM indicated that H₂O₂ precondition reduce increasing in rate of apoptosis induced by high dose, TPCK administered before H₂O₂ precondition significantly increased rate of apoptosis in PC 12 cell.5. NF-κB affects apoptosis in caspase-dependant manner H₂O₂ precondition significantly decreased the activity of caspase-3 induced by high dose H₂O₂.TPCK administered before H₂O₂ precondition obviously increased the activity of caspase-3.6. H₂O₂ precondition induces expression of heat shock protein （HSP） 70 and 90 via NF-κB pathwayH₂O₂ precondition up-regulated the expression of HSP 70 and HSP 90 induced by 300μM H₂O₂, and TPCK administered before H₂O₂ precondition inhibited the expression of HSP 70 and HSP 90.Conclusion1. Oxidative stress and H₂O₂ precondition induced overexpression of NF-κB, and H₂O₂ precondition enhance nuclear translocation and transcriptional activity of NF-κB.2. Expression and activation of NF-κB participate in adaptive cytoprotection induced by H₂O₂ precondition. The mechanisms of anti-apoptosis of H₂O₂ precondition involved in caspase-dependant pathway.3. HSP 70 and 90 served as effective protein after NF-κB triger transcription, and may be one of mechanism in H₂O₂ precondition induced-cytoprotection.Part two Role of PI3K/Akt signal pathway in H₂O₂ preconditioning-induced adaptive cytoprotectionObjectiveTo discover whether PI3K/Akt signal pathway participate in H₂O₂ precondition induced-cytoprotection.Methods1. Estimation of damages on cells: MTT was used to detect the viability of PC12 cell, and the cytotoxicity was quantitatively assessed by measuring the amount of lactate dehydrogenase （LDH） released from damaged cells into the cell culture medium.2. Assessment of cell apoptosis: morphological changes were determined by using Hoechst 33258 staining, and flow cytometry （FCM） analysis was used to measure the rate of apoptosis.3. Activity of caspase-3 was measured by colorimetry.4. Western blot analysis was used to measure the expression of protein.5. All data are expressed as mean + SD. The differences between groups were evaluated by one-way analyses of variance （ANOVA） using SPSS 14.0. Unless indicated, all experiments were performed at least three times with similar results. Differences were considered significant at P<0.05.Results1. H₂O₂ precondition induces expression of p-akt in a PI3K-dependant manner Analysis of western blot indicated H₂O₂ precondition induced expression of p-akt in PC 12 cells. Inhibitor of PI3K, ly294002 reduced the expression of p-akt induced by H₂O₂ precondition.2. PI3K/Akt pathway mediates adaptive cytoprotection induced by H₂O₂ precondition①Ly294002 antagonize relieving in cell damages induced by H₂O₂ precondition: measurement of MTT showed that H₂O₂ precondition resist decreasing in viability induced by high dose H₂O₂ in PC 12 cells, and ly294002 administered before H₂O₂ precondition significantly reduced the viability of cell. Assessment of LDH in cell culture medium showed that H₂O₂ precondition decrease release of LDH induced by high dose H₂O₂ in PC 12 cells, and ly294002 administered before H₂O₂ precondition significantly increased the release of LDH in PC 12 cell.②Ly294002 antagonize relieving in cell apoptosis induced by H₂O₂ precondition: Hoechst 33258 staining showed that H₂O₂ precondition reduce numbers of apoptic cell, but ly294002 administered before H₂O₂ precondition significantly increase numbers of apoptotic cell. Analysis of FCM indicated that H₂O₂ precondition reduce increasing in rate of apoptosis induced by high dose, ly294002 administered before H₂O₂ precondition significantly increased rate of apoptosis in PC 12 cell.3. PI3K/Akt pathway affects apoptosis in a caspase-dependant manner H₂O₂ precondition significantly decreased the activity of caspase-3 induced by high dose H₂O₂. Ly294002 administered before H₂O₂ precondition obviously increased the activity of caspase-3.4. H₂O₂ precondition induces expression of heat shock protein （HSP） 70 and 90 via PI3K/Akt pathway H₂O₂ precondition up-regulated the expression of HSP 70 and HSP 90 induced by 300μM H₂O₂, ly294002 administered before H₂O₂ precondition inhibited the expression of HSP 70 and HSP 90.5. H₂O₂ precondition activates NF-кB pathway via PI3K/Akt pathway It is showed that ly294002, inhibtor of PI3K block the activation of NF-кB induced by H₂O₂, which indicated that PI3K/Akt pathway lies in upstream of NF-кB in H₂O₂ precondition-induced adaptive cytoprotection.Conclusion 1. H₂O₂ precondition induced activation of Akt via PI3K-dependant pathway.2. Activation of PI3K/Akt pathway participated in adaptive cytoprotection induced by H₂O₂ precondition. The mechanisms of anti-apoptosis of H₂O₂ precondition involved in caspase-dependant pathway.3. HSP 70 and 90 served as effective protein after activation of PI3K/Akt triger transcription, and may be one of mechanisms in H₂O₂ precondition induced-cytoprotection.4. PI3K/Akt pathway lies in upstream of NF-кB in H₂O₂ precondition-induced adaptive cytoprotection.Our study demonstrated that both PI3K/Ak and NF-κB signal pathway mediate H₂O₂ precondition-induced adaptive cytoprotection. H₂O₂ precondition activates Akt in PI3K-dependant manner, and activation of Akt induce activate of NF-κB signal pathway, which antagonize apoptosis via inhibition caspase-3 activity. Simultaneously NF-κB signal pathway promote expression of some gene related with stress and synthesize protective protein HSP 90 and HSP 70, which confer cell ability to resist subsequent serous injury.更多还原