【作者】 袁华；

【导师】 赵秀兰；

【作者基本信息】 山东大学 ， 劳动卫生与环境卫生学， 2014， 硕士

【摘要】 研究目的1-溴丙烷(1-bromopropane,1-BP),又名溴丙烷、正丙基溴等,由于其化学性质稳定、易挥发、不易燃烧、在大气中的半衰期短,对臭氧层的破坏较小等特点作为臭氧层破坏物质的替代剂,被国内外广泛应用。随着1-BP的广泛应用,职业接触人群不断增多,职业中毒也随之出现。流行病学调查和实验室研究显示1-BP具有神经毒性,相对周围神经而言,中枢神经系统(Central nervous.system,CNS)对1-BP的毒性更加敏感。目前1-BP中毒机制的研究还未完全阐明,临床上亦缺乏特效治疗药物,因此研究1-BP致CNS损伤的中毒机制,对有效预防和治疗1-BP中毒有重要的意义。二十二碳六烯酸(Docosahexaenoic acid, DHA)是人体必不可少的n-3多不饱和脂肪酸,主要来自鱼油和微藻油。DHA本身的结构特点使其能够调节机体对氧化应激的生理反应,降低氧化应激损伤。研究表明DHA能够促进神经发育,具有神经保护作用。新近的研究证实,DHA有较强的抗炎作用。神经酸(Nervonic acid, NA)是一种长链单不饱和脂肪酸,主要来源于鲨鱼脑及鲨鱼油,是大脑白质中细胞膜的重要组成成分,能有效地促进神经组织的修复和再生,防止大脑神经衰老。NA具有较强的抗氧化能力,能增加SOD活性,降低MDA含量。研究显示NA对帕金森、老年痴呆等疾病也具有一定的治疗作用。本实验中,大鼠经口染毒1-BP,采用Morris水迷宫试验评价1-BP对大鼠学习记忆能力的影响；并分别给予DHA和NA干预,观察DHA和NA对1-BP导致的CNS损伤的拮抗作用。通过检测动物大脑皮层的氧化应激状态、星形胶质细胞激活情况、核因子-κB (nucler factor kappa B, NF-κB)、NF-E2相关因子2(transcription factor NF-E2-related factor-2, Nrf2)蛋白的表达初步探讨1-BP对CNS损伤的机制。研究方法1DH[A和NA对1-BP导致中枢神经系统损伤的拮抗作用40只雄性Wistar大鼠适应性喂养5d后,随机分为对照组、1-BP染毒组、DHA干预组和NA干预组,每组10只。1-BP染毒组、DHA干预组、NA干预组每天经灌胃给予800mg/kg.bw1-BP,对照组给予等体积玉米油。在给予1-BP4小时后,分别经口给予500mg/kg.bw DHA和150mg/kg.bw NA,连续12d。给予相应受试物的第8d,采用Morris水迷宫检测大鼠学习记忆能力,持续5d。水迷宫结束后次日断头处死大鼠,迅速取出大脑皮层和海马。生化法检测大脑皮层中还原型谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)的含量,谷胱甘肽还原酶(glutathione reductase, GR)和γ-谷氨酰半胱氨酸连接酶(y-Glutamate cysteine ligase, y-GCL)的活性。2DHA对1-BP导致中枢神经系统损伤的拮抗机制雄性Wistar大鼠48只,适应性喂养5d后,随机分为对照组、1-BP染毒组、低剂量DHA干预组和高剂量DHA干预组,每组12只。低剂量DHA干预组和高剂量DHA干预组分别经灌胃250mg/kg.bw、500mg/kg.bw的DHA,其余各组给予等体积玉米油,连续7d。在给予DHA的第8d,1-BP组、低剂量DHA干预组、高剂量DHA干预组分别经灌胃给予800mg/kg.bw1-BP,对照组给予等体积的玉米油。给予1-BP的第8d,采用Morris水迷宫检测大鼠学习能力。连续4d,期间继续按照原剂量给予相应受试物。水迷宫结束后,立即处死动物,分离出大脑皮层进行各指标测定。生化法检测大脑皮层中GSH、MDA的含量,免疫荧光法检测大鼠大脑皮层中胶质原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)观察星形胶质细胞的激活情况,采用western blotting检测动物大脑皮层胞浆和胞核中NF-κB、Nrf2蛋白的表达,观察其活化的变化。研究结果1DHA和NA对1-BP导致中枢神经系统损伤的拮抗作用1.1Morris水迷宫实验结果Morris水迷宫结果显示,与对照组相比,1-BP染毒组的游泳总路程和逃避潜伏期明显延长(P<0.01,P<0.05),穿越平台次数及平台周边时间／总时间明显降低(P<0.05),提示1-BP能够明显损伤实验动物的学习记忆能力。与1-BP染毒组相比,补充NA和DHA均可使大鼠的逃避潜伏期和游泳总路程明显缩短(P<0.05)；补充DHA后,动物的穿越平台次数和平台周边时间明显增加(P<0.05),NA干预组的穿越平台次数明显增加(P<0.05)；提示补充NA和DHA分别在一定程度上能够拮抗1-BP对CNS的损伤。1.2实验动物大脑皮层GSH, MDA含量和GR、γ-GCL活性与正常对照组相比,1-BP染毒组大鼠GSH含量下降了18.1%,DHA和NA能升高动物大脑皮层GSH的含量,DHA干预组与1-BP组差异具有统计学意义(P<0.05)。大鼠暴露1-BP后,MDA含量较对照组增加了14.3%,补充NA和DHA后,MDA含量明显降低(P<0.05)。与对照组相比,1-BP染毒组的GR酶活力下降,DHA干预组活力明显高于1-BP染毒组,差异具有统计学意义(P<0.05)。与1-BP染毒组相比,NA染毒组的y-GCL活力明显升高(P<0.05)。1DHA对1-BP致中枢神经系统损伤的拮抗机制通过以上实验,我们观察到DHA对1-BP导致的CNS损伤具有明显的保护作用。因此,另取一批动物观察了不同剂量DHA对CNS的保护作用,并初步探讨其作用机制。1.1Morris水迷宫实验结果定位导航实验中,1-BP染毒组动物的游泳总路程和逃避潜伏期比对照组明显延长(P<0.05),与1-BP染毒组相比,低剂量DHA干预组和高剂量DHA干预组均能明显缩短游泳总路程和逃避潜伏期(P<0.01)。2.2实验动物大脑皮层中GSH, MDA含量1-BP染毒组GSH含量明显降低,与对照组相比,1-BP染毒组的GSH含量下降了48.1%(P<0.01),与1-BP染毒组相比,低剂量DHA干预组和高剂量DHA干预组大脑皮层GSH含量分别升高了59.9%(P<0.01)、81.9%(P<0.01)。与对照组相比,1-BP染毒组动物MDA含量增加了27.0%(P<0.01),低剂量DHA干预组和高剂量DHA干预组的MDA含量分别降低了19.5%(P<0.01)、29.1%(P<0.01)。2.3实验动物大脑皮层星形胶质细胞的活化情况免疫荧光结果显示,正常对照组大鼠大脑中GFAP表达很低,1-BP染毒组大鼠大脑皮层中荧光强度升高明显,提示1-BP能够大量激活大脑的星形胶质细胞。低剂量DHA和高剂量DHA均能明显抑制1-BP导致的大脑星形胶质细胞的激活。实验同时采用Western blotting验证了大脑皮层GFAP的表达。检测结果与免疫荧光结果一致。与对照相比,1-BP大鼠皮层中GFAP蛋白表达显著升高(P<0.05),提前给予低剂量DHA和高剂量DHA干预后,与1-BP染毒组相比,大鼠大脑皮层中GFAP蛋白表达明显降低(P<0.05,P<0.01)。2.4实验动物大脑皮层中NF-κB、Nrf2蛋白的表达采用Western blotting分别检测了大脑皮层细胞质和细胞核中NF-κB、Nrf2蛋白的表达,观察1-BP对动物CNS中NF-κB、Nrf2的激活情况。结果显示,1-BP染毒能够明显升高细胞质和细胞核中的NF-κB。与1-BP染毒组相比,DHA干预组大鼠大脑皮层细胞质和细胞核中NF-κB显著降低(P<0.05),其中高剂量DHA干预组细胞质NF-κB的表达比1-BP染毒组降低了53.4%(P<0.05)。1-BP暴露亦能激活Nrf2,大脑皮层的胞质和胞核中Nrf2的表达有所增加,与对照组相比,差异有统计学意义(P<0.05)。与1-BP染毒组相比,低剂量DHA干预组和高剂量DHA干预组大脑皮层的胞质和胞核中Nrf2蛋白表达升高,与1-BP染毒组相比,差异具有统计学意义(P<0.05,P<0.01)。结论11-BP能够导致实验动物CNS损伤,使学习记忆能力明显下降。NA(150mg/kg.bw)和DHA (500mg/kg.bw)均能拮抗1-BP导致的CNS的损伤,DHA的效果优于NA。21-BP暴露使大脑GSH含量下降,GSH生成酶GR和y-GCL的活性明显降低。NA和DHA均可增强GR和y-GCL的活性,显著升高GSH,降低1-BP导致的氧化应激。31-BP对CNS的损伤可能与AS激活相关,DHA可能通过抑制NF-κB的活化、激活Nrf2通路减轻1-BP的CNS毒性。更多还原

【Abstract】 Objective1-Bromopropane (1-BP), also named brompropane. Due to its noninflammability, volatility, and lower ozone-depleting potency, it is now used as an alternative to ozone-depleting solvents. With the widely use of1-BP, the number of worker exposed occupationally to1-BP is increasing. The occupational poisoning also emerged. Epidemiological and laboratory studies indicate that the nervous systems are the one targets of the1-BP toxicity, the central nervous system (CNS) is demonstrated the more sensitive than the peripheral nervous system (PNS). However, the exactly underlying toxicology mechanisms of1-BP remain unclear, and there is no effective therapeutic remedy for1-BP so far as well. Therefore, the study addressed on the mechanisms of CNS and tried to provide some useful information for preventing and curing1-BP toxicityDocosahexaenoic acid (DHA) is an necessary n-3polyunsaturated fatty acids, mainly derived from fish oil and micro algae oil. DHA is able to influence the activity of cells, regluate physiological reaction to oxidative stress, and reduce the oxidative stress injury. Previous research indicated that DHA has neutoprotective effection, and DHA is improtant for the development of nervous system. Recently research showed that DHA has antioxidant and anti-inflammatory effect.1-BP induced toxicity of CNS is related to oxidative stress and inflammation. Therefore we hypothesis that DHA could exert protection for1-BP induced injury of nervous system.Nervonic acid is a kind of long chain monounsaturated fatty acids which mainly comes from shark brain and shark oil. NA is a major component of cell membrane of white matter, which can promote recovery of nervous system effectively, and prevent brain aging. Studies found that NA has an important role in treatment of Parkinson and alzheimer. With its strong antioxidant ability, NA could increase the activity of SOD, and decrease the level of MDA. We hypothesized that NA maybe has a certain degree protection for1-BP caused central nervous system injury.In order to choose the optimal intervention and to study the underlying mechanism, the rats were given corresponding test material by gavage, and the Morris water maze was applied to evaluate learning and memory of rats. The biochemical parameters were used to determine the function of DHA and NA. After intervention, we adopt Morris water maze to evalutate the intervention effect. We detected astrocytes activation, the expression of nucler factor-kappa B (NF-κB) and transcription factor NF-E2-related factor-2(Nrf2) to illustrate the mechanism of1-BP.Methods1DHA and NA against1-BP neurotoxicity on the central nervous system40Wistar male rats were randomly divided into4groups with10in each, i.e. control group,1-BP group, NA intervention group and DHA intervention group. The rats of1-BP group, NA intervention, and DHA intervention group received800mg/kg.bw1-BP by gavage, while the rats in control group received eauivalent corn oil. After4hours of giving1-BP, NA intervention group and DHA intervention group orally administered150mg/kg.bw NA and500mg/kg.bw DHA respectively, for consecutive12days. Rats in each group were given in accordance with their material for7days, and then Morris water maze was used to detect learning and memory ability of rats in each group. After the Morris water maze test, rats were sacrificed; the cerebral cortex and hippocampus were quickly dissected. The cerebral cortexes were homogenized, and levels of glutathione (GSH) and malondialdehyde (MDA), the activity of glutathione reductase (GR) and y-Glutamate cysteine ligase (y-GCL) were determined by biochemical method.2The antagonistic effects of DHA on1-BP neurotoxicity48male healthy Wistar rats were randomly divided into4groups with12in each group, i.e. control group,1-BP group, low-dose DHA intervention and high-dose DHA intervention. Low-dose DHA intervention and high-dose DHA intervention group were treated250mg/kg.bw and500mg/kg.bw DHA respectively, while control group and1-BP group received equivalent corn oil. On day8of DHA treatment,1-BP group, low-dose DHA intervention and high-dose DHA intervention were given800mg/kg.bw1-BP, whereas control received corn oil. The Morris water maze test was applied to detect the ability of learning and memory fuction. After the Morris water maze, rats were sacrificed and the cerebral cortexes were removed. Biochemical methods detected the levels of GSH and MDA. Using immunofluorescence and western blotting determine the protein expression of glial fibrillary acidic protein (GFAP), NF-κB and Nrf2.Results1DHA and NA against1-BP neurotoxicity on the central nervous system1.1The results of the Morris water mazeCompared to control group, the latency and swim distance of rats in1-BP group were prolonged significantly (P<0.05,P<0.01). In the spatial probe test, the number of crossing the platform in1-BP group decreased evidently (P<0.05). While the latency and swim distance of rats treated with NA and DHA were much less than the value of1-BP group, and the number of crossing the platform in NA intervention group and DHA intervention group increased evidently (P<0.05).1.2The changes of biochemical indicatorsIn our study, the level of GSH in1-BP group which compared with control group was decreased by18.1%. While compared with the1-BP group, the GSH content in NA intervention group and DHA intervention group were increased significantly (P<0.05).Results showed that exposed1-BP resulted in lipid peroxidation of cortex. Compared with control group, the MDA content in1-BP group was increased by14.3%. While the levels of MDA in NA treatment group and DHA treatment group were decreased significantly, when compared with1-BP group.In addition, the activity of GR and y-GCL in1-BP group reduced evidently, when compared with the value of control group, while DHA treatment could increase the GR activity (P<0.05) and NA administration could increase y-GCL activity (P<0.05) respectively.2The antagonistic effects of DHA on1-BP neurotoxicity2.1The result of Morris water mazeIn place navigation test, compared with control group, the latency and total swim distance of rats in1-BP group were prolonged significantly (P<0.05). While compared to1-BP group, low-dose DHA intervention group and high-dose DHA intervention group both could decrease the latency and swim distance evidently (P<0.01). 2.2The change of biochemical indicatorsIn our study, the level of GSH of rats in1-BP group was much lower than of control group. Compared with control group, the GSH content in1-BP group was decreased by48.1%(P<0.01). While compared with1-BP group, the GSH content of cerebral cortex in low and high dose DHA intervention group were increased by59.9%(P<0.01) and81.9%(P<0.01) respectively.The content of MDA of cortex in1-BP group was increased by27.0%(P<0.01), when compared with control group. After supplement of low and high dose DHA, the levels of MDA were decresased significantly (P<0.01).2.3Expression of GFAP in cerebral cortexImmunofluorescence showed that astrocytes in control group express very low levels. But the GFAP protein expression in the cerebral cortex of1-BP group was significantly larger, when compared to the control group. As expected, DHA treatment exhibited the astroctytes activation, indicated by the reduction of GFAP, with stronger effect in high dose group.The reactive astrocytes were also evidenced by Western blotting analysis. The results showed that the levels of GFAP in1-BP group were significantly increased, compared to that control group (P<0.05). While low-dose and high-dose DHA treatment could inhibit this increase (P<0.05,P<0.01).2.4Expression of NF-κB and Nrf2in cerebral cortexIn this study, the protein expressions of NF-κB in1-BP group were higher than that of control group in cytoplasm and nuclear extraction of cerebral cortex. The levels of NF-κB in DHA treatment group in cell nuclear were significantly decreased (P<0.05), compared to that of1-BP group.Western blotting for Nrf2protein showed that both1-BP treatment and DHA intervention group were factors that significantly increased Nrf2protein content. As a result, the high-dose DHA treatment group, which had both the1-BP and the DHA factors, had the highest brain content of Nrf2protein of all the groups. Furthermore, treatment with DHA not only increased the total content of Nrf2protein but also increased its nuclear translocation in with1-BP treatment rats, as shown by increased Nrf2nuclear accumulation. Conclusion11-BP could result in the CNS damage, evidenced by the disorder of Morris Water Maze appearances. NA (150mg/kg.bw) and DHA (500mg/kg.bw) both could effectively attenuate1-BP (800mg/kg.bw) neurtoxicity on central nevours system.21-BP exposure could decrease the GSH contents, GR and y-GCL activities. NA and DHA could significantly increase the content of GSH and upregulate the activity of GR andy-GCL, which were two important enzymes in GSH synthesis process.3The1-BP neurotoxicity might be related to the astrocyte activation mediated by the NF-κB pathway of the brain in rats. DHA could increase the protein level of Nrf2and decrease the expression of NF-κB, which could ameliorate the activation of astocytes.更多还原