【作者】 王旭；

【导师】 张小强； 宋爱菊；

【作者基本信息】 东南大学 ， 公共卫生（专业学位）， 2019， 硕士

【摘要】 Tau蛋白作为一种微管相关蛋白,是微管骨架装配和稳定所必需的分子之一。正常脑中tau蛋白与微管蛋白结合促进其聚合形成微管;并具有维持微管稳定性,降低微管蛋白分子的解离,并诱导微管成束的作用。但tau蛋白的异常磷酸化却会降低与微管的结合能力,并丧失维持微管稳定性的作用。而且异常磷酸化的tau蛋白在细胞内的相互聚集将促进双螺旋样纤维（PHFs）的形成,进一步导致了神经元纤维缠结（neurofibrillary tangles,NFTs）的产生,引起神经元功能损伤。NFTs与细胞外β-淀粉样蛋白的异常沉积是阿尔兹海默症（Alzheimer’s disease,AD）的典型病理特征。原花青素是一种多酚类化合物,具有很强的抗氧化、抗炎、调节神经递质等生物学作用。目前发现,原花青素可有效改善β-淀粉样蛋白的异常沉积所造成的神经损伤,然而有关其对tau蛋白异常磷酸化的积极作用尚不明确。为了探讨原花青素对tau蛋白磷酸化的调控作用,本研究以Aβ_25-35诱导SH-SY5Y细胞的tau蛋白磷酸化,并给予不同浓度原花青素干预,通过检测SOD、MDA、tau蛋白表达水平探讨原花青素对调控tau蛋白磷酸化所发挥的积极作用。同时,通过给予原花青素或相应的抑制剂后检测GSK-3β和JNK/p38/ERK MAPK的蛋白表达水平以探究原花青素对tau蛋白磷酸化调控的可能途径,为进一步研究原花青素为阿尔兹海默症治疗提供新策略的可能性提供理论依据。第一部分原花青素对Aβ_25-35诱导的SH-SY5Y细胞的氧化应激及tau蛋白磷酸化的作用研究目的:以Aβ_25-35诱导SH-SY5Y细胞,建立阿尔茨海默病tau蛋白磷酸化模型,探索PC对Aβ_25-35介导的SH-SY5Y细胞tau蛋白磷酸化的作用。研究方法:1.给予不同浓度原花青素（0.1、0.5、1.0、2.5、5.0μg/mL）干预SH-SY5Y细胞,并设立对照组,应用噻唑蓝比色法对细胞存活率进行检测。2.以不同浓度Aβ_25-35（0.1、1.0、2.5、10.0、20.0μM）诱导损伤SH-SY5Y细胞,应用MTT法对细胞活力进行检测,选择适宜浓度的Aβ_25-35用于构建体外tau蛋白磷酸化模型。3.以1.0μM Aβ_25-35介导SH-SY5Y细胞,同时应用不同剂量浓度原花青素（0.1、1.0、2.5、5.0μg/mL）处理,MTT法测定细胞存活率。4.以1.0μM Aβ_25-35诱导SH-SY5Y细胞,并应用不同剂量浓度原花青素（0.1、1.0、2.5、5.0μg/mL）进行干预,硫代巴比妥酸法检测SH-SY5Y内MDA含量、WST-8法检测SH-SY5Y内总SOD活力、Western blot法检测p-tau（Ser396）与tau蛋白表达水平。研究结果:1.相比于对照组,不同浓度剂量原花青素对细胞活力的影响均无统计学意义（P>0.05）。2.相比于对照组,不同浓度剂量Aβ_25-35均可降低SH-SY5Y细胞活力（P<0.05）,其中1.0μM Aβ_25-35对细胞存活率的抑制作用最为显著。3.相比于Aβ_25-35诱导组,给予不同浓度剂量的原花青素（0.1、1.0、2.5、5.0μg/mL）干预,均可升高细胞活力（P<0.05）。4.相比于对照组,1.0μM Aβ_25-35可升高细胞内MDA含量（P<0.05）,减少细胞内总SOD活力（P<0.05）。与1.0μM Aβ_25-35组相比,5.0μg/mL的原花青素可减少细胞内MDA含量（P<0.05）,0.1、1.0、2.5、5.0μg/mL组均可提高SH-SY5Y细胞内总SOD活力（P<0.05）。5.相比于对照组,1.0μM Aβ_25-35组p-tau（Ser396）蛋白表达水平明显升高（P<0.05）,与1.0μM Aβ_25-35组相比,1.0、2.5、5.0μg/mL原花青素均可使SH-SY5Y细胞tau蛋白的磷酸化水平降低（P<0.05）。研究结论:研究剂量范围内的原花青素对SH-SY5Y细胞活力无显著作用,且可抑制Aβ_25-35诱导的SH-SY5Y细胞的tau蛋白磷酸化,提高细胞生存率,降低氧化应激水平。第二部分原花青素对Aβ_25-35诱导SH-SY5Y细胞的MAPK与GSK3β信号通路的影响研究目的:探索原花青素对Aβ_25-35诱导的SH-SY5Y细胞的MAPK与GSK3β通路的影响,应用MAPK与GSK3β各通路相应的特异性抑制剂干预细胞,观察MAPK与GSK3β各通路对Aβ_25-35诱导SH-SY5Y细胞tau蛋白磷酸化的影响。研究方法:1.以1.0μM Aβ_25-35诱导SH-SY5Y细胞,给予不同剂量原花青素（0.1、1.0、2.5、5.0μg/mL）进行干预,Western blot法测定p-p38、p38、p-ERK、ERK、p-JNK、JNK、p-GSK3β、GSK3β蛋白表达水平变化情况。2.设立对照组、抑制剂对照组、1.0μM Aβ_25-35组、SB203580+1.0μM Aβ_25-35组、SP600125+1.0μM Aβ_25-35组、U0126+1.0μM Aβ_25-35组、1.0μM Aβ_25-35+5.0μg/mL原花青素组,Western blot法检测p-p38、p38、p-ERK、ERK、p-JNK、JNK、p-GSK3β、GSK3β、p-tau、tau蛋白表达水平的变化。Image J对蛋白条带灰度值进行分析。研究结果:1.与对照组相比,1.0μM Aβ_25-35可提高SH-SY5Y细胞p-p38、p-ERK、p-JNK、p-GSK3β蛋白表达水平（P<0.05）;与1.0μM Aβ_25-35组相比,1.0、2.5、5.0μg/mL原花青素均可降低p38、ERK、JNK、GSK3β蛋白磷酸化水平（P<0.05）。2.与对照组相比,Aβ_25-35可诱导SH-SY5Y细胞表达p-p38、p-ERK、p-JNK、p-GSK3β、p-tau水平增加（P<0.05）,与1.0μM Aβ_25-3组相比,特异性通路抑制剂组可明显抑制Aβ_25-35诱导的相应蛋白磷酸化水平（P<0.05）,同时5.0μg/mL原花青素可抑制Aβ_25-35诱导的p38、ERK、JNK、GSK3β通路激活（P<0.05）。研究结论:Aβ_25-35可诱导SH-SY5Y细胞中tau蛋白磷酸化水平的改变,这一作用的发挥可能涉及p38、ERK、JNK、GSK3β通路的激活。原花青素在抑制Aβ_25-35介导的p38、ERK、JNK、GSK3β通路激活从而改善tau蛋白的磷酸化的过程中可能发挥了作用。更多还原

【Abstract】 As a microtubule-related protein,tau protein is one of the molecules necessary for microtubule skeleton assembly and stability.In normal brain,tau protein binds to microtubule protein and promotes its polymerization to form microtubules.In addition,it can maintain the stability of microtubules,reduce the dissociation of microtubule protein molecules,and induce microtubule bunching.However,abnormal phosphorylation of tau protein will reduce the ability to bind to microtubules and lose the function of maintaining microtubule stability.Moreover,the mutual aggregation of abnormal phosphorylated tau protein in the cell will promote the formation of double helical fibers（PHFs）,which further leads to the generation of neurofibrillary tangles（NFTs）and causes functional damage of neurons.Abnormal deposition of NFTs and extracellular-β-amyloid protein are typical pathological features of Alzheimer’s disease（AD）.Proanthocyanidins are polyphenols with strong biological effects of antioxidant,anti-inflammatory and neurotransmitter regulation.At present,proanthocyanidins have been found to effectively ameliorate nerve damage caused by abnormal deposition ofβ-amyloid.However,its positive effect on abnormal phosphorylation of tau protein remains unclear.To investigate the effect of proanthocyanidin on phosphorylation of tau protein,tau protein phosphorylation was induced by Aβ_25-355-35 in SH-SY5Y cells,and proanthocyanidin treatment was given to cells.The positive effect of proanthocyanidin on tau protein phosphorylation was explored by detecting the expression levels of SOD,MDA and tau protein.At the same time,after treatment of SH-SY5Y cells with proanthocyanidin or corresponding inhibitors,the protein expression levels of GSK-3βand JNK/p38/ERK MAPK were detected to explore the possible regulation of proanthocyanidins on tau protein phosphorylation.The study may provide a theoretical basis for further research on proanthocyanidin as a new strategy for AD treatment.Part Ⅰ:Effect of proanthocyanidin on oxidative stress and tau protein phosphorylation induced by Aβ_25-355-35 in SH-SY5Y cellsObjective:To study the protective effects of proanthocyanidin on tau protein phosphorylation in SH-SY5Y cells induced by Aβ_25-35,a vitro model of Alzheimer’s disease was produced and SH-SY5Y cells were treated with proanthocyanidin.Methods:1.Treatment of SH-SY5Y cells with different concentrations of proanthocyanidin（0.1,1.0,2.5,5.0μg/mL）,control group was set up,cell viability was determined by MTT assay.2.Treatment of SH-SY5Y cells with different concentrations of Aβ_25-35（0.1,1.0,2.5,10.0,20.0μM）,cell viability was determined by MTT assay,and a proper concentration of Aβ_25-355-35 was selected to build the model of tau protein phosphorylation in vitro.3.Treatment of SH-SY5Y cells with 1.0μM Aβ_25-35.Different concetrations of proanthocyanidin（0.1,0.5,1.0,2.5,5.0μg/mL）were added to cell cultures as intervention.Cell viability was detected by MTT assay.4.Treatment of SH-SY5Y cells with 1.0μM Aβ_25-35 and proanthocyanidin（0.1,1.0,2.5,5.0μg/mL）.The intracellular MDA and total SOD vitality were determined by the method of TBA measurement and WST-8 measurement respectively,The expression levels of p-tau（Ser396）and tau were detected by Western blotting.Results:1.Compared with the controls,the effects of different concentrations of proanthocyanidin on cell viability was not significant（P>0.05）.2.Compared with the controls,different concentrations of Aβ_25-35 declined cell viability（P<0.05）,and 1.0μM Aβ_25-35 declined cell viability obviously.3.Compared with Aβ_25-35 treated group,proanthocyanidin at different concentrations（0.1,1.0,2.5,5.0μg/mL）improved cell viability（P<0.05）.4.Compared with the controls,1.0μM Aβ_25-35 increased intracellular MDA（P<0.05）,and decreased total SOD vitality（P<0.05）.While compared with Aβ_25-35 injury cell group,5.0μg/mL proanthocyanidin decreased intracellular MDA（P<0.05）,and proanthocyanidin（1.0,2.5,5.0μg/mL）increased total SOD vitality（P<0.05）.5.Compared with the controls,1.0μM Aβ_25-355-35 induced the expression level of p-tau（Ser396）protein was significantly increased in SH-SY5Y cells.Compared with Aβ_25-355-35 treated group,proanthocyanidin（1.0,2.5,5.0μg/mL）decreased the phosphorylation level of tau protein in SH-SY5Y cells（P<0.05）.Conclusion:At the level of treated concentrations,proanthocyanidin has no effects on cell viability in SH-SY5Y cells,and can improve the vitality of cells,decreased the oxidative stress level and inhibit hyperphosphorylation of tau protein mediated by Aβ_25-35 in SH-SY5Y cells.PartⅡ:Mechanism of proanthocyanidins on MAPK and GSK3βsignaling pathways induced by Aβ_25-355-35 in SH-SY5Y cellsObjective:To investigate the effect of proanthocyanidin on MAPK and GSK3βsignaling pathways induced by Aβ_25-35 in SH-SY5Y cells,we use MAPK and GSK3βcorresponding specific blockers to intervene in cells.The cells were treated with specific blockers of MAPK and GSK3βpathways to explore the role of tau phosphorylation by Aβ_25-35 in SH-SY5Y cells.Methods:1.SH-SY5Y cells were treated with 1.0μM Aβ_25-355-35 and proanthocyanidin（0.1,1.0,2.5,5.0μg/mL）the expression of p-p38/p38,p-ERK/ERK,p-JNK/JNK and p-GSK3β/GSK3βwere examined by using Western blotting.2.The research groups were designed to Control group,inhibited control group,1.0μM Aβ_25-35 group,SB203580+1.0μM Aβ_25-35 group,SP600125+1.0μM Aβ_25-35 group,U0126+1.0μM Aβ_25-35 group and 1.0μM Aβ_25-35+5.0μg/mL PC group.Western Blot assay was used to determine the changes of p38,p-p38,JNK,p-JNK,ERK,p-ERK,p-tau,tau,GSK3βand p-GSK3βproteins in each group.Image J was used to analyze the gray value of the protein band.Results:1.Compared with the control group,1.0μM Aβ_25-35 increased expression level of p-p38,p-ERK,p-JNK and p-GSK3β（P<0.05）.Compared with 1.0μM Aβ_25-35 group,proanthocyanidin（1.0,2.5,5.0μg/mL）decreased the expression of phosphorylation of p38,ERK,JNK and GSK3β（P<0.05）.2.Compared with the control group,1.0μM Aβ_25-35 increased the expression of p-p38,p-ERK,p-JNK,p-GSK3βand p-tau in SH-SY5Y cells（P<0.05）.Compared with cells treated with 1.0μM Aβ_25-35,the specific pathway inhibitor group could significantly inhibit the phosphorylation level of the corresponding protein induced by Aβ_25-35（P<0.05）,and 5.0μg/mL proanthocyanidin could inhibit the activation of the p38,ERK,JNK,GSK3βpathway induced by Aβ_25-35（P<0.05）.Conclusion:Aβ_25-35 can stimulate the phosphorylation levels of tau protein in SH-SY5Y cells,and activation of p38,ERK,JNK,and GSK3βpathways may be involved in this effect.Proanthocyanidins may improve tau phosphorylation by inhibiting the activation of p38,ERK,JNK,and GSK3βpathways induced by Aβ_25-35.更多还原