【作者】 钟玉芳；

【导师】 石年；

【作者基本信息】 华中科技大学 ， 卫生毒理学， 2006， 硕士

【摘要】 溴氰菊酯（Deltamethrin,DM）是一种使用广泛的杀虫剂,属于Ⅱ型拟除虫菊酯（Pyrethroids,Py）类,因其对环境的污染导致哺乳动物和人类潜在的神经毒性而倍受关注。近年来拟除虫菊酯类杀虫剂被认为对神经-内分泌-免疫系统有干扰作用,可能通过内分泌或免疫系统途径影响其神经毒性。神经-内分泌-免疫三大系统是一个相互调节相互影响的网络,多种内分泌和免疫因子可在神经系统中发挥作用,其中免疫因子白细胞介素1β（interleukin 1β, IL-1β）在神经系统的多种生理和病理过程（如睡眠、发热、缺血缺氧等）中具有重要的作用。IL-1β的成熟需要白介素转化酶（interleukin 1 converting enzyme, ICE）的作用,而其功能可被ICE的抑制剂以及其内源性受体拮抗剂（interleukin 1 receptor antagonist, IL-1ra）所阻断。溴氰菊酯对中枢神经系统的兴奋性神经损害同脑缺血缺氧等过程有相似的病理机制,但尚未有相关研究揭示溴氰菊酯对神经免疫功能的影响以及IL-1β与溴氰菊酯神经毒性间的关系。本研究拟先观察整体动物中溴氰菊酯对神经细胞中IL-1β表达的影响,再利用体外实验模型,从添加外源性IL-1β、IL-1ra和ICE抑制剂等因素,观察溴氰菊酯神经毒性是否发生改变,深入探讨IL-1β在溴氰菊酯神经毒性中发挥的可能作用,研究结果如下:第一部分整体实验:观察DM对大鼠不同脑区IL-1β的影响及其与DM神经毒性间的关系一、DM对大鼠脑组织IL-1β的影响（一）对大鼠不同脑区IL-1βmRNA表达的影响-RT-PCR法目的:观察不同作用时间,DM对神经细胞IL-1βmRNA的影响方法:15只雄性SD大鼠随机分为一个对照组和4个DM作用不同时间的实验组,实验组大鼠一次性腹腔注射12.5 mg?kg-1 DM,分别于1 h、6 h、24 h、48 h后提取皮层、海马和下丘脑总RNA进行RT-PCR,扩增IL-1β,并以GAPDH为内参,测定IL-1β/GAPDH相对表达量。结果:皮层IL-1β的基因转录在给药后1 h开始持续升高;海马IL-1β转录水平在24 h时表现出一过性升高;下丘脑IL-1β的转录水平1 h即明显升高并于6 h时达峰值,48 h时恢复至对照组水平。结论:DM可快速诱导皮层和下丘脑IL-1βmRNA转录,而海马IL-1βmRNA转录较迟被诱导。（二）DM对大鼠不同脑区IL-1β蛋白含量的影响-免疫组化法目的:观察不同作用时间下DM对神经细胞IL-1β蛋白含量的影响方法:25只雄性SD大鼠随机分为一个对照组和4个DM作用不同时间的实验组,实验组大鼠一次性腹腔注射12.5 mg?kg-1 DM,测定各组1 h、6 h、24 h、48 h皮层、海马CA1、CA2、CA3、DG区和下丘脑IL-1β阳性细胞平均积分光密度。结果:皮层对照组阳性细胞仅在胞浆中有少量IL-1β表达,给药后1 h及6 h含量显著增加;海马CA2、CA3区在给药后48 h呈强阳性表达,而DG区给药后1 h即升高,但整个海马则表现为给药后1 h到48 hIL-1β表达均比对照组高;下丘脑对照组阳性细胞也仅在胞浆有少量IL-1β表达,而24 h和48 h后阳性表达显著增强。结论:皮层和海马IL-1β基因转录的同时伴有IL-1β蛋白的大量表达,而下丘脑则在IL-1βmRNA表达高峰后18 h才出现IL-1β蛋白含量的增加。二、DM对大鼠神经组织的毒性作用-ATPase活力测定目的:以ATPase活力为神经毒性指标,观察不同作用时间下DM对神经细胞的毒性方法:25只雄性SD大鼠随机分为5组:一个对照组和4个DM作用不同时间的实验组,实验组大鼠一次性腹腔注射12.5 mg?kg-1 DM,尔后1 h、6 h、24 h、48 h断头取脑,分别测定皮层、海马和下丘脑Na+-K+-ATPase和Ca2+-Mg2+-ATPase的活力。结果:海马Na+-K+-ATPase活力在给药后6 h和48 h显著下降,但在24 h时出现暂时的恢复;下丘脑的Na+-K+-ATPase活力在1 h比对照组降低。各脑区Ca2+-Mg2+-ATPase活力变化趋势与Na+-K+-ATPase一致。结论:DM对各脑区的作用不同,对海马和下丘脑的毒性较大,而对大脑皮层的毒性作用不明显。在整体水平上,DM诱导IL-1β表达的时相变化在三个脑区各有不同,DM对皮层与下丘脑IL-1β的诱导迅速而对海马IL-1β的诱导较迟缓,分析各脑区IL-1β的诱导与ATPase活力变化的关系,推测给予DM后IL-1β被诱导可能对皮层神经细胞中毒有一定保护作用,而对海马则表现为加剧DM的毒作用,对下丘脑也许也具有一定保护作用,也可能同时参与了诱导远端皮层IL-1β的表达。第二部分利用体外模型,探讨IL-1β在DM神经毒性中的意义在整体实验中,DM对IL-1β的诱导在不同脑区表现不一致,与DM的神经毒性之间的关系不同脑区也有所不同,为深入探讨IL-1β在DM神经毒性中的意义,在这一部分实验中,拟采用体外培养的神经细胞单细胞悬液为模型,观察给予外源性IL-1β和IL-1β受体的天然抑制剂IL-1ra对DM的神经毒性的影响。一、大鼠神经单细胞悬液实验模型的建立目的:制备有活性的体外培养神经细胞方法:成年雄性SD大鼠断头取脑,分离皮层、海马和下丘脑,眼科剪剪碎组织,加0.125%的胰酶于37℃消化30min,每隔5～6min轻柔吹打一次,尔后加等体积血清终止消化,1500rpm离心10min, Hank’s液重悬细胞,置37℃CO2培养箱内培养,1 h和6 h后台盼兰染色各计数一次细胞存活率。结果:1 h内细胞存活率>90%,6 h内细胞存活率>70%结论:成功制备有活性的体外培养神经细胞模型。二、体外实验染毒剂量和染毒时间的确定目的:观察不同浓度DM作用不同时间对离体大鼠神经细胞ATPase活力的影响,以选择体外实验染毒剂量和染毒时间。方法:制备大鼠皮层、海马和下丘脑三个脑区的单细胞悬液,调整细胞数至1×106个/ml,随机分为6个组:对照组、2×10-6mol/L DM组和2×10-5mol/L DM组,DM作用1 h和6 h后分别测定Na+-K+-ATPase的活力。结果:6 h对照组的ATPase活力较1 h对照组明显降低,低剂量与高剂量DM组均显著降低三个脑区神经细胞ATPase的活力。结论:以下体外单细胞悬液实验中,DM剂量选用2×10-6mol/L,作用时间1 h。三、外源性IL-1β对DM作用后的大鼠神经细胞ATPase活力的影响目的:观察给予外源性IL-1β以及其天然抑制剂IL-1ra后对DM损伤ATPase活力的影响方法:皮层、海马和下丘脑三个脑区的单细胞悬液均随机分为5组:对照组、2×10-6mol/L DM组、100U/ml IL-1β组、DM+IL-1β组和0.1μg/ml IL-1ra+DM组; DM作用后15min加入IL-1β,IL-1ra预孵育30min后加DM,DM作用细胞1 h后测Na+-K+-ATPase的活力。结果:DM、IL-1β以及二者的共同作用均使皮层神经细胞ATPase活力降低。预先给予IL-1ra,观察到皮层和下丘脑细胞ATPase活力可部分恢复,而对海马细胞没有观察到有保护作用。结论:该剂量条件下IL-1β并不能加重DM对神经细胞ATPase活力的损伤;但IL-1β的受体拮抗剂IL-1ra对皮层和下丘脑细胞ATPase活力损伤有一定保护作用,说明IL-1ra可阻断IL-1β的作用,减少DM的毒性。四、外源性IL-1β对DM作用后的大鼠神经细胞NO释放的影响目的:观察给予外源性IL-1β以及其天然抑制剂IL-1ra后DM诱导神经细胞NO释放的影响方法:皮层、海马和下丘脑单细胞悬液均随机分为5组:对照组、2×10^-6mol/L DM组、100U/ml IL-1β组、DM+IL-1β组和0.1μg/ml IL-1ra+DM组,DM作用后15min加入IL-1β,IL-1ra预孵育30min后加DM,DM作用细胞1 h后测定细胞培养上清中NO的含量。结果:与对照组相比,DM增加皮层和海马神经细胞NO的释放,IL-1β增加了皮层神经细胞NO的释放,DM与IL-1β联合作用使三个脑区细胞的NO释放均比DM单独作用高,而IL-1ra对DM诱导皮层和海马神经细胞NO释放有抑制作用。结论:IL-1β可能通过NO介导DM对皮层和海马神经毒性中的作用。在本部分实验中, 100U/ml剂量条件下的IL-1β不能加重DM对ATPase活力的损伤,但能促进DM诱导的皮层和海马离体神经细胞NO的释放,此作用可被其内源性受体阻断剂IL-1ra抑制,同时IL-1ra对DM导致的皮层和下丘脑ATPase活力的损伤有一定保护作用,推测IL-1β促进了DM的毒性,但在不同脑区表现不一。第三部分IL-1β转化酶抑制剂对DM神经毒性的影响第二部分里,我们观察到IL-1ra封闭IL-1β受体的作用可部分减轻DM的毒性,本部分实验则欲利用IL-1β转化酶抑制剂减少IL-1β的产生,来观察对DM神经毒性的影响。一、IL-1β转化酶抑制剂对DM作用神经细胞的ATPase活力的影响目的:观察IL-1β转化酶抑制剂对神经细胞的ATPase活力的影响。方法:皮层、海马和下丘脑单细胞悬液均随机分为8个组:对照组、抑制剂1组（10μmol/L Ac-YVAD-CHO）、抑制剂2组（15μmol/L Ac-YVAD-CHO ）、2×10-6mol/L DM组、DM1组（10μmol/L Ac-YVAD-CHO同时加入2×10-6DM）、DM2组（给予2×10-6DM后15min加10μmol/L Ac-YVAD-CHO）、DM3组（15μmol/L Ac-YVAD-CHO同时加入2×10-6DM）和DM4组（给予2×10-6DM后15min加15μmol/L Ac-YVAD-CHO）,DM作用细胞1 h后测Na+-K+-ATPase的活力。结果:皮层细胞DM1、DM2和DM3组ATPase活力较DM组高,下丘脑细胞DM4组ATPase活力较DM组高。结论:本实验剂量和干预时间条件下ICE抑制剂对DM引起的皮层和下丘脑的ATPase损伤有一定保护作用。二、IL-1β转化酶抑制剂对DM作用神经细胞的NO释放的影响目的:观察不同干预时间和剂量的IL-1β转化酶抑制剂对神经细胞的NO释放的影响。方法:分组同第三部分第一小部分,NO测定同第二部分结果: DM促进皮层和海马细胞的NO释放却抑制下丘脑细胞NO的释放;ICE抑制剂可使皮层和海马NO的释放较DM组减少,但对下丘脑细胞,DM4组较对照组NO释放量低而与DM组无差异,其余各抑制剂组NO的释放量均比DM组高。结论:ICE抑制剂对DM诱导皮层和海马细胞NO的释放有拮抗作用。三、IL-1β转化酶抑制剂对DM作用神经细胞的谷氨酸（Glu）释放的影响——HPLC法目的:观察不同干预时间和剂量的IL-1β转化酶抑制剂Ac-YVAD-CHO对神经细胞的谷氨酸释放的影响。方法:分组同第三部分第一小部分,HPLC法测定细胞培养上清液中谷氨酸的含量。结果:DM可显著促进皮层和海马神经细胞的Glu释放,但抑制下丘脑细胞Glu释放;而各抑制剂组对3个脑区的神经细胞均表现为抑制Glu释放。结论:IL-1β可能通过Glu介导参与兴奋性神经毒作用。由本部分研究可见,ICE抑制剂对皮层和下丘脑细胞ATPase活力有一定的保护作用,同时可拮抗DM对皮层、海马神经细胞NO和Glu的释放。推测对皮层和海马的神经细胞,IL-1β在DM的兴奋性神经毒性中可能通过促进NO和Glu介导的途径发挥作用,但并不通过该途径影响DM对下丘脑的作用。更多还原

【Abstract】 Deltamethrin (DM), one of the type II pyrethroids, is employed widely as composition of insecticide, and has drawn widely attention of more and more toxicologists for its latent mammal and human neural toxicity . In recent decades, pyrethroids are thought to be a kind of interfering substance to the web of nerve-endocrine-immune system. Nerve system, endocrine system and immune system interacts each other, many endocrine and immune factors play important roles in the function of nerve system, such as interleukin 1β(IL-1β), which involved in many physical and pathological process (e.g. sleeping, fever, ischemia and hypoxia). The pre-protein of IL-1βis converted to mature form after the dissection by interleukin converting enzyme (ICE) and then is secreted. Nature antagonist of IL-1β(interleukin 1 receptor antagonist, IL-1ra) and the inhibitor of ICE can block the function of IL-1β. The mechanism of neural exciting toxicity induced by DM is similar to ischemia and hypoxia, but the relationship between IL-1βand DM is still unknown. We measured the expression of IL-1βafter DM intoxication, the effect of exogenous IL-1β, IL-1ra and inhibitor of ICE co-operated with DM on the activities of ATPase and the releasing of NO and aminoglutaminic acid (GLU) to explore the role of IL-1βin the nervous lesion induced by DM. Result as follow:Section 1 Expression of IL-1βinduced by DM1. Expression of IL-1βinduced by DM(1). IL-1βmRNA transcription by RT-PCR Aim: To measured the relative expression of IL-1β/ GAPDH. Methods: 15 male SD rats were separated randomly into 5 groups: 1 control group and 4 DM treated groups, 1 h, 6 h, 24 h, 48 h after intraperitoneal injection of 12.5 mg?kg-1 DM, total RNA was extracted from cortex, hippocampus and hypothalamus, the RNA was reverse transcribed to cDNA , IL-1βgene and GAPDH gene were replicated by PCR., and then we measured the relative expression of IL-1β/ GAPDH.Results: The expression of IL-1βmRNA increased in 1 h in cortex, and in hippocampus it showed a transient up-regulation at 24 h, while in hypothalamus it reached maximum at 6 h. Conclusions: DM up-regulated IL-1βmRNA in cortex and hypothalamus rapidly, and IL-1βmRNA expression increased 18 h later in hippocampus.(2). IL-1βprotein content by immunohistochemistryAim: To compared the content of IL-1βin different time after DM injection. Methods: 25 male SD rats were separated randomly into 5 groups: 1 control group and 4 DM treated groups, 1 h, 6 h, 24 h, 48 h after intraperitoneal injection of 12.5 mg?kg-1 DM, we used immunohistochemistry to detect the OD of IL-1βpositive cells in cortex, CA1,CA2,CA3 and DG regions of hippocampus and hypothalamus.Results: In cortex, IL-1βof 1 h and 6 h groups were higher than the control group; in hippocampus, IL-1βwas up-regulated in 1 h, and kept higher than the control group till 48 h, while in the CA2 and CA3 regions of hippocampus, IL-1βwas elevated at 48 h, and in DG IL-1βexpression was up-regulated in 1 h; in hypothalamus IL-1βshowed higher after 24 h compared with the control group.Conclusions: The mRNA and protein expression of IL-1βwere up-regulated simultaneously in cortex and hippocampus; and in hypothalamus, molecular of IL-1βprotein augmented 18 h after its gene expression.2. The changing of the activities of ATPase after DM administration Aim: To detect the activities of ATPase in different times after DM administration. Methods: 25 male SD rats were separated randomly into 5 groups: 1 control group and 4 DM treated groups, 1 h, 6 h, 24 h, 48 h after intraperitoneal injection of 12.5 mg·kg-1 DM, we measured the activities of ATPase in cortex, hippocampus and hypothalamus. Results: The activities of Na+-K+-ATPase in hippocampus decreased in 6 h group and 48 h group, and showed a transient recovery in 24 h group; the activities of Na+-K+-ATPase of hypothalamus decreased in 1 h group.Conclusions: Na+-K+-ATPase in hippocampus and hypothalamus tended to be damaged by DM but cortex seemed be tolerant to DM. In this section, IL-1βinduced by DM showed different peak point times in different cerebral regions, and when we analyzed relationship between IL-1βand the lesion of ATPase we concluded that IL-1βmay play a protective role in cortex and hypothalamus, while may aggravate the hippocampus lesion.Section 2 Effect of exogenous IL-1βon the neural toxicity of DMAs the IL-1βshowed different roles involved in different CNS regions in section 1, we wanted to know whether exogenous IL-1βand the nature antagonist of IL-1β, IL-1ra, could intensify or block the DM toxicity in vitro. Cultured neural cells were applied. 1. Prepared compt. cultured neural cellsAim: To establish the model of compt. cultured neural cells for the next experiment in vitro. Methods: Male adult rats were decapitated, cortex, hippocampus and hypothalamus were digested by 0.125% trypsogen at 37℃for 30min, blew the dish per 5～6min, then added 1:1 volume of serum to stop digest, centrifuged at 1500rpm for 10min, cells were suspended with Hank’s buffer and cultured in CO2 incubator, after 1 h and 6 h, the unicells were dyed with trypan blue to detect the cell survival rates.Results: The cell survival rate in 1 h was >90%, and at 6 h, the cell survival rate was >70%. Conclusion: Cultured neural cells model was established successfully.2. Choose the dose of DM administration and its action time Aim: Observing the effect of different dose of DM on the activities of Na+-K+-ATPase to choose the dose and the action time of DM.Methods: Cortex, hippocampus and hypothalamus were separated from cerebrum and dissected to unicells, the neural cells were treated with 2×10-6mol/L and 2×10-5mol/L DM, after 1 h and 6 h the cells were harvest for activities of Na+-K+-ATPase.Results: The activities of Na+-K+-ATPase of all the three brain region cells’were damaged by different dose DM, even the 6 h control group showed lower activity of Na+-K+-ATPase than the 1 h control group. Conclusions: Dose 2×10-6mol/L and 1 h time point were chosen to the next experiments in vitro.3. Effect of exogenous IL-1βand IL-1ra on the activities of ATPase Aim: To observe the effect of exogenous IL-1βand IL-1ra on the activities of Na+-K+-ATPase Methods: Cells of three encephalic regions were separated into 5 groups: control group, 2×10-6mol/L DM group, 100U/ml IL-1βgroup, DM+IL-1βgroup and 0.1μg/ml IL-1ra+DM group, 1 h after DM treated, cells were collected for activities of Na+-K+-ATPase.Results: DM group, IL-1βgroup and DM+IL-1βgroup manifested decreasing of the activities of ATPase of cortex cells, IL-1ra+DM group showed higher activities of ATPase compared with the DM group of cortex and hypothalamus cells.Conclusion: 100U/ml IL-1βand DM had no synergistic effect on ATPase damage, but IL-1ra showed protective effect on the activities of ATPase of cortex and hypothalamus cells.4. Effect of exogenous IL-1βand IL-1ra on the releasing of NOAim: To observe the effect of exogenous IL-1βand IL-1ra on the releasing of NO. Methods: Cells of three encephalic regions were separated into 5 groups: control group, 2×10-6mol/L DM group, 100U/ml IL-1βgroup, DM+IL-1βgroup and 0.1μg/ml IL-1ra+DM group, 1 h after DM treating, supernatant were collected for the NO content.Results: The NO release of DM group in cortex and hippocampus neural cells were higher than the control group; and IL-1βgroup of cortex was higher than the control group also, but the NO release of hypothalamus was lower than the control group; DM+IL-1βgroups of cortex, hippocampus and hypothalamus manifested a drastic up-regulation compared with the DM group; IL-1ra showed suppressed effect on the NO release of cortex and hippocampus cells. Conclusions: IL-1βmay be involved in the function of DM neural toxicity in cortex and hippocampus by causing NO releasing.Though the exogenous IL-1βof dose 100U/ml couldn’t aggravate the activities losing of ATPase, IL-1ra could protect the ATPase in cortex and hypothalamus, and it could decrease the releasing of NO in cortex and hippocampus, we concluded IL-1βmay involved in DM neural toxicity through NO pathway.Section 3 Effect of the inhibitor of interleukin converting enzyme on the neural toxicity of DMWe had observed the effect of exogenous IL-1βand IL-1ra on the DM neural toxicity, in this section, we wanted to applied inhibitor of interleukin converting enzyme to inhibit the maturation of IL-1βand observed the effect of DM on neural cells. 1. Effect of the ICE inhibitor on the activities of ATPaseAim: To observe the effect of ICE inhibitor on the activities of Na+-K+-ATPase.Methods: Cells of three encephalic regions were separated into 8 groups: control group, 10μmol/LAc-YVAD-CHO group, 15μmol/L Ac-YVAD-CHO group, 2×10-6mol/L DM group, DM1 group(cells were treated with 10μmol/L Ac-YVAD-CHO and 2×10-6DM simultaneously), DM2 group(15min after DM treating 10μmol/L Ac-YVAD-CHO was added into culture),DM3 group(cells were treated with 15μmol/L Ac-YVAD-CHO and 2×10-6DM simultaneously)and DM4 group(15min after DM treating 10μmol/L Ac-YVAD-CHO was added into culture), 1 h after DM treating, cells were collected for activities of Na+-K+-ATPase.Results: The activities of Na+-K+-ATPase of DM1, DM2, DM3 groups of cortex cells were higher than the DM group, while those of DM4 group of hypothalamus cells were higher than the DM group.Conclusions: Ac-YVAD-CHO had protective effect on the activities of ATPase of cortex and hypothalamus.2. Effect of ICE inhibitor on the releasing of NO Aim: To observe the effect of ICE inhibitor on the releasing of NO. Methods: Cells of three encephalic regions were separated into 8 groups: control group, 10μmol/L Ac-YVAD-CHO group, 15μmol/L Ac-YVAD-CHO group, 2×10-6mol/L DM group, DM1 group, DM2 group, DM3 group and DM4 group, 1 h after DM treating, supernatant were collected for the NO content.Results: DM increased the release of NO of cortex and hippocampus cells but reduced NO release of hypothalamus cells compared with the control group; inhibitor of ICE suppressed the release of NO of cortex and hippocampus but induced NO release of hypothalamus. Conclusions: Inhibitor of ICE could block the effect of DM on releasing of NO of cortex and hippocampus cells.3. Effect of ICE inhibitor on the releasing of GLUAim: To observe the effect of ICE inhibitor on the releasing of GLU. Methods: Cells of three encephalic regions were separated into 8 groups: control group, 10μmol/L Ac-YVAD-CHO group, 15μmol/L Ac-YVAD-CHO group, 2×10-6mol/L DM group, DM1 group, DM2 group, DM3 group and DM4 group, 1 h after DM treating, supernatant were collected for the Glu content.Results: DM increased the release of GLU of cortex and hippocampus, while reduced that of hypothalamus; DM1, DM2, DM3 and DM4 groups all showed blocking effect of releasing of GLU induced by DM.Conclusions: IL-1βmay participates in the DM neuronal toxicity by GLU.In this section, we found that the ICE inhibitor can protect the activities of ATPase of cortex and hypothalamus cells, and it can antagonize the effect of DM on the NO and GLU release of cortex and hippocampus.As mentioned above, to cortex and hippocampus, NO and Glu might involved in the function of IL-1βin nerve lesion induced by DM , but to hypothalamus, there might be some other factors playing roles in neural toxicity of DM.更多还原