【作者】 韩晓英；

【导师】 谢克勤；

【作者基本信息】 山东大学 ， 卫生毒理学， 2014， 博士

【摘要】 研究目的正己烷[n-hexane]是己烷的五种同分异构体中的一种,分子式为CH3(CH2)4CH3,属于直链饱和脂肪烃类,由原油裂解及分馏获得。外观无色,有汽油味,分子量为86.17,易燃,易挥发(沸点68.74℃)。正己烷的急性毒性较低,大鼠经口LD50为15-30g/kg。在工业上主要用作溶剂,常用于配制粘胶以粘合鞋革、箱包,电子信息产业中常用于生产过程中的擦拭清洗作业,食品制造业中常用于粗油浸提,另外还用于塑料制造业的丙烯溶剂回收、化学实验中的萃取剂(如：光气实验)以及日用化学品生产时的花香溶剂萃取等。若使用不当,极易造成中毒。因正己烷易挥发,其挥发速度比酒精还要快,所以常经呼吸道和皮肤进入人体。短时吸入高浓度正己烷可出现眼及上呼吸道刺激症状,出现色觉、视觉障碍,并有麻醉症状,如眩晕、头痛、恶心、共济失调等。严重者可致昏迷乃至死亡。长期接触一定量后可出现多发性周围神经病,可表现为乏力、四肢远端(尤其下肢)进行性感觉异常、感觉减退。而后有双下肢无力、肌肉疼痛、萎缩及运动障碍,严重者可致瘫痪。毒代动力学研究表明,2,5-己二酮(2,5-Hexanedione,2,5-HD)是正己烷在体内的最终代谢产物,介导了中毒性周围神经病的发生。形态学研究证实：轴突萎缩是正己烷中毒性周围神经病的特异性病理改变,据此Lopachin等人提出了“轴突萎缩”假说。该假说认为,2,5-HD可作用于骨架蛋白,如神经丝(neurofilament,NF)等,引起轴突萎缩,最终导致周围神经受损。而轴突作为神经元的突起,必须依赖轴浆转运从神经元的胞体部分得到其所需的各种成分。轴浆转运的物质基础是运输器利用ATP水解释放的化学能,携带被转运物质沿微管向不同方向(即顺向、逆向)进行移动。简单地讲,轴浆转运的完成离不开以下几方面的内容：cargo,即需要运输的“货物”；动力蛋白；轨道；连接蛋白,连接cargo和动力蛋白；辅助分子。越来越多的证据表明：轴浆转运缺陷是多种神经退行性疾病的发病机制之一。本研究用2,5-HD建立了体外培养的新生乳大鼠背根神经节(dorsal root ganglia, DRG)细胞和亚慢性中毒大鼠二种模型,以神经丝亚单位NF-M的轴浆转运为切入点,从体内体外二种角度观察中毒性神经病发生过程中神经丝的轴浆运输速度、动力蛋白的含量、ATP的合成以及细胞骨架NFs、微管(α-tubulin、 β-tubulin)和微丝(β-actin)蛋白相对含量的变化,确定2,5-HD对神经丝轴浆运输的影响,为正己烷职业中毒的预防和治疗提供可以借鉴的基础资料。研究方法1.出生48小时内的SD乳大鼠,无菌条件下取背根神经节,酶法消化后建立DRG细胞体外培养模型。2.MTT法检测2,5-HD对细胞存活率的影响,确定2,5-HD的染毒剂量。3.利用电转染法,将pPA-GFP-NF-M和pDsRed二种质粒同时转入体外培养的DRG神经元中。4.2,5-HD染毒24h后,荧光显微镜下用活细胞成像(Live-cell imaging)技术,检测NF-M转运量的变化。5.收集培养的DRG细胞,加入预冷的RIPA裂解液(50mM Tris-HCl, pH7.4,150mM NaCl,1%Triton X-100,1%脱氧胆酸钠,0.1%SDS,1%Protease Inhibitor Cocktail,磷酸酶抑制剂(5mM Na3VO4,10mM Na4P2O7and1mM碘乙酸))中,裂解细胞。收集裂解液,4℃,12,000×g离心10min。吸出上清,用BCA法测定蛋白含量。6. Western-blotting法测定2,5-HD对DRG细胞中细胞骨架蛋白(NF-H、NF-M、 NF-L、α-tubuln、β-tubulin和β-actin),动力蛋白(kinesin heavy chain (KHC)、 dynein和dynactin p50),以及μ-calpain和FK2含量的影响。7.使用ATP检测试剂盒(ATP Assay Kit)测定2,5-HD对DRG细胞内ATP含量的影响。8.雄性Wistar大鼠用300mg/kg-bw的2,5-HD腹腔注射染毒,染毒时间8周(每周染毒5d,每日1次),建立2,5-HD中毒性周围神经病模型。9.取大鼠大脑、小脑、脊髓和坐骨神经组织在匀浆缓冲液(50mM Tris-HCl,pH7.4,150mM NaCl,1%Triton X-100,1%脱氧胆酸钠,0.1%SDS,1%Protease Inhibitor Cocktail,磷酸酶抑制剂(5mM Na3VO4,10mM Na4P2O7和1mM碘乙酸))中制备匀浆,10,000×g、4℃离心10min,留取上清。采用Western-Blotting技术研究大鼠大脑、小脑、脊髓和坐骨神经组织中细胞骨架蛋白(NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin)和动力蛋白(kinesin heavy chain (KHC)、和dynein)含量的变化。研究结果1.成功建立了乳大鼠背根神经节细胞的体外培养模型。2.根据MTT试验结果,在16mM以下,2,5-HD对DRG细胞未显示明显毒性作用,因此,选定3个作用剂量,分别为4mM、8mM、6mM,加对照组OmM,共4个组别,在后续试验中应用。3.活细胞观察实验120min后实验终止时,0,4,8,16mM2,5-HD组的荧光光密度剩余量分别为64.40±11.04%,71.52±11.64%,77.42±9.24%,81.50±8.58%。结果说明：2,5-HD给药组荧光强度减少的量均低于对照组(0mM2,5-HD组),且存在浓度-反应关系,提示：2,5-HD降低了NF-M的转运量,即2,5-HD减慢了NF-M的转运。4.2,5-HD对DRG细胞中细胞骨架蛋白含量的影响与对照组相比,4,8,16mM2,5-HD染毒组的细胞中NF-H的蛋白含量分别下降了7%、21%*和33%**,NF-M分别下降了17%*、38%**和41%**,NF-L仅在16mM组下降了25%**(*P<0.05,**P<0.01)。a-tubulin、βP-tubulin和β-actin的含量与对照组相比无明显差别(P>0.05)。5.2,5-HD对DRG细胞中轴浆运输相关蛋白的影响与对照组相比,4,8,16mM2,5-HD染毒组的细胞中KHC的蛋白含量分别下降了10%、24%*和36%**,dynactinp50分别增加了43%**、62%**和28%*(*P<0.05,**P<0.01),dynein含量无明显改变(P>0.05)。6.2,5-HD对DRG细胞中μ-calpain和FK2蛋白含量的影响2,5-HD染毒后,DRG细胞中μ-calpain和FK2的含量与对照组相比无明显差别(P>0.05)。7.2,5-HD对DRG细胞中ATP含量的影响染毒24h后,对照组的ATP含量为15.9nmol/mg protein,4,8,16mM2,5-HD组则分别为10.7,8.3,7.8nmol/mg protein,有明显的浓度-反应关系。8.2,5-HD对大鼠体重增长的影响在实验过程中,对照组大鼠体重稳定增长,到实验结束时动物平均体重增加了77.6%。2,5-HD染毒组大鼠体重增长明显比对照组慢,至8周实验结束时,染毒组大鼠体重仅增加了11.8%。组间动物体重差别达到了58.9%,有显著统计学差异(P<0.01)。9.大鼠步态的观察与对照组相比,染毒组大鼠的步态随着实验的进行不断发生改变,而且个体间差异极小(标准差很小)。第5周时,染毒组大鼠的步态评分达到2分(轻度异常)；第7周时,染毒组大鼠已经行走困难(平均为3.3分)；第8周实验结束时,染毒组大鼠几乎全部瘫痪(平均为3.93分)。10.2,5-HD对大鼠大脑组织中细胞骨架蛋白含量的影响5个对照组间,NF-H的含量无明显差异(P>0.05)。与对照组相比,2,5-HD染毒组大鼠在第2,4,6和8周时,NF-H的蛋白含量分别下降了7%、38%**,61%**和65%**(*P<0.05,**P<0.01)。随着实验的进行,不同年龄对照组的大鼠大脑组织中NF-M的含量在不断地增长,以实验起始时0周(con0周)为100%,第2,4,6,8周对照组大鼠大脑组织中NF-M的含量分别增加了62%,98%,102%和114%,与con0周比,均有极显著性差异P<0.01)。2,5-HD染毒4,6,8周大鼠与同年龄对照组相比,大脑组织中NF-M的含量均显著降低(P<0.01)；2,5-HD染毒2周大鼠与con2周相比,NF-M含量明显增加(P<0.01)。5个对照组间,NF-L的含量无明显差异(P>0.05)。与对照组相比,2,5-HD染毒组大鼠在第4,6和8周时,NF-L的蛋白含量分别下降了18%**,34%**和36%**(*P<0.05,**P<0.01)。5个对照组间,α-tubulin和β-tubulin的含量无明显差异(P>0.05)。与对照组相比,2,5-HD染毒组大鼠在第8周时,α-tubulin的蛋白含量下降了13%*(*P<0.05)；β-tubulin的含量无明显改变(P>0.05)。随着实验的进行,不同年龄对照组的大鼠大脑组织中β-actin的含量在不断地增长,以实验起始时0周为100%,第2,4,6,8周对照组大鼠大脑组织中β-actin的含量分别增加了0.65%,11%*,15%*和27%**(*P<0.05,**P<0.01)。2,5-HD染毒2,4,6,8周大鼠与同年龄对照组相比大脑组织中,β-actin含量变化分别是+21%a(增加),+7%a,-12%a(降低)和-34%aa(aP<0.05,aaP<0.01)。(增加)11.2,5-HD对大脑组织中轴浆运输相关蛋白含量的影响Con6周和con8周与其它对照组相比,KHC的含量略有降低(分别为27%**和20%*),2,5-HD染毒组与对照组100%相比,KHC的含量也略有降低。因变化趋势不明显,所以,组间差异有可能是实验误差所致(*P<0.05,**P<0.01)。5个对照组间,dynein的含量无明显差异(P>0.05)。与对照组相比,2,5-HD染毒组大鼠在第4,6和8周时,dynein的蛋白含量分别下降了24%*,23%*和12.5%(*P<0.05,**P<0.01)。12.2,5-HD对大鼠脊髓组织中细胞骨架蛋白含量的影响大鼠脊髓组织中细胞骨架蛋白含量的变化趋势与大脑组织中的变化基本一致,幅度略有区别。β-actin的含量在脊髓组织各组间无差异(P>0.05)。13.2,5-HD对脊髓组织中轴浆运输相关蛋白含量的影响在脊髓组织中,5个对照组间,KHC和dynein的含量均无明显差异(P>0.05)。与对照组相比,2,5-HD染毒组大鼠在第4,6和8周时,脊髓组织中dynein的蛋白含量分别下降了16%*,33%**和35%**；KHC的蛋白含量分别下降了4.5%,14%,15%和31%*(*P<0.05,**P<0.01)。14.2,5-HD对大鼠小脑组织中细胞骨架蛋白含量的影响小脑组织中,5个对照组间,NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin的含量无明显差异(P>0.05)。与对照组相比,2,5-HD染毒组大鼠小脑组织中3个神经丝亚单位NF-H、NF-M、NF-L和β-tubulin、β-actin的蛋白含量发生了不同程度的下降；但α-tubulin在2,5-HD染毒后相对含量有所上升。15.2,5-HD对小脑组织中轴浆运输相关蛋白含量的影响小脑组织中KHC和dynein的含量在各组之间无明显差异(P>0.05)。16.2,5-HD对大鼠坐骨神经组织中细胞骨架蛋白含量的影响坐骨神经组织中NF-H和NF-M蛋白含量的变化与大脑组织中的变化基本一致；NF-L和β-actin的相对含量在各组间无明显差异(P>0.05)；β-tubulin的含量略有增加。17.2,5-HD对大鼠坐骨神经组织中轴浆运输相关蛋白含量的影响在坐骨神经组织中,5个对照组间,KHC和dynein的含量均无明显差异(P>0.05)。与对照组相比,2,5-HD染毒组大鼠在第4,6和8周时,脊髓组织中dynein的蛋白含量分别下降了30%**,44%**和28%**；KHC的蛋白含量第2,4,6和8周分别下降了14%、30%**,35%**和57%**(*P<0.05,**P<0.01)。结论1.2,5-HD明显减慢了NF-M在体外培养的DRG神经元轴突中的转运。2.在体外培养的DRG细胞中,2,5-HD染毒可导致轴浆运输动力蛋白和ATP的含量发生降低,同时神经丝蛋白的相对含量也明显降低,这些因素的改变可能和NF-M的运输减慢有关。3.动物在体实验证实：2,5-HD染毒可以导致大鼠大脑、小脑、脊髓和坐骨神经组织中KHC和dynein的含量降低,且越接近神经的远端,这种变化越明显。同时在上述神经组织中神经丝的含量也明显降低,这可能与2,5-HD中毒性周围神经病表现的轴浆运输障碍有关。更多还原

【Abstract】 ObjectiveThe organic solvent n-hexane belongs to one of the most important aliphatic glue industry, paints, varnishes, printing inks, shoe manufacturing, shoe repair, and the food industry. Occupational or experimental exposure to n-hexane induces a central-peripheral nervous system damage. Chronic exposure to n-hexane produces loss of sensory and motor function in arms and legs, which is the result of the nerve damages characterized as a central-peripheral distal axonopathy. Initial clinical manifestations include numbness and tingling sensation in the toes and fingers, followed by progressive weakness and areflexia, particularly in the distal limbs. Severely affected patients may develop sequelae of muscle wasting, foot drop, and spasticity, even paralysis. Electrophysiological studies demonstrate prominent prolongation of distal latencies, slowing of nerve conduction velocities, and conduction block with temporal dispersion particularly in severely intoxicated patients.Toxicokinetic studies have shown that2,5-Hexanedione (2,5-HD) is the active metabolite of n-hexane and mediates the neurotoxicity of the parent compound. However, the molecular mechanisms for2,5-HD-induced neuropathy remains unclear. The primary morphologic feature of2,5-HD-induced neuropathy is axon atrophy in peripheral and central nerves. Therefore, Lopachin and his partners proposed that axon atrophy in2,5-HD-induced neuropathy is mediated by a reduction in axonal NF contents. Axon atrophy involves disruption of the neurophysiological processes responsible for maintaining axon caliber in neurons. And it is affirmed that changes of axon caliber could impact the cable properties and nerve conduction velocity. And these descriptions are consistent with the electrophysiological alterations of n-hexane intoxicated patients. Axon is one important part of the neuron, its materials must be synthesizied and assembled in the neuronal cell body and transported along the axon toward the distal tip. This transportation is known as’axonal transport’. Growing evidence supports the idea that deficits in axonal transport contribute to pathogenesis in multiple neurodegenerative diseases, for example, Amyotrophic Lateral Sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, and so on.In the present study, we investigated the effects of2,5-HD on slow axonal transport of GFP-tagged NF-M and partial factors involved in NF-M transport, as well as the cytoskeletal proteins in cultured DRG cells and subchronic poisoning rats, and studied the role of axonal transport played in2,5-HD-induced neuropathy.MethodsNeurons were dissociated from dorsal root ganglia(DRG) of postnatal2day old Sprague Dawley rats and cultured in vitro. After the cells were treated with0,4,8,16mM2,5-HD for24h, live-cell imaging experiment was performed. Under fluorescence microscope, PA-GFP-NFM was observed and pictured. The PA-GFP-NFM fluorescence intensity of each image was analyzed and compared. The amount of NF-M transported was measured by the decrease of the fluorescence intensity.The cultured DRG cells were harvested and crushed in lysis buffer (10mM Tris-HCl, pH7.5,150mM NaCl,1mM EDTA,1%Triton X-100,1%sodium deoxycholate,0.1%SDS, and10μl/ml proteinase inhibitor Cocktail) after24h of2,5-HD exposure. The lysate was sonicated and centrifuged at12,000g for10min at4℃. The supernatants were used for immunoblotting analysis and determination of ATP.Adult male Wistar rats were treated with2,5-HD by intraperitoneal injection at dosages of300mg/kg-bw for8weeks. The onset and development of neurotoxicity were determined by neurological testing (gait abnormality). The tissues of cerebrum, cerebellum, spinal cord and sciatic nerve, were homogenized in ice-cold homogenizing buffer containing1%X-100,50mM Tris (pH7.5),25mM KC1,2mM MgCl2,5mM EGTA,5mM dithiothreitol, Protease Inhibitor Cocktail (50ul/g tissue) and phosphatase inhibitors (5mM Na3VO4,10mM Na4P2O7and1mM iodoacetic acid) and then centrifuged at10,000xg for10min at4℃. The relative levels of NF-H, NF-M、NF-L、α-tubulin, β-tubulin, β-actin, kniesin heavy chain(KHC) and dynein in the supernatant of nerve tissues were determined by immunoblotting.Results1Cytotoxicity test2,5-HD inhibited the viability of cultured DRG cells, different concentration of2,5-HD shown different decrease of cell viability. There was an apparent concentration-response relationship. Finally,0,4,8,16mM2,5-HD were confirmed to be applied in the following experiments.22,5-HD decelerated NF-M transport in cultured DRG neuronsDuring the experiments, the density of fluorescence became weaker. Compared with the initial (set asl00%), the particular values were64.40±11.04%,71.52±11.64%,77.42±9.24%, and81.50±8.58%in0,4,8, and16mM2,5-HD-treated groups at120min, respectively. The decrease of the fluorescence density in2,5-HD-treated groups were all less than that of the control, indicating that the transport of NF-M was decelerated in2,5-HD groups.3Levels of cytoskeletal proteins in cultured DRG cells treated with2,5-HDThe prominent proteins in neurons (neurofilament subunits) were all reduced in2,5-HD groups. Among the three subunits, the decline of NF-M was the most evident. Compared with vehicle controls, a17%(P<0.05),38%(P<0.01) and41%(P<0.01) decrease of NF-M levels was observed in4,8,16mM2,5-HD-treated DRG cells, respectively, while the NF-H protein levels were decreased only by7%,21%(P<0.05) and33%(P<0.01), respectively. The decrease of NF-L was only observed in cells treated with16mM2,5-HD. In contrast to the obvious reduction of the NFs, no significant alteration of the levels of microtubules and microfilaments was observed.4Levels of motor proteins in cultured DRG cells treated with2,5-HDCompared with the control, kinesin heavy chain (KHC) protein levels in2,5-HD-treated DRG cells decreased by9.75%,24.02%(P<0.05) and36.06%(P<0.01) in4,8,16mM2,5-HD group, respectively. By contrast, the levels of dynactin p50were increased by43.37%(P<0.01),62.10%(P<0.01), and28.10%(P<0.05), respectively. However,2,5-HD intoxication failed to change the levels of dynein.5ATP concentration in cultured DRG cells treated with2,5-HD After24h intoxication, cells were collected and ATP content was determined. The ATP content in control cells was about15.9nmol/mg protein, and were10.7,8.3,7.8nmol/mg protein in cells treated with4,8,16mM2,5-HD, respectively.6Levels of μ-calpain and FK2in cultured DRG cells treated with2,5-HDIn cultured DRG cells, the levels of μ-calpain and FK2showed no significant changes after treated by2,5-HD (P>0.05)7The establishment of the animal modelAge-matched control rats had a starting mean body weight of272.3g, which increase steadily to483g at endpoint (8weeks). This represents a77.6%increase in body weight during the experiment period. Rats in the2,5-HD-treated groups had a similar starting weight, but gained only11.8%of their original weight, and, at the8weeks endpoint,2,5-HD-treated rats weighed63.1%of control.Intoxicated rats showed a progressive development of gait abnormalities. In the5th weekend, rats developed an unsteady walking pattern with a slight ataxia, and hindlimb weakness (2.05±0.23of mean gait score), and in the8th weekend, rats developed frank hindlimb weakness and an inability to rear (3.93±0.18of mean gait score).8Changes of cytoskeletal proteins in2,5-HD-intoxicated rats’nerve tissuesThe exposure to2,5-HD resulted in a significant decrease in NF-H, NF-M and NF-L in the homogenate of nerve tissues. In comparison with the control rats, the levels of NF-H significantly decreased (P<0.01) by65%,62%,66%and48%in cerebrum, cerebellum, spinal cord and sciatic nerve at the end of8weeks. Those of NF-L decreased by36%,66%, and45%in cerebrum, cerebellum and spinal cord respectively (P<0.01). The content of NF-M increased as the growth of rats, and compared with the age-matched control rats, those reductions were169%,73%,170%and60%in cerebrum, cerebellum, spinal cord and sciatic nerve at the end of8weeks (P<0.01).In cerebrum, β-tubulin showed no changes by2,5-HD-treated, α-tubulin and P-actin decreased slightly at the end of8weeks. In cerebellum, a-tubulin increased56%**, P-tubulin and β-actin reduced33%*and39%**, respectively. In sciatic nerve, a-tubulin showed a33%*reduction, but β-tubulin showed a78%**augment (*P <..05,**P<0.01) 9Changes of motor proteins in2,5-HD-intoxicated rats’ nerve tissuesThe motor protein, KHC declined by31%and57%, dynein dropped by35%and28%in spinal cord and sciatic nerve, respectively (P<0.01). There is no significant changes in cerebrum and cerebellum tissues.Conclusions1.2,5-HD significantly decelerated the axonal transport of NF-M in cultured DRG neurons.2. In cultured DRG cells, the levels of motor proteins and the content of ATP were decreased, as well as the levels of neurofilaments, after2,5-HD intoxication. This may be contributing to the deceleration of NF-M transport.3. The exposure to2,5-HD can lead to the reduction of KHC and dynein in rats’ cerebrum, cerebellum, spinal cord and sciatic nerve, and this reduction was more evident in the distal end of the axons. The alteration of neurofilaments had an similar tendency as KHC and dynein in the above nerve tissues. These may be involved in the onset and development of2,5-HD-induced neuropathy.更多还原