【作者】 刘超；

【导师】 王少辉；

【作者基本信息】 华中师范大学 ， 动物学， 2018， 硕士

【摘要】 三(β-氯乙基)磷酸酯(Tris(β-chloroethyl)phosphate,简称TCEP)因具有良好的阻燃兼增塑性能,广泛用于各种生活制品中,如家具、电子产品、食品包装以及建筑材料等,有效提高产品的安全性能。TCEP作为添加剂与材料以非共价键方式结合,经磨损和挥发进入周围环境,再通过呼吸和饮食等方式进入机体并在生物体内富集。已有研究表明,TCEP具有一定的致癌性,可导致氧化应激、内分泌功能失调以及生殖和发育异常等。此外,还有研究发现大鼠长时间暴露于高剂量TCEP后,大脑海马CA1区神经元缺失。然而,海马与学习记忆功能密切相关,TCEP对学习与记忆的影响及其机制有待进一步研究。本研究以雄性昆明小鼠为对象,探讨TCEP对小鼠学习与记忆功能的影响及其潜在机制。我们首先每天通过灌胃给予小鼠不同剂量(75 mg/kg或150 mg/kg)TCEP处理 3 周,即75 mg/kg TCEP 组和 150 mg/kg TCEP 组;或给予 150 mg/kg TCEP 处理1周后再给予20 mg/kg TubastatinA(简称TBSA)共同处理2周,即TCEP+TBSA组;同时,对照组给予这两种药物的溶剂处理。药物处理之后,我们再利用行为学实验,如Morris水迷宫、新异物体识别实验和电跳台实验检测TCEP及TBSA对小鼠学习与记忆能力的影响,并进一步通过免疫组化和免疫印迹探究其潜在分子机制。结果发现:(1)75 mg/kg TCEP、150 mg/kgTCEP 及 20 mg/kg TBSA 处理后,小鼠未出现急性反应且各组间小鼠平均体重无明显差异。(2)在Morris水迷宫中:定位航行实验阶段,与对照组相比,75 mg/kg TCEP组的逃避潜伏期明显延长(第2天,p<0.05;第5天,p<0.05);150mg/kg TCEP组的逃避潜伏期明显延长(第4天,p<0.05;第5天,p<0.001);TCEP+TBSA处理组较150mg/kg TCEP组,逃避潜伏期明显缩短(第4天,p<0.01;第5天,p<0.05)。在空间探索实验阶段:与对照组相比,150 mg/kg TCEP组的目标象限停留时间(p<0.05)和穿越平台次数(p<0.05)均明显降低;而TCEP+TBSA处理组较150mg/kg TCEP组的目标象限停留时间(p<0.05)和穿越平台次数(p<0.05)均明显增加;两TCEP处理组小鼠寻找隐藏平台的游泳轨迹无目的性,杂乱。由此,表明TCEP诱导小鼠空间学习与记忆发生障碍。(3)新异物体识别实验:与对照组相比,150mg/kg TCEP组探索总时间稍有增加(p<0.05);与对照组相比,TCEP组的探索新异物体的时间均显著减少(75 mg/kg TCEP 组,p<0.05;150 mg/kg TCEP 组,p<0.05),表明 TCEP 处理引起小鼠新异识别记忆异常。(4)电跳台实验:与对照组相比,两TCEP处理组跳台潜伏期均显著降低(75mg/kg TCEP组,p<0.05;150mg/kg TCEP组,p<0.05),同时错误次数均显著增加(p<0.05),表明TCEP诱导小鼠情绪性记忆下降。(5)我们先通过免疫印迹检测TCEP处理对Akt/GSK-3β信号通路的影响,发现这两种总蛋白无明显差异,但其磷酸化水平有差异。两TCEP组的p-Akt(Ser 473)均明显降低(75 mg/kg TCEP 组,p<0.05;150 mg/kg TCEP 组,p<0.001),以及 150mg/kg TCEP组的p-GSK-3β(Ser9)明显降低(p<0.05),表明TCEP处理可通过磷酸化作用调控Akt/GSK-3β信号通路。(6)不同处理组的HDAC6蛋白水平无明显差异,进一步检测对HDAC6下游底物微管蛋白(α-tubulin)的乙酰化程度影响。与对照组相比,150 mg/kg TCEP 组的 α-tubulin 水平显著降低(p<0.05),而 TCEP+TBSA 组较150 mg/kg TCEP组明显增加。我们再通过免疫组化观察海马CA1区锥体细胞内的轴突微管,观察到对照组微管较长且整齐;75 mg/kgTCEP组的微管较少、缩短且杂乱,150 mg/kg TCEP组的微管解聚呈点状,且TCEP+TBSA处理组较150 mg/kg TCEP组的微管解聚情况有所改善,提示TCEP处理导致微管解聚、缩短甚至崩解。(7)与记忆密切相关的突触蛋白表达:与对照组相比,两TCEP组的Synapsin-1蛋白水平明显降低(75 mg/kg TCEP 组,p<0.05;150 mg/kg TCEP 组,p<0.05);150 mg/kg TCEP 组的 PSD-95 明显降低(p<0.05);且 TCEP+TBSA 组较 150 mg/kg TCEP组有所增加。我们进一步通过免疫组化观察上述两种蛋白质在海马区域的表达情况,与对照组相比,两TCEP组的Synapsin-1在CA1和CA3染色均变浅;且PSD-95在CA3区染色变浅,进一步说明TCEP处理引起海马内此类蛋白水平降低,且TCEP+TBSA组较150mg/kg TCEP组的染色均加深,表明该区的蛋白表达增加。以上结果表明:TCEP处理诱导小鼠空间学习与记忆、新异识别记忆及情绪性记忆功能异常,而TBSA处理可显著改善小鼠上述行为学障碍。Akt/GSK-3β信号通路、HDAC6、微管稳定性、突触蛋白在TCEP诱导的认知障碍中发挥潜在作用。本研究期望为TCEP的环境预警策略提供新信息,以及TCEP的神经毒性尤其是对中枢神经系统相关的学习与记忆功能的损伤提供一定实验依据,并进一步为Tubastatin A在此类治疗中的应用提高参考。更多还原

【Abstract】 Tris(P-chloroethyl)phosphate(TCEP)is an excellect flame retardant and plasticizer.To effectively improve the safety performance in products,TCEP is widely used in various living products,such as furniture,electronic products,food packaging bags and building materials,etc.As an additive,because of the weaker combination of non-covalent manner with material,it can enter into the environment by abrasion and volatilization and has a certain environmental persistence.It can be intaken into body through breathing,diet and other ways,then enriches in the organisms.Studies have shown that TCEP had the potential toxicity and induce oxidative stress,endocrine disruption,and had certain carcinogenic,reproductive and developmental toxicity.In addition,some evidences showed that when the rats were exposed to high doses of TCEP for a long time,the neurons were loss in CA1 area of the hippocampus.The hippocampus is closely related to learning and memory function.However,the effect of TCEP on learning and memory and its mechanism is not clearly.In this study,we choosen male Kunming mice as experimental subjects through gastric infusion to study effect of TCEP on learning and memory ability.Firstly,mice anderwent two different dose TCEP treatment for consecutive three weeks,75mg/kg TCEP group and 150mg/kg TCEP group respectively;TCEP+TBSA group was given 20mg/kg Tubastatin A(TBSA)cotreatment after 150mg/kg TCEP treatment for one week;The control group was given solvent treatment of the two drugs.Then we used Morris water maze,Novel object recognition and Electrical step-down to detect the the effects of TCEP on learning and memory abilities.We further test the potential molecular mechanism by Western blotting and Immunohistochemistry.The results showed that:(1)After the treatment at a dose of 75 mg/kg,150 mg/kg TCEP and 20 mg/kg TBSA,there was no acute reaction and significant change in body weight among the four groups.(2)In the Morris water maze test:the escape latency was prolonged at 75 m/kg TCEP group compared with the control group(p<0.05 for day 2;p<0.05 for day 5);and 150 mg/kg TCEP group was significant prolonged(p<0.05 for day 4;p<0.001 for day 5);the escape latency was significantly shorter(p<0.01 for day 4;P<0.05 for day 5)at TCEP+TBSA group compared with 150mg/kg TCEP group.In the space exploration phase,the time spent in the target quadrant and number of platform area crossing at 150 mg/kg TCEP group were significantly reduced(p<0.05,respectively)compared with the control group.Swimming trajectories for looking for hidden platform at the two different dose TCEP treatment groups are aimless and messy.(3)In the novel object recognition test:the total exploration time was slightly increased at 150 mg/kg TCEP group compared with the control group(p<0.05);the time exploring the novel object was significantly decreased at 75 mg/kg TCEP group and 150 mg/kg TCEP groups compared with the control group(p<0.05 respectively).(4)In Eelectrical step-down test:Compared with the control group,the step down latency were significantly lower at 75 mg/kg TCEP group and 150 mg/kg TCEP groups(p<0.05 respectively),while the error times increased significantly(p<0.05 for day 2-5 respectively).(5)We first examined the effect of Akt/GSK-3βsignaling pathway by immunohistochemistry and found that there was no change in the each total proteins,but their phosphorylation levels had some differences.Compared with the control group,the p-Akt(Ser 473)was significantly lower in these two TCEP groups(75 mg/kg TCEP group,p<0.05;150 mg/kg TCEP group,p<0.001)and p-GSK-3(3(Ser 9)was significantly decreased at 150 mg/kg TCEP group(p<0.05),and indicating that TCEP regulates Akt/GSK-3β pathway through phosphorylation.(6)There was no significant difference in HDAC6 protein level among the four groups.However,the protein level of ac-tubulin,a deacetylated substrate of HDAC6,was significantly decreased in the mice hippocampus at 150 mg/kg TCEP group(p<0.05)compared with the control group.We further examined the microtubules of neuronal axonal in hippocampal CA1 pyramidal neurons by immunohistochemistry,and revealed that the microtubules at the control group were long and tidy;and 75 mg/kg TCEP group were fewer,shorter,and cluttered;150 mg/kg TCEP group were short and dotted,and suggesting that the TCEP treatment resulted in the depolymerization of microtubules to being shortened,and even disintegratied.(7)Synaptic protein expression closely related to memory in the hippocampus:compared with the control group,Synapsin-1 was significantly decreased(75 mg/kg TCEP group,p<0.05;150 mg/kg TCEP group,p<0.05);PSD-95 was significantly decreased at 150 mg/kg TCEP group(p<0.05).We further observed the protein-rich area in the hippocampus by immunohistochemistry.Compared with the control group,Synapsin-1 had lighter staining in CA1 and CA3 area at 75 mg/kg TCEP group and 150mg/kg TCEP group,and PSD-95 showed lighter staining in CA3 region,indicating that the protein expression in the region is less after TCEP treatment.The staining was deeper at TCEP+TBSA group compared with 150 mg/kg TCEP group,indicating that these proteins expression increased in this area.The above results indicate that TCEP treatment can impaired spatial learning and memory,novel recognition memory and emotional memory in mice.The TBSA treatment significantly ameliorate the memory impairment by TCEP in the above behavioral changes.Further biochemical date suggested Akt/GSK-3β pathway,HDAC6,microtubule stability and synaptic proteins play a potential role in cognitive impairment induced by TCEP.This study provides a certain experimental basis for the impairment of learning and memory related to central nervous system induced by TCEP,and offer the references for the application of Tubastatin A in preventing the congitive disorders caused by TCEP.更多还原