【作者】 宋福永；

【导师】 谢克勤；

【作者基本信息】 山东大学 ， 生理学， 2007， 博士

【摘要】 研究目的正己烷（n-hexane）和二硫化碳（carbon disulfide，CS₂）是常用的工业溶剂。正已烷主要应用于粘胶配制、除污、干洗、植物油提取、制鞋、印刷、油漆、制药、家具制造及电子元件制造等行业，而CS₂则广泛应用于粘胶纤维、玻璃纸、四氯化碳和杀虫剂等生产过程。长期职业接触正己烷、CS₂可引起以感觉运动型周围神经病为主要临床特征的慢性中毒。早期表现为手足部感觉异常，痛觉、触觉减退呈手套、袜套样分布，进一步发展可表现为四肢无力、行走困难以至瘫痪。主要的病理改变发生在肢体远端最长、最粗的感觉和运动神经轴索，并在郎飞氏节附近形成巨大的充满神经丝（neurofilament，NF）的肿胀，郎飞氏节解剖结构扭曲且髓鞘从肿胀处回缩，有时伴有节段性脱髓鞘，最后轴索发生变性。正己烷、CS₂中毒周围神经病的发生机制尚不清楚，但由于二者在神经行为学改变和病理改变上都非常相似，提示它们可能分享相同或相似的分子机制。同时，病理形态学研究提示NF、微管（microtubule，MT）等骨架蛋白可能参与了中毒性轴索病变的发生。国内外的研究已经报道，2，5-己二酮（2，5-hexanedione，HD）、CS₂亚慢性染毒可引起大鼠神经组织中NF含量的减少，但对这种变化发生的原因以及在它们在中毒性神经病中的作用没有进行深入探讨。因此，细胞骨架在中毒性周围神经病中的作用并没有真正阐明。本文通过建立亚慢性中毒动物模型，观察正己烷、CS₂中毒性神经病发生过程中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等细胞骨架蛋白相对含量的变化，在此基础上进一步研究毒物对细胞骨架蛋白的基因转录、蛋白降解的影响，以探讨有机溶剂中毒性周围神经病的发病机制。研究方法1雄性Wistar大鼠用400mg／kg．bw的HD腹腔注射染毒，染毒时间分别为2、4和8周，建立正己烷中毒性周围神经病模型，并取一半8周染毒动物继续观察至24周。雄性Wistar大鼠分别用300、500mg／kg．bw的CS₂灌胃染毒，连续12周，建立CS₂中毒性周围神经病模型。2取大鼠大脑、脊髓和坐骨神经组织制备匀浆，100，000×g、4℃离心60min。利用Western-Blotting技术研究大鼠大脑、脊髓、坐骨神经上清和沉淀中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等骨架蛋白以及钙激活的中性蛋白酶m-calpain和μ-calpain含量的变化。3取大鼠大脑和脊髓组织，提取总RNA，利用RT-PCR技术研究HD和CS₂染毒对大鼠中枢神经组织中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin mRNA表达水平的影响。4取大鼠大脑、脊髓和坐骨神经组织制备匀浆，8，000×g，4℃离心30min。利用Calbiochem（?） InnoZymeTM Calpain 1&2 Activity Kit检测大鼠大脑、脊髓、坐骨神经组织中calpains总活性的变化。研究结果1动物模型的建立以及神经行为学指标的变化给予HD、CS₂染毒后，动物逐渐出现肌肉松弛、肌张力降低、步态异常以至瘫痪等症状，神经行为学测试表明染毒大鼠呈现典型的中毒性周围神经病特征，这提示正己烷、CS₂中毒性周围神经病模型建立成功。同时HD染毒停止后，染毒大鼠的感觉运动指标能够逐渐改善。2 HD、CS₂染毒大鼠神经组织中细胞骨架蛋白含量的变化2．1 HD染毒大鼠神经组织中细胞骨架蛋白含量的时间变化趋势2．1．1大脑NF-H：与对照相比，大脑上清中NF-H的含量在HD染毒第2、4和8周时分别下降31.6％、40.3％和42％（P＜0.01），大脑沉淀中NF-H的含量分别下降11.1％（P＜0.05）、51.8％和53.2％（P＜0.01）。NF-M：与对照相比，大脑上清中NF-M的含量在HD染毒第2、4和8周时分别下降34.7％、58.2％和77.2％（P＜0.01），大脑沉淀中NF-M的含量分别下降29.4％、50.6％和72.5％（P＜0.01）。NF-L：与对照相比，大脑上清中NF-L的含量在HD染毒第2、4和8周时分别下降28％、35.4％和46.3％（P＜0.01），大脑沉淀中NF-L的含量分别下降18％、34.7％和34.7％（P＜0.01）。β-tubulin：与对照相比，大脑上清中β-tubulin的含量在HD染毒4周和8周时分别升高29％和33％（P＜0.01），大脑沉淀中β-tubulin的含量在HD染毒8周时也升高16.3％（P＜0.05）。α-tubulin和β-actin：与对照相比，大脑上清和沉淀中α-tubulin和β-actin的含量在HD染毒后未发生明显变化。2．1．2脊髓NF-H：与对照相比，脊髓上清中NF-H的含量在HD染毒第2、4和8周时分别下降16％、57％和58％（P＜0.01），脊髓沉淀中NF-H的含量在HD染毒第4和8周时分别下降33％和43％（P＜0.01）。停药16周后，NF-H的含量恢复正常。NF-M：与对照相比，脊髓上清中NF-M的含量在HD染毒第2、4和8周时分别下降36％、61％和65％（P＜0.01），脊髓沉淀中NF-M的含量分别下降23％、34％和64％（P＜0.01）。停药16周后，NF-M含量又恢复正常。NF-L：与对照相比，脊髓上清中NF-L的含量在HD染毒第2、4和8周时分别下降21％、44％和45％（P＜0.01），脊髓沉淀中NF-L含量在第4和8周时分别下降26％和42％（P＜0.01）。停药16周后，NF-L含量又恢复正常。α-tubulin、β-tubulin和β-actin：除HD染毒第8周时脊髓上清中β-tubulin的含量升高34.8％（P＜0.01）外，其余均未发生明显变化。2．1．3坐骨神经NF-H：与对照相比，坐骨神经上清中NF-H的含量在HD染毒第2、4和8周时分别下降19％、22％和43％（P＜0.01），坐骨神经沉淀中NF-H含量在HD染毒第4和8周时分别下降12％和51％（P＜0.01）。停药16周后，NF-H含量又恢复正常。NF-M：与年龄匹配的对照相比，坐骨神经上清中NF-M的含量在HD染毒第2、4、8周以及停止染毒16周时分别下降15％、24％、88％和83％（P＜0.01），坐骨神经沉淀中NF-M的含量分别下降12％、19％、54％和41％（P＜0.01）。NF-L：与年龄匹配的对照相比，坐骨神经上清中NF-L的含量在HD染毒第2、4、8周以及停止染毒16周时分别下降15％、40％、58％和34％（P＜0.01）；坐骨神经沉淀中NF-L的含量在HD染毒第4和8周时分别下降27％和53％（P＜0.01），停药16周后恢复至正常水平。a-tubulin：与对照相比，坐骨神经上清中a-tubulin的含量在HD染毒2、4、8周以及停药16周时均无显著性差异（P＞0.05），坐骨神经沉淀中a-tubulin的含量在HD染毒2、4、8周以及停药16周后分别下降40.6％，41.2％、34.5％和14.2％（P＜0.01）。β-tubulin：与对照相比，坐骨神经上清中β-tubulin的含量在HD染毒2、4、8周以及停药16周后β-tubulin的含量分别升高16％、44％、88.9％和86.1％（P＜0.01），坐骨神经沉淀中β-tubulin的含量在HD染毒8周及停药16周后分别升高102.1％和99.2％（P＜0.01）。β-actin：与对照相比，坐骨神经上清中β-actin的含量在染毒8周以及停药16周后分别下降27.1％和24.2％（P＜0.01）；坐骨神经沉淀中β-actin的含量在HD染毒2、4、8周时β-actin的含量分别升高24.8％、66.6％和103.1％（P＜0.01），停药16周后恢复正常。2．2 HD染毒大鼠神经组织中NFs含量变化与神经行为学改变的相关性分析HD染毒诱导的大鼠神经组织NFs含量变化与其神经行为学改变之间存在明显的相关性。大脑中NF-H、NF-M和NF-L的含量与步态改变之间的复相关系数分别为0.8651（P＜0.0001）、0.9644（P＜0.0001）和0.8872（P＜0.0001），脊髓中NF-H、NF-M和NF-L的含量与步态改变之间的复相关系数分别为0.8912（P＜0.0001）、0.9282（P＜0.0001）和0.8981（P＜0.0001），坐骨神经中NF-H、NF-M和NF-L的含量与步态改变的复相关系数分别为0.9133（P＜0.0001）、0.9156（P＜0.0001）和0.9446（P＜0.0001）。2．3 CS₂染毒大鼠神经组织中细胞骨架蛋白含量的变化2．3．1大脑与对照相比，300、500mg／kg CS₂染毒组大脑上清中NF-H含量分别下降31％和33％（P＜0.01），大脑沉淀中分别下降37％和45％（P＜0.01）；300、500mg／kgCS₂组大脑上清中NF-M含量分别下降52％和58％（P＜0.01），大脑沉淀中分别下降37％和75％（P＜0.01）；300、500mg／kg CS₂组大脑上清中NF-L的含量分别下降60％和83％（P＜0.01），大脑沉淀中分别下降57％和60％（P＜0.01）。与对照相比，α-tubulin的含量在300、500mg／kgCS₂染毒组大脑上清中分别升高20％和27％（P＜0.01），大脑沉淀中无显著性变化；β-tubulin的含量在300、500mg／kgCS₂染毒组大脑上清中分别升高63％和71％（P＜0.01），大脑沉淀中分别下降20％、26％（P＜0.01）；此外，β-actin的含量在500mg／kgCS₂染毒组大脑上清中升高31％（P＜0.01）。2．3．2脊髓与对照相比，NF-H含量在500 mg／kg CS₂染毒组脊髓上清中下降37％（P＜0.01），而在300、500mg／kg CS₂染毒组脊髓沉淀中分别下降20％、24％（P＜0.01）；NF-M含量在500mg／kg CS₂染毒组脊髓上清中下降33％（P＜0.01），而在300、500mg／kgCS₂染毒组脊髓沉淀中分别下降16％、65％（P＜0.01）；NF-L含量在500mg／kgCS₂染毒组脊髓上清中下降68％（P＜0.01），脊髓沉淀中下降40％（P＜0.01）。与对照相比，β-tubulin的含量在300、500mg／kgCS₂组脊髓上清中分别升高141％、158％（P＜0.01），脊髓沉淀中分别升高107％、118％（P＜0.01）；β-actin的含量在300、500mg／kgCS₂组脊髓上清中分别升高19％、31％（p＜0.01），脊髓沉淀中无显著性变化；此外，α-tubulin的含量均无显著性变化。2．3．3坐骨神经与对照相比，300、500mg／kgCS₂染毒组坐骨神经上清中NF-H的含量分别下降21.3％和30.9％（P＜0.01），沉淀中分别下降12.6％（P＞0.05）、21.4％（P＜0.01）；300、500 mg／kgCS₂染毒组坐骨神经上清中NF-M的含量分别升高21.8％、42.5％（P＜0.01），沉淀中分别升高41％、43.4％（P＜0.01）；300、500mg／kgCS₂染毒组坐骨神经上清中NF-L的含量分别下降24.4％、26.8％（P＜0.01），沉淀中分别下降4.6％（P＞0.05）、45.4％（P＜0.01）。与对照相比，300、500 mg／kgCS₂组坐骨神经上清中β-tubulin含量分别升高18.3％、40.4％（P＜0.01），沉淀中分别下降18.4％和26.8％（P＜0.01）；此外，500 mg／kg CS₂组坐骨神经上清中α-tubulin、β-actin含量分别升高74％和17.3％（P＜0.01），坐骨神经沉淀中分别下降19.7％和30.5％（P＜0.01）。3 HD和CS₂染毒大鼠神经组织中细胞骨架mRNA表达的变化3．1 HD染毒大鼠神经组织中细胞骨架mRNA表达的时间变化趋势3．1．1大脑与对照相比，大脑组织中NF-L、NF-H、α-tubulin、β-tubulin、β-actin的mRNA水平在HD染毒2、4和8周时均无显著性改变，但NF-M的mRNA水平在HD染毒2、4周时分别下降13.5％和24.8％（P＜0.01），3．1．2脊髓与对照相比，脊髓组织中NF-L、NF-M、NF-H、β-actin、α-tubulin、β-tubulin的mRNA水平在HD染毒2、4和8周时均无显著性变化。3．2 CS₂染毒大鼠神经组织中细胞骨架mRNA表达的变化3．2．1大脑与对照相比，NF-M的mRNA水平在300、500 mg／kg染毒组中分别下降了21.6％和21.8％（p＜0.01），NF-L的mRNA水平仅在500mg／kg染毒组中下降18.1％（p＜0.01）。然而，NF-H的mRNA表达未发生显著性变化。与对照相比，α-tubulin和β-actin的mRNA水平无显著性变化，而β-tubulin的mRNA水平在500 mg／kg染毒组下降13.5％（p＜0.01）。3．2．2脊髓与对照相比，300、500 mg／kg CS₂染毒组NF-H的mRNA水平分别升高33.3％和46.2％（P＜0.01），NF-M的mRNA水平分别升高119.8％和181.1％（P＜0.01），NF-L的mRNA水平分别升高101.7％和130.3％（P＜0.01）。与对照相比，300、500 mg／kg CS₂染毒组β-tubulin的mRNA水平分别升高207％和212％（P＜0.01），β-actin的mRNA水平分别升高94％和91％（P＜0.01），而α-tubulin的mRNA水平无显著性变化。4．HD和CS₂染毒大鼠神经组织中calpains表达的变化4．1 HD染毒大鼠神经组织中calpains表达的时间变化趋势4．1．1大脑m-calpain：与对照相比，大脑上清中m-calpain的含量在HD染毒第4、8周时分别下降15.3％和21.8％（P＜0.01），大脑沉淀中m-calpain的含量仅在HD染毒第8周时下降17.4％（P＜0.01）。μ-calpain：与对照相比，大脑上清中μ-calpain的含量在HD染毒第4、8周时分别升高24.6％和64.4％（P＜0.01），大脑沉淀中μ-calpain的含量仅在HD染毒第8周时升高72.7％（P＜0.01）。4．1．2脊髓m-calpain：与对照相比，HD染毒第2、4和8周时脊髓上清中m-calpain含量分别下降11.6％（P＞0.05）、19.9％（P＜0.05）和20.4％（P＜0.05），脊髓沉淀中分别下降15.8％、35.9％和54.6％（P＜0.01）。停止染毒16周后，脊髓中m-calpain的含量又恢复正常。μ-calpain：与对照相比，脊髓上清中μ-calpain含量在HD染毒第2、4周时分别下降12.9％和24.3％（P＜0.01），而染毒第8周时却升高62.3％（P＜0.01），停止染毒16周后仍然升高33.6％（P＜0.01）；脊髓沉淀中μ-calpain含量在HD染毒第2、4时分别下降15.4％（P＞0.05）和23％（P＜0.01），而染毒第8周时却升高25.2％（P＜0.01），停止染毒16周后恢复正常。4．1．3坐骨神经m-calpain：与对照相比，坐骨神经上清中m-calpain的含量在HD染毒第8周以及停药16周时分别升高46％和44％（P＜0.01）；坐骨神经沉淀中m-calpain的含量在HD染毒第8周时升高61％（P＜0.01），停药16周后恢复正常。μ-calpain：与对照相比，坐骨神经上清中μ-calpain的含量在HD染毒第4、8周以及停止染毒16周时分别升高30％、63％和54％（P＜0.01），而坐骨神经沉淀中分别升高31％、59％和34％（P＜0.01）。4．2 CS₂染毒大鼠神经组织中calpains表达的变化4．2．1大脑组织m-calpain：与对照相比，300、500 mg／kg CS₂染毒组大脑组织中m-calpain的含量均无显著变化。μ-calpain：与对照相比，300、500 mg／kg CS₂染毒组大脑上清中μ-calpain的含量分别升高34％、53％（P＜0.01），大脑沉淀中分别升高74％、49％（P＜0.01）。4．2．2脊髓组织m-calpain：与对照相比，300、500 mg／kgCS₂染毒组脊髓上清中m-calpain的含量分别下降46％和47.8％（P＜0.01），脊髓沉淀中无显著变化。μ-calpain：与对照相比，300、500 mg／kg CS₂染毒组脊髓上清中μ-calpain含量分别升高39.3％和47.2％（P＜0.01），脊髓沉淀中分别升高37.5％和36.2％（P＜0.01）。4．2．3坐骨神经m-calpain：与对照相比，300、500 mg／kg CS₂染毒组坐骨神经上清中m-calpain的含量分别升高18.3％和42.7％（P＜0.01），500 mg／kg CS₂染毒组坐骨神经沉淀中m-calpain的含量升高19.3％（P＜0.05）。μ-calpain：与对照相比，300 mg／kg CS₂染毒组坐骨神经上清中μ-calpain的含量下降19.2％（P＜0.01），500 mg／kg染毒组坐骨神经上清中无变化；300mg／kgCS₂染毒组坐骨神经沉淀中μ-calpain的含量下降36.8％（P＜0.01），而500mg／kg染毒组中升高35.9％（P＜0.01）。5．HD和CS₂染毒大鼠神经组织中calpains总活性的变化5．1 HD染毒大鼠神经组织中calpains总活性的变化与对照相比，大脑组织中calpains总活性仅在HD染毒第8周时下降37.2％（P＜0.01）；脊髓组织中calpains总活性在HD染毒第2、4和8周时分别下降22.8％、26.9％和27.5％（P＜0.01），停止染毒16周后calpain活性恢复正常；坐骨神经中calpains总活性在HD染毒第2、4和8周以及停止染毒第16周后分别升高14％、20％（P＜0.05）、34％（P＜0.01）和18％（P＜0.05）。5.2 CS₂染毒大鼠神经组织中calpains总活性的变化与对照相比，300、500 mg／kgCS₂染毒组脊髓组织中calpains总活性分别下降18.5％和20.6％（P＜0.01），坐骨神经组织中calpains总活性分别上升15.5％和20.7％（P＜0.01），而大脑中calpains总活性未发生显著变化。结论1、HD、CS₂染毒引起大鼠中枢和周围神经组织中3种NF亚单位含量发生明显变化，其中NF-M和NF-L的变化较NF-H更加显著。2、HD诱导的大鼠神经组织NF亚单位含量变化与其神经行为学改变之间存在高度相关性。3、HD诱导的神经组织NF含量下降具有可逆性，并与HD中毒性神经病症状的恢复有关。4、HD、CS₂染毒对不同神经组织中α-tubulin、β-tubulin和β-actin亚单位的影响不同。HD主要影响坐骨神经中α-tubulin、β-tubulin和β-actin的含量，而CS₂对中枢和周围神经组织中α-tubulin、β-mbulin和β-actin的含量均产生明显影响，其中β-tubulin的变化较α-tubulin和β-actin更加显著。5、HD、CS₂对细胞骨架的六种成分的影响程度不同，其中NFs含量的变化比tubulins、β-actin显著。6、HD染毒对神经组织中六种细胞骨架蛋白的mRNA表达未产生明显影响，而CS₂染毒影响了神经组织中六种细胞骨架蛋白的mRNA表达水平，表现为大脑组织中NF-L、NF-H和β-tubulin mRNA的转录水平下调，而脊髓组织中NF-H、NF-M、NF-L、β-tubulin和β-actin mRNA的转录水平上调。7、HD、CS₂染毒影响了神经组织中calpains表达和活性，表现为中枢神经组织中calpains活性下降，坐骨神经中calpains活性升高。末梢轴索中calpains的激活可能与HD、CS₂诱导的神经丝含量下降有关。8、毒物引起的神经组织中骨架蛋白改变参与了正己烷和CS₂中毒性周围神经病的发生。更多还原

【Abstract】 Objectiven-hexane and carbon disulfide （CS₂） are organic solvents widely used in many industrial processes, n-hexane is used widely in the preparation of fabrics, adhesives, lacquers and other coatings, etc. CS₂ is widely used in the vulcanization of rubber and synthesis of carbon tetrachloride and the manufacture of cellophane, plywood and viscose rayon.Continued exposure to hexane or CS₂ 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. The corresponding pathological alterations observed in this axonopathy include the accumulation of neurolfilaments （NFs） proximal to the nodes of Ranvier, a Wallerian-type degeneration consisting of accumulation of mitochondria, vesicles, microtubules （MT） and dense bodies distal to the axonal swellings, retraction of myelin from Ranvier nodes and segmental demyelination, and distal axon atrophy.The mechanisms of the axonopathy induced by hexane or CS₂ are currently unclear, although the pathological alterations indicate that NF and MT are involved in this axonapathy. In the present investigation, the rat models of HD and CS₂-induced axonopathy were established, and the relative levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin, andβ-actin in the tissues of sciatic nerves, spinal cords and cerebrums were determined to investigate their corresponding alterations during the axonopathy. Furthermore, the levels of gene expression of NF-H, NF-M, and NF-L subunit,α-tubulin,β-tubulin, andβ-actin mRNA were quantified using reverse transcription-polymerase chain reaction （RT-PCR）. In addition, the expression and activity of calpains, ca²⁺-activated neutral protease responsible for the degradation of cytoskeleton, were investigated to explore the possible mechanism of neuropathy induced by n-hexane and CS₂.Methods Adult male Wistar rats were treated with HD by intraperitoneal injection at dosages of 400 mg/kg/day HD respectively for 2, 4 or 8 weeks. To assess the recovery of HD-treated rats, half of 8-week treated animals continued to be observed for 16 weeks after 8-week exposure ended. Furthmore, the rats were treated with CS₂ by gavage at dosages of 300 and 500 mg/kg/day respectively for continuous 12 weeks to establish the model of CS2-induced neuropathy. The onset and development of neurotoxicity were determined by neurological testing.The tissues of sciatic nerves, spinal cords and cerebrums were homogenized and then centrifuged at 100, 000×g for 30min at 4℃. The relative levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin,β-actin, m-calpain andμ-calpain in the supernatant and pellet of nervous tissues were determined by immunoblotting.The tissues of spinal cords and cerebrums were excised, and the total mRNA was isolated from flash frozen tissues using Trizol reagent. The mRNA levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin, andβ-actin genes were quantified using RT-PCR.The excised nervous tissues of the sciatic nerves, spinal cords and cerebrums were homogenized and centrifuged at 8000 rpm for 20 min at 4℃Calpain activity in the supernatant was assayed using a Calbiochem^? InnoZyme^TM Calpain 1 & 2 Activity Kit.Results1 The establishment of the animal modelRats exposed to HD produced progressive neurobehavioural abnormalities. After 2, 4, and 8 weeks of HD treatment at 400 mg/kg dose-rate, the rats were at three different levels of neurological defects respectively, i.e. slightly abnormal gait, moderately abnormal gait, or severely abnormal gait. After stopping the treatment, the neurological defects of treated animals displayed obvious recovery. Furthermore, CS₂ intoxication could result in significant neurological defects. After 12 weeks of CS₂ treatment at 300 or 500 mg/kg dose-rate, the rats were at two different levels of slightly or moderately neurological abnormalities respectively. The results above suggested that the rat model of toxicant-induced neurotoxicity were established successfully.2 The alterations of cytoskeletal proteins in rat nervous tissues2.1 HD-induced alterations of cytoskeletal proteins in rat nervous tissues 2.1.1 CerebrumCompared to the control, the contents of NF-H decreased by 31.6%, 40.3%, and 42% respectively in the supernatant, and decreased by 11.1%, 51.8%, and 53.2% respectively in the pellet at 2, 4, and 8 weeks time point of treatment with HD; NF-M decreased by 34.7%, 58.2%, and 77.2% respectively in the supernatant, and decreased by 29.4%, 50.6%, and 72.5% respectively in the pellet at 2, 4, and 8 weeks time point of HD exposure; NF-L decreased by 28%, 35.4%, and 46.3% respectively in the supernatant, and decreased by 18%, 34.7%, and 34.7% respectively in the pellet at 2,4, and 8 weeks time point.Compared to the control, the levels ofα-tubulin andβ-actin in both fractions kept unaffected during the period of HD exposure. However, the level ofβ-tubulin increased by 29% and 33% respectively in the supernatant at 4, 8 weeks time point of HD exposure, and increased by 16.3% in the pellet at 8-week time point following HD.2.1.2 Spinal cordCompared to the control, the level of NF-H in the supernatant decreased by 16%, 57%, and 58% at 2, 4, and 8 weeks time point of treatment with HD respectively, and that in the pellet decreased by 33% and 35% at 4 and 8 weeks time point of treatment respectively; Accordingly, NF-M decreased by 36%, 61%, and 65% respectively in the supernatant, and decreased by 23%, 34%, and 44% respectively in the pellet at 2, 4, and 8 weeks time point of HD exposure. Furthermore, NF-L in the supernatant decreased by 21%, 44%, and 45% respectively at 2, 4, and 8 weeks time point following HD, and that in the pellet decreased by 26% and 35% at 4 and 8-week time point respectively. However, after a 16-week recovery, the levels of three NF subunits in spinal cord of 8-week treated rats returned to normal.Compared to the control, the levels ofα-tubulin andβ-actin in both fractions remained unchangeable throughout HD exposure. However, the level ofβ-tubulin was increased by 34.8% in the supernatant at 8-week time point of HD exposure.2.1.3 Sciatic nerveIn the pellet fraction, NF-H content decreased by 19%, 22%, and 43% respectively after 2,4, and 8-week treatment of HD. Accordingly, in the supernatant fraction, the contents of NF-H in groups of 4 and 8 weeks’ exposure decreased by 12% and 52%. Furthermore, after treated animals being allowed to recover for 16 weeks, the level of NF-H in sciatic nerves returned to normal.When compared to the age-matched control, the level of NF-M decreased by 15%, 24%, 88% and 83% at 2, 4, 8, and 24 weeks of the experiment in the supernatant, and accordingly decreased by 12%, 19%, 54% and 41% in the pelletWhen compared to the age-matched control, the level of NF-L in the supernatant decreased by 15%, 40%, 58% and 34% at 2, 4, 8, and 24 weeks of the experiment. Furthermore, in the pellet, a significant decrease of NF-L was observed in 4 and 8-week treated group, which decreased by 27% and 53%. However, after a 16-week recovery, the level of NF-L in the pellet returned to normal.When compared to the control, the level of a-tubulin in the pellet decreased by 40.6%, 41.2%, 34.5% and 14.2% respectively at 2, 4, 8, and 24 weeks of the experiment. However, a-tubulin content in the supernatant showed no significant alteration throughout the 24-week experiment.When compared to the control, the level ofβ-tubulin in the supernatant increased by 16%, 44%, 88.9% and 86.1% respectively at 2, 4, 8, and 24 weeks of the experiment. Accordingly, in the pellet fraction, the contents ofβ-tubulin increased by 15.3%, 9.5%, 102.1% and 99.2% respectively.When compared to the control, the level ofβ-actin in the supernatant decreased by 27.1% and 24.2% respectively at 8 and 24 weeks of the experiment. However,β-actin in the pellet increased by 24.8%, 66.6%, and 103.1% respectively at 2, 4, and 8 weeks of the experiment.2.2 Correlation relationship of NFs alteration in nerve tissues and neurobehavioral abnormality of HD-treated ratsTo investigate the contribution of NFs alteration to HD-induced neuropathy, the relationship between gait score and NFs content in rat nerve tissues was determined by multiple correlation analysis. Multiple correlation coefficients of NF-H, NF-M, or NF-L content in cerebrum with rat’s gait score are 0.8872 （P<0.0001）, 0.9644 （P<0.0001）, and 0.8651 （P<0.0001） respectively, those of NF-H, NF-M, or NF-L content in spinal cord with rat’s gait score are 0.8912 （P<0.0001）, 0.9282 （P<0.0001）, and 0.8981 （P<0.0001） respectively, and those in sciatic nerve are 0.9133 （P<0.0001）, 0.9156 （P<0.0001）, and 0.9446 （P<0.0001） respectively.2.3 CS2-induced alterations of cytoskeletal proteins in rat nervous tissues23.1 CerebrumCompared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS₂-treated groups decreased by 31% and 33% respectively, NF-M by 52% and 58% respectively, and NF-L by 60% and 83% respectively. Furthermore, in the pellet fraction, NF-H levels in 300 and 500 mg/kg CS₂-treated groups decreased by 37% and 45% respectively, NF-M by 37% and 75% respectively, and NF-L by 57% and 60% respectively.In the supernatant fraction, a -tubulin levels in 300 and 500 mg/kg CS₂-treated groups increased by 20% and 27% respectively,β-tubulin increased by 63% and 71% respectively. Furthermore, the level ofβ-actin in rats treated with 500 mg/kg CS2 increase by 31%. In the pellet fraction,β-tubulin levels in 300 and 500 mg/kg CS2-treated groups decreased by 20 and 26% respectively. However, the levels of a -tubulin andβ-actin remained unchangeable in both treated groups.2.3.2 Spinal cordIn the supernatant fraction, the levels of NF-H, NF-M, and NF-L in 300 mg/kg CS₂-treated group remained unaffected, however, their levels in 500 mg/kg CS₂-treated group decreased by 37%, 33% and 68% respectively. In the pellet, the levels of NF-H in 300 and 500 mg/kg CS₂-treated groups decreased by 20% and 24% respectively, and NF-M decreased by 16% and 65% respectively. Furthermore, the content of NF-L in 500 mg/kg CS₂ group decreased by 40%.When compared to the controls,β-tubulin levels in 300 and 500 mg/kg CS₂-treated groups increased by 141% and 158% respectively in the supernatant, and increased by 107% and 118% respectively in the pellet. Furthermore, in the supernatant,β-actin levels in 300 and 500 mg/kg CS₂-treated groups increased by 19% and 31% respectively. However, the levels ofα-tubulin remained unchangeable in both treated groups.2.3.3 Sciatic nerveCompared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS₂-treated groups decreased by 21.3% and 30.9% respectively, NF-L by 24.4% and 26.8% respectively. In the pellet fraction, the levels of NF-H levels in 300 and 500 mg/kg CS₂-treated groups decreased by 12.6% and 21.4% respectively, and NF-L by 4.6% and 45.4% respectively. As far as NF-M was concerned, its levels in 300 and 500 mg/kg CS₂-treated groups increased by 21.8% and 41.5% respectively in the supernatant fraction, and increased by 41% and 43.4% respectively in the pellet.When compared to the controls, the levels ofα-tubulin andβ-actin in 500 mg/kg CS₂-treated groups increased by 74% and 17.3% respectively in the supernatant fraction. However, in the pellet, the levels ofα-tubulin andβ-actin in 500 mg/kg CS₂-treated groups decreased by 19.7% and 30.5% respectively. As far asβ-tubulin was concerned, its levels in 300 and 500 mg/kg CS₂-treated groups increased by 18.3% and 40.4% respectively in the supernatant. However,β-tubulin decreased by 18.4% and 26.8% respectively in the pellet.3 The alterations of cytoskeleton mRNA in rat nervous tissues3.1 HD-induced alterations of cytoskeleton mRNA in rat nervous tissues3.1.1 CerebrumWhen compared to the age-matched controls, the mRNA levels of NF-H, NF-L,α-tubulin,β-tubulin andβ-actin stayed at the level of control groups throughout the intoxication process of HD, while the mRNA levels of NF-M demonstrated obvious fluctuation, which decreased by 13.5% and 24.8% respectively at weeks 2 and 4 of HD treatment, and returned to the level of control by the end of 8 weeks treatment.3.1.2 Spinal cordWhen compared to the age-matched controls, the mRNA levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin andβ-actin showed no significant alteration throughoutthe HD exposure.3.2 CS₂-induced alterations of cytoskeleton mRNA in rat nervous tissues 3.2.1 CerebrumWhen compared to the control, the mRNA level of NF-M in rats treated with 300 mg/kg CS₂ decreased by 21.6%, and that in rats treated with 500 mg/kg CS₂ by 21.8%. Furthermore, the mRNA levels of NF-L andβ-tubulin in rats treated with 300 mg/kg CS₂ decreased by 18.1% and 13.5% respectively. However, the mRNA levels of NF-H,α-tubulin andβ-actin showed no significant alteration in both treated groups.3.2.2 Spinal cordWhen compared to the control, the mRNA levels of NF-H, NF-M and NF-L in rats treated with 300 mg/kg CS₂ were elevated by 32%, 120%, and 102% respectively, and those in rats treated with 500 mg/kg CS₂ by 46%, 181%, and 130% respectively. Furthermore, the mRNA levels ofβ-tubulin andβ-actin in rats treated with 300 mg/kg CS₂ were elevated by 207% and 94% respectively, and those in rats treated with 500 mg/kg CS₂ by 212% and 91% respectively. However, the mRNA level ofα-tubulin showed no significant alteration in both CS₂-treated groups.4 The alterations of calpains content in rat nervous tissues4.1 HD-induced alterations of calpains content in rat nervous tissues4.1.1 CerebrumWhen compared to the controls, the contents of m-calpain decreased by 15.3% and 21.8% at time point of week 4 and 8 respectively in the supernatant, and decreased by 17.4% only at week 8 in the pellet,.As far asμ-calpain was concerned, in the supernatant,μ-calpain levels increased by 24.6% and 64.4% at time point of week 4 and 8 respectively. In the pellet,μ-calpain increased by 72.7% only at week 8.4.1.2 Spinal cordWhen compared to the control, the contents of m-calpain decreased by 11.6%, 19.9% and 20.4% respectively on week 2, 4 and 8 in the supernatant, and decreased by 15.8%, 35.9%, and 54.6% respectively in the pellet. After a 16-week recovery, the level of m-calpain in spinal cord of 8-week treated rats returned to normal.When compared to the control, the contents ofμ-calpain decreased by 15.4% （P>0.05） and 23% （P<0.05） respectively on week 2 and 4 in the supernatant, and decreased by 12.9% （P>0.05） and 24.3% respectively in the pellet. However, the level ofμ-calpam was elevated by 62.3% and 33.6% respectively on week 8 and 24 in the supernatant. Furthermore, the level ofμ-calpain was elevated by 25.2% on week 8 in the pellet.4.1.3 Sciatic nerveWhen compared to the controls, the level of m-calpain increased by 46% and 44% at time-point of week 8 and 24 respectively in the supernatant, and increased by 61% only at 8-week time point in the pellet fraction.As far asμ-calpain was concerned, in the supernatant fraction, the content ofμ-calpain increased by 30%, 63% and 54% at time point of week 4, 8 and 24 respectively. Correspondingly,μ-calpain increased by 31%, 59% and 34% respectively in the pellet fraction.4.2 CS2-induced alterations of calpains content in rat nervous tissues4.2.1 CerebrumWhen compared to the control, the levels of m-calpain in both fractions showed no significant alteration in both CS₂-treated groups. As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300, 500mg/kg CS₂ were elevated by 34% and 53% in the supernatant fraction respectively. In the pellet fraction, its levels increased by 74% and 49% respectively.4.2.2 Spinal cordIn the supernatant fraction, the levels of m-calpain in rats treated with 300, 500 mg/kg CS₂ were decreased by 46% and 48% respectively. However, the level of m-calpain in the pellet fraction stayed at the level of the controls. As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300, 500 mg/kg CS2 were elevated by 39% and 47% in the supernatant fraction respectively when compared to control group. In the pellet fraction, its levels increased by 37.5% and 36.2% respectively.4.2.3 Sciatic nerveWhen compared to the control, the levels of m-calpain in rats treated with 300, 500 mg/kg CS₂ were elevated by 18.3% and 42.7% in the supernatant fraction respectively. In the pellet fraction, its levels increased by 19.3% only in 500 mg/kg CS₂-treated group.As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300 mg/kg CS₂ decreased by 19.2% and 36.8% in the supernatant and pellet fractions respectively, and those in rats treated with 500 mg/kg CS₂ increased by 35.9% only in the pellet fraction.5 The alteration of calpains activity in rat nervous tissues5.1 HD-induced alteration of calpains activity in rat nervous tissuesWhen compared to age-matched controls, the calpains activity of spinal cords decreased by 22.8%, 26.9% and 27.5% respectively on week 2, 4, 8 after HD exposure; that of cerebrums decreased by 4.2%, 8% and 37.2% respectively on week 2, 4, 8 after HD exposure. However, calpains activity in sciatic nerves increased by 14%, 20%, 34% and 18% respectively on week 2, 4, 8 and 24.5.2 CS₂-induced alteration of calpains activity in rat nervous tissuesWhen compared to age-matched controls, the calpains activity of spinal cords decreased by 18.5% and 20.6% in 300 and 500 mg/kg CS₂-treated group respectively. Furthermore, the calpains activity of sciatic nerves increased by 15.5% and 20.7% in 300 and 500 mg/kg CS₂-treated group respectively. However, calpains activity of cerebrums remained unchangeable in both CS₂-treated groups.Conclusions1 The intoxication of HD and CS₂ resulted in significant alterations of NFs contents in nerve tissues of rats, among which the alterations of NF-M and NF-L appeared to be more noticeable than NF-H.2 HD-induced NFs alterations in rat nerve tissues were highly correlated with neurobehavioral abnormality of HD-treated rats.3 The reversibility of NF decline induced by HD is associated with the recovery of HD-treated rats.4 The intoxication of HD and CS2 resulted in significant alterations ofα-tubulin,β-tubulin andβ-actin in nerve tissues, which was mainly observed in the peripheral nerves of rats treated with HD, and observed in central and peripheral nervous tissues treated with CS2. Among them, the alterations ofβ-tubulin appeared to be more noticeable than those ofα-tubulin andβ-actin.5 The effect of HD and CS₂ on NFs,β-tubulins andβ-actin showed some diversity, among which the alterations of NFs appeared to be more marked than tubulins andβ-actin.6 CS₂ induced significant alterations in the gene expression of cytoskeleton in rat nerve tissues, i.e. the expression of NF-H, NF-M, NF-L,β-tubulin andβ-actin mRNA was upregulated significantly in spinal cord, and the expression of NF-H, NF-L andβ-tubulin mRNA was downregulated significantly in cerebrum. However, HD had no significant effect on the expression of cytoskeleton gene.7 HD and CS₂ induced significant alterations in the expression and activity of calpains in rat nervous tissues. Among them, the activity of calpains decreased in the CNS, and increased in the PNS significantly. Calpains activation in distal axons might be responsible for the decline of NFs induced by HD and CS₂.8 The alteration of cytoskeleton induced by HD and CS₂ was involved in the development of HD or CS2- induced neuropathy.更多还原