【作者】 欧超燕；

【导师】 姜岳明；

【作者基本信息】 广西医科大学 ， 流行病与卫生统计学， 2012， 博士

【摘要】 锰(Mn)是人体必需微量元素,是某些正常细胞动态平衡金属酶的必需辅酶。然而,长期吸入较高浓度的锰烟尘可引起中毒,出现帕金森病(PD)样临床表现。磁共振成像(MRI)和氢质子磁共振波谱(1H-MRS)研究发现,锰暴露工人苍白球锰蓄积明显,丘脑及其邻近基底核GABA-感兴趣区(VOI)γ-氨基丁酸(GABA)水平明显升高。锰暴露可改变动物纹状体及其所在基底核的GABA浓度改变,改变脑内GAD、GABA-T活性及GABA受体与转运载体的mRNA及蛋白表达。我室的较早系列研究显示,对氨基水杨酸钠(PAS-Na)有促排锰作用,可使染锰4周的大鼠轻度受损的神经细胞病理改变恢复正常,PAS-Na对锰致海马神经元损害有拮抗作用。广西医科大学纪淑琴等率先在临床上应用PAS-Na治疗锰中毒患者取得较好疗效(临床表现基本恢复正常或改善、尿锰排泄增加、不良反应较少等),贵州、江西、包头、上海和重庆也有类似的临床报道。但是,PAS-Na防治锰中毒机制尚未清楚。因此,本研究旨在探讨PAS-Na对短期及亚慢性锰暴露大鼠GABA能神经元损伤是否有拮抗作用,以期为锰中毒防治提供科学依据。目的：探讨PAS-Na对短期及亚慢性锰暴露大鼠生长发育、学习记忆、基底核超微结构、GABA能神经递质及其有关代谢过程的影响。材料与方法：1.动物分组及处理：雄性SD大鼠适应性喂养1周后,①短期实验：随机分为染锰组、低、高剂量PAS-Na (L-、H-PAS)治疗组和正常对照组(对照组),共4组。观察期为7周(染锰4周+PAS治疗3周)或10周(染锰4周+PAS治疗6周),每期每组15只大鼠(后同)。染锰、PAS治疗组腹腔注射(ip) MnCl2·4H2015mg/kg,对照组ip等容量生理盐水,每日1次,每周5天,连续4周。然后,PAS治疗组大鼠背部皮下注射(sc)PAS-Na100或200mg/kg,其余组背部sc等容量生理盐水,每日1次,连续3周或6周。②亚慢性实验：随机分为对照、染锰、PAS-Na预防性干预(预防组)和低、中、高剂量PAS-Na (L-、M-、H-PAS)治疗组,观察期为4、8、12、18周。给染锰、预防、PAS治疗组ip MnCl2·4H2015mg/kg,对照组ip等容量生理盐水,每日1次,每周5天,连续4、8、12周,其中预防组在染锰的同时,每天背部sc PAS-Na200mg/kg(每周3天),连续8、12周。然后,给L-、M-、H-PAS治疗组分别sc PAS-Na100、200、300mg/kg,其余组背部sc等容量生理盐水,每日1次,连续6周。2.实验指标测定：①生长发育指标检测：每周末称动物体重1次；实验结束时,处死动物,取心、肝、脾、肺、肾、睾丸等脏器,并称其重量,算其脏体比；②学习记忆能力检测：实验结束前1周,每组10只动物进行Morris水迷宫实验以测试其学习记忆能力改变；③基底核超微结构观察：实验结束时,每组取5只动物进行2%多聚甲醛、2.5%戊二醛混合固定液经心脏内灌注固定,后分离基底核,进行透射电镜观察基底核超微结构改变；④GABA能神经递质改变检测：实验结束时,每组取10只动物,处死动物,取基底核迅速于液氮速冻,并于-80℃冰箱保存,高效液相色谱荧光检测法检测GABA、Glu、Gln、Gly氨基酸类神经递质含量,高效液相色谱法及荧光分光光度计法检测其GAD与GABA-T活性；实时荧光定量PCR(RT-PCR)与蛋白免疫印迹(WB)检测GABA转运载体(GAT-1)及GABA受体(GABAAR) mRNA与蛋白表达。结果：1. PAS-Na对锰暴露大鼠生长发育的影响(1)锰暴露第一至第四周、第八周、第十周,大鼠体重比对照组轻,差异具统计学意义；预防组大鼠在第一致第三周较染锰组重,差异具统计学意义。(2)观察期4周,染锰组脾脏与睾丸脏体比比对照组大,差异具统计学意义；观察期8周,染锰组脾脏脏体比比对照组大,差异具统计学意义；观察期12周,染锰组脾脏与肝脏脏体比比对着组大,预防组脾脏与肝脏脏体比比染锰组小,差异均具统计学意义；观察期18周,L-与H-PAS组肝脏脏体比比染锰组小,脾脏脏体比比染锰组大,差异均具统计学意义。2. PAS-Na对锰暴露大鼠学习记忆能力的影响(1)学习能力的影响：在观察期4、10、12、18周,染锰组大鼠的逃避潜伏期和或游泳路程均比对照长。锰暴露4或12周后,给予PAS-Na为期6周的治疗,各治疗剂量组均可恢复锰暴露所延长了的逃避潜伏期及游泳路程。(2)记忆能力的影响：观察期7周,染锰大鼠平台所在象限时间或路程除总时间或路程均比对照组长；高-PAS组平台所在象限时间或路程除总时间或路程均比染锰组短。观察期8周,平台所在象限时间或路程除总时间或路程染锰组与对照比较减少,PAS-Na预防组与染锰组比较增多。3. PAS-Na对锰暴露大鼠基底核超微结构的影响：短期或亚慢性锰暴露可损伤大鼠脑基底核的神经元、神经毡及星形胶质细胞,使得其神经元出现变性、凋亡及坏死等现象,神经元树突、轴突终末肿胀,星形胶质细胞增生、凋亡退变,且随染锰期限的延长,其损伤越发严重；此外,即使终止染锰这些损伤仍在继续,且随时间的延长而越发严重。与锰中毒病人的临床表现相一致。PAS-Na对染锰大鼠所致的基底核改变都有一定的拮抗作用,会使得神经元变性、凋亡、坏死现象得到好转,突触间隙重变清晰,但对于染锰12周大鼠的基底核改变,改善作用不是特别明显。4. PAS-Na对锰暴露大鼠基底核GABA能神经递质的影响(1)短期及亚慢性锰暴露对大鼠基底核GABA能神经递质的影响：观察期4周,Glu、Gln与GABA含量,GAD活性及GABAAR mRNA表达均升高。观察期7周,Gly含量减少,GABAAR蛋白表达升高；观察期10周,Glu、Gln与Gly含量,GABAAR与GAT-1mRNA表达均降低。观察期8周,Glu/GABA比值及GABAAR mRNA表达均降低。观察期12周,GABA-T活性及GABAAR蛋白表达升高；观察期18周,基底核Glu、Gln、与GABA含量及GABAAR mRNA表达均降低,而GAD与GABA-T活性及GAT-1mRNA表达均升高。(2)PAS-Na对短期及亚慢性锰暴露大鼠基底核GABA能神经递质的预防或治疗效果：①治疗效果观察：观察期10周,L-PAS与H-PAS组的Glu、Gln含量及GAT-1mRNA表达与H-PAS组Gly含量比染锰组升高；观察期18周,M-PAS及H-PAS组的GAD活性与H-PAS组的GABA-T活性及GAT-1mRNA表达降低。②预防效果观察：观察期8周,预防组的Glu/GABA比值与GABAAR mRNA表达比染锰组升高；观察期12周,预防组的GABAAR蛋白表达比染锰组降低。结论：(1)短期及亚慢性锰暴露使大鼠生长发育迟缓,体重增加较少,脾脏、睾丸及肝脏脏器系数增加,提示锰具生长发育及器官整体毒性,PAS-Na对锰所致的生长发育及器官整体毒性具一定拮抗作用。(2)短期及亚慢性锰暴露均可影响大鼠的学习能力,其记忆功能的改变仅出现在亚慢性的锰暴露。PAS-Na治疗或预防均可拮抗锰暴露所致的学习及记忆功能改变。(3)短期及亚慢性锰暴露均可对大鼠基底核超微结构产生影响,PAS-Na预防和/或治疗对其病理改变具有一定的拮抗作用,使其改变得到一定的恢复,表现为早期治疗效果较佳,且预防效果优于治疗效果。(4)短期及亚慢性锰暴露对大鼠基底核Glu、Gln、Gly与GABA含量,GAD与GABA-T活性,GABAAR mRNA与蛋白表达及GAT-1mRNA表达均有影响。PAS-Na预防及治疗对不同期限锰暴露所致的以上改变均有一定的拮抗作用,PAS-Na对亚慢性锰暴露致GABAAR mRNA或蛋白表达改变有预防性干预作用,对锰致大鼠基底核Glu、Gln及Gly含量、GAD活性与GAT-1mRNA表达改变有治疗性干预作用。更多还原

【Abstract】 Manganese (Mn) is a naturally occurring element that is an essential nutrient, and cofactor for certain metalloenzymes required for normal cellular homeostasis. Although Mn consumption is necessary in humans, excessive exposure is associated with body’s adverse effects, in especial the psychiatric, cognitive, and motor adverse effects. Magnetic resonance imaging studies are in agreement that Mn accumulates to a large extent in the globus pallidus. The globus pallidus and related basal ganglia structures have therefore been a focus of the preponderance of the research efforts on Mn neurotoxicity. it is now believed that the GABAergic system is probably perturbed first in the etiology of manganism. Several reports have linked Mn exposure to decrease or increase in the concentration of brain GABA.Addressing a need for a mechanism to explain the apparent reduction in GABA concentration. Reported decreases in overall brain Glu decarboxylase (GAD) and GABA-transaminase (GABA-T) levels subsequent to intraperitoneal injections of MnCl2, and alterations in expression of GABA transport and receptor proteins.Sodium para-aminosalicylic acid (PAS-Na) is effective in reducing Mn concentrations in body fluids and brain to physiological level, thus acting as a Mn-chelating agent. In clinical studies and obtained animal studies have found that the PAS-Na can get rid of brain manganese, repair manganese-induced brain damage and ultrastructural changes, the body’s enzymes returned to normal, the antagonistic manganese-induced learning and memory changes, oxidative stress change, neural microfilament protein(NF) positive cells of the dentate gyrus granulosa cells and the glial fiber acid protein (GFAP) positive in the hippocampus.Meanwhile the PAS-Na used in the treatment of clinical manganism patients were also found in patients with a variety of symptoms and signs such asneurasthenic symptoms, eyes closed, elite refers to nose test, refer to the test write barriers have improved markedly, and the efficacy of the consolidation of follow-up after17years of clinical effect is still good. However,The mechanism of PAS-Na for manganese exposure caused the GABAergic damage still unknowed. This study intends to explore the intervention mechanism of PAS-Na exposed to manganese for the basal ganglia GABAergic by short-term and subchronic Mn exposed rats in vivo.[Objective] To study the role of sodium aminosalicylate (PAS-Na) on growthing development, learning and memory, the ultrastructure of the basal ganglia and the concentration of GAB A, activity of GAD and GABA-T, GABA A receptor (GABAAR) and GABA transporter (GAT) expression in basal ganglia of short-term and subchronic Mn exposed rats.[Methods and materials](1) Animals grouping and exposure. Sprague-Dawley male rats received daily intraperitoneally (i.p.) injections of15mgMn/kg,5days/week for4weeks, followed by a daily subcutaneously (s.c.) dose of PAS-Na (100and200mg/kg as the PAS-L and PAS-H group, respectively) for another3or6weeks. Sprague-Dawley rats received daily i.p. injections of15mg Mn/kg,5days/week for4,8orl2weeks, followingby a daily s.c. dose of PAS-Na200mg/kg as the PAS-P group,3days/week at the same time for8and12weeks, followed by a daily s.c. dose of PAS-Na (100,200and300mg/kg as the PAS-L, PAS-M and PAS-H group, respectively) for another6weeks. the rest of the groups sc an equal volume of saline.(2) determination of indicators①Growthing development indicators detection. Weighing the anminal weight one time every week. Obtaining the organ of heart, liver,spleen,lungs,kidneys,testis, and weighing the weight of them,than calculate its organ body ratio.②The learning and memory ability test. Before the anatomy, Morris watermaze test lasting for6days was used to detect learning and memory ability of rats.③The Ultrastructural of the basal ganglia observation. The end of theexperiment, five animals of each group perfused the mixed liquor of2%paraformaldehyde and2.5%glutaraldehyde through the heart, than separated the basal ganglia, observed ultrastructure changs by transmission electron microscopy④GABAergic neurotransmitters detection. At the end of the experiment,the animals were sacrificed to take the basal ganglia rapidly and froze in liquid nitrogen and stored at-80℃. To survey the content of GAB A, Glu, Gln and Gly by high performance liquid chromatography (HPLC) with fluorescence detection.To examine the activity of GAD and GABA-T by high performance liquid chromatography and fluorescence spec trophotometer detection.To probe the mRNA and protein expression of GABA transporters and GABA receptors (GABAAR) by real-time fluorescent quantitative PCR (RT-PCR) and Western blot (WB).[Results]1. Effects of PAS-Na to growthing development of Mn exposure rats.①Weight gain changes. The weight of the rats in each group with time growthing in all of our experimental period. Manganese exposed rats growth slower than the control rats. From exposed to manganese to the fourth week, eighth and tenth week, the manganese exposed rats body weight growed signafiance slower than the control rats. The weight of preven rats growed signafiance faster than Mn exposure rats from the first to the third week. There are no no significant change in rats bod’s weight by the remedy of PAS-Na after Mn exposure, have nothing to do with the length of the period of PAS-Na treatment and Mn exposure.②The ratio of organ to body. Regardless of the length of period exposed to manganese, the rat showed the increasing of spleen quality. The rat exposed to manganese for4weeks, not only caused the increasesing of spleen quality but also the testis. When menganese exposure for12weeks, in addition to the spleen quality significantly increased, the quality of liver also significantly increased. Prevention with PAS-Na for12weeks to12weeks manganese-exposed rats, may recove the quality of liver and spleen. Treatment with L-PAS and H-PAS for6weeks to12weeks manganese-exposed may restore the liver body ratio.2. Effects of PAS-Na to learning and memory abilities of Mn exposure rats.①The effect on learning capability.The rats whether exposed to Mn for4weeks,8weeks,12weeks or were Mn-exposed for4weeks then stop3weeks,6weeks and were Mn-exposed for12weeks then stop6weeks, their incubation period and swimming journey increased compare with control. The effects of PAS-Na on learning and memory capabilities in Mn-exposed rats Whether in exposed to Mn for4weeks then treated with PAS-Na for3weeks or6weeks,or were Mn-exposed and PAS-Na prevented at the same time for8weeks or12weeks,or exposed to Mn for12weeks then treated with PAS-Na for6weeks the incubation period and swimming journey were decreased.②The effect on memory capability. The platform quadrant time or distance to total time or total distance ratio. With4weeks of Mn-expose then PAS treated for3weeks increased compare with control; contrary, in H-PAS group decreased compare with Mn-exposed rats. Mn-exposed and PAS-Na prevented at the same time for8weeks, the platform quadrant time or distance to total time or total distance ratio in Mn-exposed group rats decreased compare with control,and PAS-prevented group increased compare with Mn-exposed group rats.3. Effects of PAS-Na on basal ganglion ultrastructure of Mn-exposed rats.Short-term and subchronic Mn-exposed may induced basal ganglion damaged, neuropil, astroglia cells damaged. caused the neurons appeared degeneration, apoptosis and necrosis, that neurons dendritic and axon terminal swelling, astroglia cells appeared hyperplasia and apoptosis degeneration, and with the extension of Mn-expose, the damage become more serious. PAS-Na could against the alteration of basal ganglion in Mn-exposed rats, restored the degeneration, apoptosis and necrosis in neurons, synaptic cleft clear, but there was no significant in the basal ganglion of rats were exposed to Mn for12weeks.4. Effects of PAS-Na on GABAergic neurotransmitter in basal ganglion of Mn-exposed rats(1)The effects of Short-term and subchronic Mn-exposed on GABAergic neurotransmitters in basal ganglion. Mn-exposed for4weeks, the concentrations of Glu,Gln and GABA, the GAD activity and GABAAR mRNA expression significant increased in basal ganglion. Mn-exposed for4weeks and then stop for3weeks, the concentrations of Gly decreased and GABAAR protein expression increased significantly. When stop exposure to Mn for6weeks, the concentrations of Glu,Gln,Gly and GABAAR and GAT-1mRNA expression significant decreased. Mn-exposed for8weeks, the ratio of Glu/GABA and GABAAR mRNA expression decreased significantly. When stop exposure to Mn for12weeks, GABA-T activity and GABAAR protein expression increased, and stop Mn-exposed for6weeks, the concentrations of Glu,Gln,GABAand the expression of GABAAR mRNA decreased, and GAD and GABA-T activities, GAT-1mRNA expression increased.(2) The prevention and/or treatment effects on Short-term and subchronic Mn-exposed rats in basal ganglion. ①The effect of treatment with PAS-Na. After Mn-exposed for4weeks then used L-PAS and H-PAS treated for3weeks, the concentrations of Gly and GABAAR protein expression alterations no recovery. When for6weeks treatment, the changes of the concentrations of Glu, Glu and GAT-1mRNA protein expression were restored by L-PAS and H-PAS, H-PAS may returned the Gly concentration, but no effects on GABAAR mRNA expression no matter by L-PAS or H-PAS. When Mn-exposed for12weeks rats than treatment withL-PAS、M-PAS and H-PAS for6weeks, the alteration of GAD activity recovered by M-PAS and H-PAS, the alterations of GABA-T activity and GAT-1mRNA expression both restored by H-PAS,but Glu, Gln and GABA content and GABAAR protein expression did not recovered by any PAS-Na dose treatment.②The effects of prevention with PAS-Na. Mn-exposed and PAS-Na prevented at the same time for8weeks, the changes of Glu/GABA ratio and GABAAR mRNA expression recovered. Mn-exposed and PAS-Na prevented at the same time for12weeks, the alter of GABAAR protein expression restored, but no the GABA-T activity.[Conclusion](1) Short-term and subchronic Mn-exposed could lead to the slower growth of weight vs the control. Results showed that Mn has the growthing development toxicity. Prevention with PAS-Na in early period of manganese exposure, could restore the weight changing caused by manganese exposure.(2) Exposure manganese in short-term could affect the rat testes quality and the quality of the spleen, and in long-term exposure not only affect the spleen quality, but also the liver quality,l ead to its organ body ratio rising. The prevention of PAS-Na in organ quality changed caused by manganese exposure is good.(3) Short-term and subchronic manganese exposure all could affect the rat learning ability, and the memory fuction change only appeares in the long term period of manganese exposure. Treatment and prevention with PAS-Na for manganese exposure made an antagonism effect in learning and memory fuction.(4) Short-term and subchronic manganese exposure all could affect basal nuclear ultrastructure. Treatment and prevention with PAS-Na for manganese exposure made an antagonism effect in basal nuclear ultrastructure.And the treatment effect is better in the early period manganese exposure,the prevention effect better than the treatment.(5) Short-term and subchronic manganese exposure could affect the content of Glu, Gln, Gly and GAB A, GAD and GABA-T activity, GABAAR mRNA and protein expression and GAT-1mRNA in basal nuclear. Treatment and prevention with PAS-Na for manganese exposure made an antagonism effect in that alteration. PAS-Na plays a prevention effect in GABAAR mRNA or protein expression of subchronic Mn-exposed rats, and a treatment effect in the content of Glu, Gln and Gly, GAD and GABA-T activity and GAT-1mRNA expression of Mn-exposed rats.更多还原