【作者】 邓祥发；

【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， 2010， 博士

【摘要】 锰是一种必需微量元素,摄入过量锰会对机体产生毒性损伤。以往锰神经毒性的研究主要关注其对锥体外系的损伤。但是,锰中毒病人不但出现锥体外系损害的临床表现,还有学习记忆、认知和空间方向感的障碍,以及易哭泣、烦躁、孤独自闭等情绪改变的症状,后者可能与锰对大脑边缘系统毒性损害有关系。应用对氨基水杨酸钠(PAS-Na)干预及治疗锰中毒,近年来不断有临床和实验室研究的报道,临床应用PAS-Na治疗锰中毒病人可取得一定疗效,但其机制仍不清楚,而有关PAS-Na对锰致大鼠大脑边缘系统损伤的干预机制的研究,报道的文献则更为稀少。本研究拟通过体内外实验,探讨PAS-Na对锰致大鼠大脑边缘系统损伤的干预机制。1.对氨基水杨酸钠(PAS-Na)对锰暴露大鼠原代培养海马神经细胞凋亡的影响【目的】探讨对氨基水杨酸钠(PAS-Na)对染锰大鼠原代培养海马神经细胞凋亡的影响。【材料和方法】(1)取新生SD大鼠海马建立体外原代海马神经细胞培养体系; (2)将培养纯化后的神经细胞分为正常对照组,染锰50μM、100μM组,PAS-Na 100μM组、PAS-Na 500μM组,染锰50μM+PAS-Na100μM和PAS-Na 500μM干预组、染锰100μM+PAS-Na100μM和PAS-Na 500μM干预组,分别进行:①MTT法检测细胞存活度;②单细胞凝胶电泳(SCGE)技术测定细胞DNA损伤;③Annexin V-FITC + PI双染流式细胞检测法测定细胞凋亡率(不含PAS-Na 100μM组及染锰50μM、100μM+PAS-Na100μM组)。【结果】染锰50μM及100μM均可致海马神经细胞的细胞存活度显著下降(P<0.05),PAS-Na干预可提高染锰细胞的存活度;染锰100μM可致海马神经细胞DNA损伤增加(P<0.05),PAS-Na干预可降低染锰对细胞DNA的损伤作用;染锰50μM及100μM可致海马神经细胞凋亡率显著升高(P<0.05),PAS-Na干预可降低染锰细胞凋亡率。2. PAS-Na对锰暴露大鼠大脑边缘系统损伤干预的体内研究2.1 PAS-Na干预对亚急性锰暴露大鼠大脑边缘系统损伤的影响【目的】探讨PAS-Na对亚急性锰暴露大鼠学习记忆功能障碍、脑海马细胞及超微结构变化的影响。【材料与方法】将64只健康雄性SD大鼠随机分为锰暴露组、PAS-Na低、高剂量治疗组(L-、H-PAS组)和正常对照组(对照组),染锰大鼠腹腔注射(ip)二氯化锰(MnCl2·4H2O )15mg/kg,对照组ip等容量生理盐水,每日1次,每周5天(染锰5天,停2天),共3周。染锰结束后L-、H-PAS组分别给大鼠背部皮下注射PAS-Na 100mg/kg、200mg/kg,每天一次,共5周。在治疗期的第5周用Morris水迷宫测试大鼠学习记忆能力;免疫组织化学法检测基底前脑斜角带水平支臂核(hDB)和斜角带垂直支臂核(vDB)胆碱乙酰基转移酶(ChAT)阳性细胞、海马齿状回颗粒细胞下层(SGZ)神经微丝蛋白(NF)和海马CA1区胶质原纤维酸性蛋白(GFAP)阳性细胞数的变化;透射电镜观察大鼠海马CA1区超微结构的改变。【结果】(1)亚急性锰暴露可显著降低大鼠学习记忆能力,以及基底前脑hDB ChAT阳性细胞数(P<0.05);L-、H-PAS组大鼠学习记忆能力比染锰组好,基底前脑hDB和vDB的ChAT阳性细胞数较染锰组增多;(2)亚急性锰暴露可显著降低大鼠SGZ的NF阳性细胞数(P<0.05),H-PAS治疗能使其提高(P<0.05);(3)亚急性锰暴露可使海马CA1区GFAP阳性细胞数显著增多(P<0.05),L-、H-PAS治疗后GFAP阳性细胞数降低,与锰暴露组差异有统计学意义(P<0.05);(4)亚急性锰暴露组大鼠海马CA1区可观察到大量变性、凋亡、坏死的神经元,线粒体的超微形态病理性改变明显,神经突起水肿、髓鞘变性,星形胶质细胞增生、肥大并伴有退行性变,血脑屏障(BBB)通透性增高,小胶质细胞活跃; H-PAS治疗可明显改善染锰大鼠线粒体的病理形态,减轻神经突起水肿和髓鞘变性的程度,并减轻胶质细胞的增生与肥大,坏死神经元胞内残存的细细胞器增多,形态表现不典型。2.2 PAS-Na干预对亚慢性锰暴露大鼠大脑边缘系统损伤的影响【目的】探讨PAS-Na干预对亚慢性锰暴露大鼠体内组织锰含量、学习记忆功能障碍、脑海马细胞、超微结构及凋亡相关基因变化的影响。【材料与方法】将144只健康雄性SD大鼠随机分对照组、染锰组、PAS-Na预防干预组(预防组)、PAS-Na治疗组(治疗组),其中对照组和染锰组在12周时再平均分为2组,共6个组,每组24只。染锰大鼠腹腔注射(ip)MnCl2·4H2O 15mg/kg,对照组ip等容量生理盐水,每日1次,每周5天,共12周。PAS-Na预防干预组在染锰同时,每天给予背部皮下注射PAS-Na 200mg/kg,共12周;治疗组则在染锰12周结束后方给予背部皮下注射PAS-Na 200mg/kg,共6周。在处死大鼠取材前一周(第12周、18周)用Morris水迷宫测试大鼠学习记忆能力。每组动物动物取材时分为三部份,第一部份每组8只处死后从心脏抽血,然后迅速断头、开颅取鲜活组织,用于如下指标测定:(1)脑海马、皮层、血锰含量测定;(2)基底前脑组织胆碱乙酰基转移酶(ChAT)活性;(3) RT-PCR法检测海马Bax、Bcl-2、Caspase-3、Caspase-9、COXI、COXIV基因mRNA的表达。第二部份每组8只采用4%多聚甲醛固定液从心脏作内灌注固定,然后开颅切取脑组织,免疫组织化学法检测:(1)基底前脑斜角带水平支臂核(hDB)、斜角带垂直支臂核(vDB)胆碱乙酰基转移酶(ChAT)阳性细胞;(2)海马齿状回颗粒细胞下层(SGZ)神经微丝蛋白(NF);(3)海马CA1区胶质原纤维酸性蛋白(GFAP)阳性细胞数的变化;第三部份每组8只采用2%多聚甲醛、2.5%戊二醛混合固定液从心脏作内灌注固定,取海马CA1区脑组织,透射电镜观察超微结构的改变。【结果】1染锰12周、预防干预12周①染锰大鼠海马、皮层、血锰含量均升高(P<0.05),PAS-Na预防干预可使血锰含量显著下降(P<0.05)。②水迷宫测试染锰大鼠第5天的逃避潜伏期、游泳总路程测量值高于对照组(P<0.05),PAS-Na预防干预可降低染锰大鼠逃避潜伏期、游泳总路程测量值(P<0.05)。③染锰大鼠基底前脑vDB、hDB的ChAT阳性细胞数明显减少(P<0.05),PAS-Na预防干预可使染锰大鼠vDB、hDB的ChAT阳性细胞数显著增加(P<0.05)。④染锰组大鼠基底前脑组织ChAT蛋白活性显著降低(P<0.05),PAS-Na预防干预可使其明显回升(P<0.05)。⑤亚慢性染锰大鼠海马超微结构改变与亚急性染锰大鼠相似,但神经毡、髓鞘、线粒体的病理改变更为明显;PAS-Na预防干预使细胞线粒体的超微形态明显回复,趋于正常,坏死神经元残存细胞器增多,神经突起水肿、髓鞘变性程度减轻,胶质细胞肥大不明显。⑥染锰大鼠GFAP阳性细胞数显著升高(P<0.05),PAS-Na预防干预后细胞数明显下降(P<0.05)。⑦染锰大鼠海马CA1区TUNEL反应的阳性细胞数显著增多(P<0.05),PAS-Na预防干预使阳性细胞数明显减少(P<0.05)。⑧RT-PCR结果显示:染锰大鼠COXI、COXIV、Bcl-2基因表达显著降低(P<0.05),Caspase-3基因表达显著升高(P<0.05);预防组Bcl-2基因表达与染锰组间差异有统计学意义(P<0.05),接近对照组;预防组COXI、Caspase-3基因表达与染锰组组间比较差异无统计学意义(P>0.05),与对照组比较差异仍存在统计学意义(P<0.05)。2染锰12周,PAS-Na治疗6周,①水迷宫测试染锰大鼠第5天的逃避潜伏期、游泳总路程测量值显著升高(P<0.05),PAS-Na预防干预可降低染锰大鼠逃避潜伏期、游泳总路程测量值(P<0.05)。②染锰组vDB的ChAT阳性细胞数较对照组下降(P<0.05),PAS-Na治疗可使染锰大鼠vDB的ChAT阳性细胞数显著提高(P<0.05)。③染锰大鼠海马超微结构的改变与亚急性染锰大鼠相似,但髓鞘的变性更为明显;PAS-Na治疗对神经突起和髓鞘变性有明显改善,趋于正常,胶质细胞无明显肥大。④染锰大鼠海马颗粒细胞下层神经微丝蛋白(NF)阳性细胞数显著降低(P<0.05),PAS-Na治疗可使NF阳性细胞数明显回升(p<0.05)。⑤染锰大鼠海马CA1区GFAP阳性细胞数显著升高(P<0.05),PAS-Na治疗组细胞数少于染锰组。⑥RT-PCR结果显示:与对照组相比,染锰大鼠COXIV、Bcl-2基因表达显著降低(P<0.05),Caspase-3基因表达显著升高(P<0.05);治疗组Bcl-2基因表达回升,与染锰组比较组间差异有统计学意义(P<0.05)。综上,本研究提示:①100μM、500μM浓度PAS-Na本身对大鼠原代培养海马神经细胞无毒性作用;②PAS-Na干预可提高锰暴露原代培养海马神经细胞存活度、对低剂量锰致DNA损伤有显著的拮抗作用,在一定程度上降低锰暴露神经细胞的凋亡率;③PAS-Na通过提高基底前脑胆碱能神经元的存活度和提高胆碱乙酰基转移酶的活性,促进乙酰胆碱生成来促进染锰大鼠受损学习记忆能力的恢复;④PAS-Na可能通过螯合体内过量的锰离子、促进锰的排泄来发挥保护神经的作用;⑤PAS-Na对染锰后星形胶质细胞反应性增生及超微结构病理改变具有明显的拮抗作用;⑥锰中毒导致大鼠海马超微结构发生广泛的病理改变,PAS-Na干预可改善组织细胞线粒体、髓鞘、神经细胞毡的病理形态,使坏死神经元残存细胞器增多;⑦锰暴露导致大鼠COXI、COXIV、Caspase-3、Bcl-2基因表达异常, PAS-Na的干预可使Bcl-2基因表达恢复至正常水平。更多还原

【Abstract】 The trace element manganese is essential for the normal human physiological function, however, high amounts of manganese will produce toxic damage to body.The long-term study of manganese neurotoxicity focused chiefly on extrapyramidal dopaminergic neurons. Clinical observation showed the victim of manganese poisoning can appear the dysfunction of learning, memory, cognition and the sense of direction, and emotional disorder including intension, anxiety, timidity and autism, which may be related to toxical damage of the limbic system induced by manganese.In recent years, it has been constantly reported that application of sodium aminosalicylate (PAS-Na) therapy for manganese–poisonging in clinical or in laboratory, in which obtained certain effect. Yet there were lacked report concerned with the damage of the limbic system induced by manganese, and mechanism of therapy for manganese–poisonging of PAS-Na still unknowed. This study intends to explore the intervention mechanism of PAS-Na for the limbic system damage of rats induced by manganese in vitro and in vivo.1 Effect of Sodium aminosalicylate (PAS-Na) to Apoptosis of Cultured Hippocampal Neurons of Manganese Exposure Rats[Objective]To explore the effect of PAS-Na to apoptosis of cultured hippocampal neurons of manganese exposure rats. [Methods and materials] (1) Hippocampal neurons of new-born SD rats were cultured to establish the hippocampal neurons culturing system; (2) Hippocampal neurons after purification were randomly divided into 6 groups, including control, 50μM Mn, 100μM Mn, PAS-Na 100μM, PAS-Na 500μM, 50μMMn+PAS-Na100μM , 50μM Mn+PAS-Na 500μM, 100μMMn+PAS-Na100μM, 100μM Mn+PAS-Na 500μM, respectively. And then, (1) MTT method was used to detect the cell viability; (2) single-celled gel electrophoresis(SCGE) was used to determine the DNA damage of neurons; (3) Annexin (FITC + V - double dye streaming cells PI was used to determine the apoptosis rate of neurons(ecxcept grouds of PAS-Na 100μM, 50μMMn+PAS-Na100μM and 100μMMn+PAS-Na100μM). [Results] (1) The cell viability of manganese exposed hippocampal neurons decreased significantly(P<0.05), and the cell viability of manganese exposed neurons increased after interfered by PAS-Na; (2) Manganese exposure increased the DNA damage of neurons(P<0.05), and the level of DNA damage of manganese exposed neurons degard after interfered by PAS-Na; (3) Manganese increased the apoptosis rate of neurons(P<0.05), and the apoptosis rate of manganese exposed neurons reduced after interfered by PAS-Na;2 Effect of PAS-Na to the Limbic System Damage of Manganese Exposed Rats in Vivo2.1 Effect of PAS-Na to the limbic system damage of subacute manganese exposed rat.[Objective] To explore the effect of PAS-Na to the limbic system damage of subacute manganese exposed rats, including learning and memory dysfunction, the changes of cells and ultrastructure of hippocampus. [Metholds and materials] 64 healthy male SD rats were randomly divided into manganese exposure group, low and high dosage of PAS-Na treatment group (L -, H - PAS group) and normal control group (control group). Rats expect for the normal control group were intraperitoneally injected (ip)MnCl2·4H2O 15mg/kg, and the normal control group was ip the same capacity of saline water per day, 5 days a week, for three weeks.Then L–PAS group and H - PAS group were given subcutaneous injection(sc) in back with PAS-Na 100mg/kg, 200mg/kg per day for 5 weeks, respectively. Morris water maze test was used to detect learning and memory of rats after 4-week treatment. Immunohistochemistry methods were used to determine the changes of number of choline acetyl transferase (ChAT) positive cells of basal forebrains bevel belt level (hDB )and oblique hits with vertical(vDB), The subgrandular zone of dentate gyrus (SGZ) neural microfilament protein (NF) positive cells and the hippocampus CA1 area glial fibrils acid protein (GFAP) positive cells.Transmission electron microscope was used to observe the hippocampal CA1 area ultrastructural changes of rats. [Results] (1) Manganese exposure significantly reduced learning and memory ability of rats(P<0.05), and decreased significantly the number of ChAT positive cells of basal forebrains nuclers of the hDB (P<0.05); After PAS-Na treatment, the learning and memory ability of the L-PAS group and H-PAS group was better than manganese exposure group, and ChAT positive cells of basal forebrains were more than those of manganese exposure group. (2) NF positive cells in SGZ were decreased in manganese exposure group(P<0.05), but were increased significantly in H-PAS treatment group(P<0.05). (3) Manganese lead to significant increase of the GFAP positive cells in hippocampal CA1 area (P<0.05), but L-PAS and H-PAS treatment groups decreased(P<0.05). (4) Manganese exposure lead to more serious neuronal degeneration, apoptosis, necrosis, neural protuberant edema and pith scabbard denaturation, astrocytes hyperplasia, hypertrophy and degeneration, microglia activation, blood brain barrier (BBB) permeability increase. H-PAS treatment could improve the pathological ultrastructure of mitochondria of manganese exposure rats, alleviate nerve protuberant edema and pith scabbard denaturation, reduce hypertrophy of glial cells, as well as increased the relic cellular organ in necrosis neurons , which showed non-typical microscopical of it.2.2 Effect of PAS-Na to the limbic system damage of chronic manganese exposed rats [Objective]To explore the effect of PAS-Na to chronic manganese exposed rats, including content of manganese in vivo tissues, learning and memory dysfunction, and the changes of ultrastructure of hippocampus and genes expression related apoptosis. [Methods and Materials] 144 healthy male SD rats were randomly divided into control group, manganese exposure group, PAS-Na prevention treatment group(prevention group) and PAS-Na treatment group(treatment group), and control group. In 12th week during experiment, The control group and the manganese exposure group were subdivided into two groud respectively, so 6 groud all together were builded . Rats expect for the control group were intraperitoneally injected (ip)MnCl2·4H2O 15mg/kg, and the control group was ip the same capacity of saline water per day, 5 days a week for 12 weeks. PAS-Na prevention treatment group(prevention group) were given subcutaneous injection(sc) in back with PAS-Na 200mg/kg during exposed to manganese. PAS-Na treatment group was given sc in back with PAS-Na 200mg/kg per day for 6 weeks after 12-week manganese exposure. Before the anatomy, Morris water maze test lasting for one week was used to detect learning and memory ability of rats, and escape incubation period and swimming distance were recorded. Then, 8 rats of each group were cervically dislocated, and blood was collected form heart, and fresh tissues was got after qucik craniotomy.These samples were used to determine (1) content of manganese in hippocampus, cortex, and red blood cells. (2)the activation of choline acetyl transferase (ChAT)in basal forebrains. (3) Bax、Bcl-2、Caspase-3、Caspase-9、COXI、COXIV mRNA expression of hippocampus with RT-PCR. The other 16 rats of each group were fixed by heart perfusion, and fixed brains were used to determine:(1) the changes of number of choline acetyl transferase (ChAT) positive cells of basal forebrains bevel belt level (hDB )and oblique hits with vertical(vDB), neural microfilament protein (NF) positive cells of the dentate gyrus granulosa cells (SGZ) and glial fibrils acid protein (GFAP) positive cells in the hippocampus CA1 area with immunohistochemistry methods; (2) the hippocampal CA1 area ultrastructural changes of rats by transmission electron microscope. [Results] (1)After 12-week manganese exposure, PAS-Na prevention treatment 12-week①the content of manganese in hippocampus, cortex and red blood cells of manganese exposure group was significantly higher than control group(P<0.05); the content of manganese in red blood cells decreased obviously in PAS-Na prevention treatment group(P<0.05);②Morris test showed escape incubation period and swimming distance of the fifth day’s test of manganese exposure group was both longer than control group(P<0.05), and PAS-Na prevention treatment could improve the learning and memory ability (P<0.05);③ChAT positive cells of basal forebrains hDB and vDB of manganese exposure group were respectively less than control group(P<0.05), and those of PAS-Na prevention group were more than manganese exposure group(P<0.05);④the activation of ChAT in basal forebrains of manganese exposure group was significantly lower than control group(P<0.05), and that of PAS-Na prevention group was higher than manganese exposure group(P<0.05);⑤Pathological changes of hippocampal ultrastructure in chronic manganese exposed were similar to those of the subacute manganese exposed rats, while the nerve felt and pith scabbard and structural changes of mitochondria was more obviously. But the ultrastructure of mitochondria recovered to the normal level after PAS-Na prevention treatment, while increased the relic cellular organ in necrosis neurons, and lessen oedema of nervors process and apomorphosis of myelin, and lessen hypertrophia of gliacyte at the same time.⑥GFAP positive cells of manganese exposure group were significantly more than those of control group (P<0.05), PAS-Na prevention treatment inhibited the increase of GFAP positive cells(P<0.05).⑦TUNEL positive cells in hippocampal CA1 area of manganese exposure group were much more, compared with control group(P<0.05), and those of PAS-Na prevention group were less than those of manganese exposure group(P<0.05).⑧The result of RT-PCR show: COXI, COXIV, Bcl-2 gene expression in manganese exposure group was significantly lower than that in control group, as well as Caspase-3 gene expression higher than that in control group(P<0.05). and that of Bcl-2 gene expression higher than that in manganese exposure group, while COXI, COXIV, Caspase-3 in PAS-Na prevention group was similar to manganese exposure group.(2) 12-week manganese exposure and following 6-week PAS-Na treatment①Morris test was used to detect the learning and memory ability of rats, escape incubation period and swimming distance of the fifth day’s test of manganese exposure group was longer than control group(P<0.05), and PAS-Na treatment could improve the learning and memory ability(P<0.05).②ChAT positive cells of basal forebrains vDB of manganese exposure group were less than control group (P<0.05), and ChAT positive cells of basal forebrains vDB of PAS-Na treatment group were more than manganese exposure group(P<0.05);③Pathological changes of hippocampal ultrastructure in chronic manganese exposed were similar to those of the subacute manganese exposed rats, while the pathological changes of myelin more serious than those in subacute manganese exposed rats. pathological changes of nervous process and myelin were recovered to the normal level after PAS-Na treatment, mast glial cells were not obvious.④NF positive cells of the SGZ of manganese exposure group were less than control group(P<0.05), and those of PAS-Na treatment group were more than manganese exposure group( P<0.05.⑤GFAP positive cells of manganese exposure group were significantly more than those of control group (P<0.05), and those of PAS-Na treatment group were more than manganese exposure group, with no significance between groups(P>0.05).⑥expression of COXI and Bcl-2 genes in manganese exposure group was less lower than that of control group(P<0.05), while the gene of Caspase-3 showed higher lever in manganese exposure group(P<0.05). Bcl-2 gene expression in PAS-Na treatment group was much higher than that of manganese exposure group(P<0.05).[conclusion]①The Sodium aminosalicylate, which concentration of 100μM or 500μM has not poisonous effect to the cultured hippocampal neurons.②The Sodium aminosalicylate may possess the ability of alleviate the damage degree of cultured hippocampal neurons, and prevents the damnification of DNA, and recedes the rate of apoptosis of maganese exposed hippocampal neurons in a certain extent.③The Sodium aminosalicylate can improve the learning and memory ability by elevate the survive of choline acetyl neurons, and enhance the activation of ChAT in basal forebrains, promoted the creating of acetylcholine.④Sodium aminosalicylate bring into neuro-protective via its ability of chelate excessive manganum, and promoted the evacuation of manganum in vivo. ⑤The Sodium aminosalicylate showed great intervention to the reactive hyperplasea and ultrastructure abnormity of gelatine.⑥Manganese exposed lead to ultrastructure pathologic change extersively of hippocampi tissue of rats, and the Intervention of Sodium aminosalicylate can reverse the pathologic morphous of mitochondria and myelin and meuropil, while lead to increase of remanetd cell organelle necrosis neurocyte, which showed non-typical in it’s ultrastructure .⑦Manganese exposed lead to abnormal expressive of genes of COXI, COXIV, Caspase-3 and Bcl-2, The Intervention of Sodium aminosalicylate can reverse expressive of gene of Bcl-2 to normal level.更多还原