【作者】 叶俊丽；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 生药学， 2010， 博士

【摘要】 氧化应激参与神经退行性疾病和神经元缺血损伤的发生发展。活性氧(Reactive oxygen species, ROS)引起神经细胞损伤的同时,细胞内会启动相应抗损伤应激反应。热休克蛋白70家族(heat shock protein70, HSP70)是多种应激原诱导细胞产生的一组保守性蛋白,HSP70(HSP72)和葡萄糖调节蛋白78(glucose-regulated protein 78, GRP78)是分别位于胞浆和内质网的HSP70家族成员。研究表明,HSP70(HSP72)除作为蛋白质分子伴侣外,还可直接抗凋亡和抗DNA损伤。丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)和生长抑制DNA损伤诱导蛋白(growth arrest and DNA damage inducible protein, GADD)是可被ROS激活并分别与细胞凋亡和DNA损伤关系密切的应激因子,神经细胞氧化应激损伤中HSP70的保护作用是否与MAPK和GADD有关尚不明确。GRP78是内质网应激反应(Endoplasmic Reticular Stress, ERS)的分子伴侣标志,过强过久ERS也可启动细胞凋亡。以GRP78表达增加为标志的ERS如何介导氧化应激损伤尚需探讨。抗氧化剂可拮抗神经细胞氧化损伤,目前关于海洋活性物质抗神经细胞氧化损伤的研究甚少。课题组前期研究表明,从海洋生物栉孔扇贝(Chlamys farreri)中提取的活性物质——扇贝多肽(polypeptide from Chlamys farreri, PCF)具有抗氧化作用,在大鼠大脑中动脉缺血模型中发挥神经保护作用,但其分子机制还不明确。基于以上背景,本研究采用MTT、ESR、透射电镜、荧光染色、Western blot、RT-PCR、酶化学和流式细胞术等研究方法,针对HSP70、GRP78介导神经细胞氧化应激和PCF的作用分三部分进行如下研究：首先,建立过氧化氢(H202)致人成神经母细胞瘤细胞(SH-SY5Y)氧化应激损伤模型,检测损伤后不同时间HSP70、MAPK、GADD153和GADD45β的表达；HSP70表达诱导剂GGA或抑制剂quercetin预孵育细胞后再行损伤,观察MAPK和GADD的表达变化,研究HSP70在H202所致SH-SY5Y细胞氧化应激损伤中的作用。其次,观察内质网超微结构、GRP78/Bip及GADD153/CHOP蛋白表达,明确H2O2是否能引起ERS。ROS清除剂N-乙酰半胱氨酸和ERS抑制剂4-PBA分别预处理细胞再进行损伤,检测细胞活性、细胞凋亡、细胞内ROS水平和内质网途径相关凋亡调控分子GRP78、CHOP、JNK和Bcl-2的表达,探讨ERS在氧化损伤致神经元凋亡中的作用。(3)观察细胞活性、ROS、脂质过氧化、DNA氧化及抗氧化酶活性,探讨PCF的抗氧化特性；检测MAPK、GRP78、CHOP/GADD153、HSP70及Bcl-2表达,研究PCF的抗凋亡分子靶点。研究结果如下：①对正常对照组相比,H2O2模型组细胞活性降低,胞内ROS水平、HSP70、GADD153/CHOP、GADD45β表达及ERK1/2和JNK磷酸化水平增加,上述效应呈时间依赖性；与H2O2模型组相比,GGA诱导HSP70高表达组细胞活性升高,DNA损伤和细胞凋亡减轻,ERK1/2、JNK磷酸化及GADD153表达减弱,GADD45P表达增强；②对正常对照组相比,H2O2模型组细胞内质网扩张脱颗粒,GRP78和CHOP表达增加,其表达呈时间依赖性；与H2O2模型组相比,NAC和4-PBA预处理组细胞活性增加,细胞凋亡减少,GRP78和CHOP表达减少,JNK活化减弱,Bcl-2表达增加；③与H2O2模型组相比,PCF预处理能提高损伤细胞活性,减少ROS和细胞凋亡,抑制脂质过氧化及DNA氧化损伤,提高GSH、SOD、CAT、GPx活性和细胞总抗氧化能力,抑制JNK磷酸化及GRP78和CHOP表达,上调HSP70和Bcl-2表达。以上结果表明：①神经细胞氧化损伤后HSP70介导的抗凋亡抗DNA损伤作用与抑制ERK1/2和JNK活化、调控GADD45β和GADD153基因表达有关；②氧化应激可诱导ERS,GRP78/CHOP/JNK/Bcl-2参与H2O2经内质网途径激活的细胞凋亡；③PCF的神经保护作用与其抗氧化特性和抗凋亡有关,PCF可上调HSP70和抑制ERS, CHOP/JNK/Bcl-2是其抗凋亡分子靶点。本研究初步探讨了HSP70家族介导神经细胞氧化应激的分子机制和PCF的神经保护作用。创新之处有：①ESR技术检测SH-SY5Y细胞损伤后ROS的经时变化；②GADD45β和GADD153是HSP70参与神经细胞DNA损伤修复的靶向下游分子；③HSP70/ERS/CHOP/JNK/Bcl-2是海洋抗氧化剂PCF神经保护作用的分子靶点。研究有待继续深入的方面有：①构建特异的HSP70高表达和沉默细胞株研究其功能；②HSP70与MAPK、GADD的直接相互作用或间接调控机制；③H202经ERS致细胞凋亡的下游途径；④PCF药代动力学和对原代神经元的抗凋亡分子机制。更多还原

【Abstract】 Oxidative stress has long been linked to cell death in many neurodegenerative conditions. Oxidative damages mediated by reactive oxygen species (ROS) could trigger cellular stress responses at the same time. Heat shock protein70 (HSP70) is a highly conserved stress proteins family expressed under pathological conditions. HSP70(HSP72) and glucose-regulated protein 78 (GRP78) are two HSP70 members respectively located in cytoplasm and endoplasmic reticulum. HSP70 (HSP72) mainly function as protein chaperones and some studies have shown its anti-apoptotic and anti-DNA damage effects. Mitogen-activated protein kinase (MAPK) and growth arrest and DNA damage inducible protein (GADD) are two stress response families which could be activated by ROS and highly related to cell apoptosis and DNA damage, however, whether the function of HSP70 in protection neurons against oxidative stress is related to MAPK and GADD is still unknown. GRP78 is the molecular chaperone mark of endoplasmic reticular stress (ERS) and severe ERS could initiate apoptosis. Whether and how ERS characterized by increased expression of GRP78 participated in the neuronal injury mediated by oxidatve stress need further study. Treatment with antioxidants is a promising approach for inhibiting neuronal oxidative damage. Owing to severe adverse effects, many synthetic antioxidants is restricted to clinical usage, therefore, searching for natural antioxidants with neuroprotective potential may provide new insight into therapeutic strategies. Until now the antioxidative effects of marine active products are rarely studied. The previous studies have demonstrated the neuroprotective effect of polypeptide from Chlamys farreri (PCF), a natural marine antioxidant, in an rat middle cerebral artery occlusion (MCAO) and reperfusion model and the molecular mechanisms need to verify. In the present study, MTT, electronspinresonance (ESR), transmission electric micscopy, fluorescence staining, western blot, RT-PCR, enzyme and biochemical assays are performed to investigate the role of HSP70 and GRP78 marked ERS in neuronal oxidative stress and the neuroprotective mechanisms of PCF. The study consist of three parts:first, the neuroblastoma SH-SY5Y cells are used as an in vitro model to established hydrogen peroxide (H2O2)-induced neuronal oxidative damage. After observing the time-course expression of HSP70, MAPK, GADD153 and GADD45βin H2O2-treated cells, the cells are pretreated with GGA (inducer of HSP70 expression) or quercetin (inhibitor of HSP70 expression) before H2O2-incubution and the difference in the expression of MAPK, GADD153 and GADD45βare assesed to explore the role of HSP70 in neuronal oxidative injury. Second, the H2O2-induced endoplasmic reticular ultrastructure changes and the expression of GRP78/Bip and GADD153/CHOP are studied to verify the ERS. Then NAC (ROS scavenger) and 4-PBA (an chemical chaperones and inhibitor of ERS) are pretreated to the medium before H2O2-incubution and cell viability, apoptosis, ROS production and the expression of GRP78, CHOP, JNK and are investigated to explore the role of ERS in neuronal oxidative stress. In the last part the antioxidative proterty of PCF on ROS production, lipid peroxidation, DNA oxidative damage and the activity of endogenous antioxidant defense components are assesed and the anti-apoptotic mechanisms of PCF on the expression of MAPK, GRP78, CHOP/GADD153, HSP70 and Bcl-2 are assesedThe results are as following:①More ROS production, increased expression of HSP70, GADD153/CHOPmRNA and GADD45βmRNA, activation of ERK1/2 and JNK accompanied by decreased cell viability in a time-dependent manner were observed in H2O2-treated cell compared to control non-treated cells; Compared to H2O2-alone-treated cells, GGA pretreatment induced high levels of HSP70 expression with DNA oxidative damage, apoptosis, phosphorylations of ERK1/2 and JNK and the expression of GADD153mRNA inhibition;②Endoplasmic reticulum dilation and increased expression of GRP78 and CHOP in a time-dependent manner were observed in H2O2-alone-treated cells, while NAC and 4-PBA pretreated cells showed more cell viability, less apoptosis and decreased expression of GRP78 and CHOP, less activation of JNK and increased level of Bcl-2 than H2O2-alone-treated cells;③PCF treatment inhibited H2O2-induced cell death, ROS production, apoptosis, LPO and DNA damage, meanwhile promoted endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, glutathione and total antioxidative capacity. PCF significantly blocked H2O2-induced phosphorylation of c-Jun N-terminal kinase (JNK) of the MAPK family and the expression of GRP78 and CHOP. The levels of HSP70 and Bcl-2 were increased in PCF-pretreated cells.These results showed that:①The anti-apoptotic function and anti-DNA damage of HSP70 is related to the inhibition of phosphorylation of ERK1/2 and JNK and the regulation of GADD45βmRNA and GADD153 mRNA expression;②Oxidative stress could trigger ERS, GRP78/CHOP/JNK/Bcl-2 participated in SH-SY5Y apoptosis mediated by H2O2-induced ERS;③The antioxidative and anti-aopototic properties contribute to the neuroprotective effects of PCF. PCF could upregulate HSP70 and inhibit ERS and CHOP/JNK/Bcl-2 may be the downstream target of PCF.In conclusion, this study mainly focuse on the molecular role of HSP70 and GRP78 mediated stress responses in neuronal oxidative stress injury and the neuroprotective effects of PCF. This study provided useful information for developing effective antioxidant with neuroprotective function. The highlight of innovation of the study are:①Time-course changes of ROS in H2O2-induced SH-SY5Y cells were measured by ESR;②GADD45βand GADD153 are the downstream signal for HSP70 participating in neuronal oxidative DNA damage and repair;③H2O2 could activate ERS and lead to SH-SY5Y cell apoptosis by CHOP/JNK pathways;④The study have first provided evidence that the neuroprotective effect of marine antioxidant PCF were mediated, at least, through scavenging oxygen free radicals, reinforcement of endogenous antioxidant defense, prevention of oxidation of macromolecules, lipids, and DNA, upregulating HSP70, and blocking the ERS and activation of downstream CHOP/JNK1/2. PCF could inhibit JNK pathway and thus inhibit SH-SY5Y cell apoptosis. In the future, further study as following should be performed:①Special stable overexpression and downexpression of HSP70 neuronal cell model should be developed to overexpress or knockdown HSP70 then to investigate the functions of HSP70;②Interaction of HSP70 and MAPK and GADD need be further verified;③the molecular targets for ERS induced apoptosis mediated by H2O2, the pharmacokinetics of PCF in neurodegeneration therapies and the molecular mechanisms of PCF on the signaling pathways of ROS-mediated primary neuronal cell growth and apoptosis.更多还原