【作者】 邢光伟；

【导师】 许文荣； 陆荣柱；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 目的丙烯腈(acrylonitrile,AN)是有机合成工业的重要原料,广泛应用于合成腈纶纤维、丁腈橡胶、ABS工程塑料、合成树脂等,因此职业和生活接触机会明显增多。为保护接触人群健康,国内外学者对其毒性特别是神经毒性进行了积极探索,深入探讨AN神经毒性的机理以及寻求有效的化学防护剂已经成为AN毒理学研究的热点。姜黄素(Curcumin,Cur)是中药姜黄中的酚类色素成分,具有广泛的生物药理活性。本研究通过急性和亚急性实验,从氧化应激和能量代谢二方面探讨AN的神经毒性的机理,并以Cur为干预物,研究其对AN神经毒性的防护效果,为探讨AN神经毒作用机制提供生化基础资料,为AN职业防护保健食品的开发提供实验依据。方法急性实验,将28只雄性Sprague-Dawley(SD)大鼠按体重随机分为四组,正常对照组、AN组、50mg/kg Cur+AN组、100mg/kgCur+AN组,每天灌胃姜黄素或橄榄油,连续七天,最后一次灌胃(ig)姜黄素2h后腹腔注射50mg/kg AN。亚急性实验,以成年健康雄性SD大鼠为研究对象,随机分为四组,正常对照组、Cur组、AN组、Cur+AN组,每天灌胃姜黄素或橄榄油,2 h后腹腔注射50 mg/kg AN或生理盐水,连续14天。观察指标:行为学指标;脑组织和肝组织脂质过氧化产物丙二醛(MDA)、还原型谷胱甘肽(GSH)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性;细胞色素C氧化酶活性(cytochrome c oxidase);脑组织中氰离子(CN~-);肝组织细胞色素P4502E1(CYP2E1)活性,CYP2E1蛋白表达,CYP2E1基因表达;组织形态学观察。结果急性实验:①AN组大鼠出现流泪,流涎,呼吸困难;②AN组在脑组织和肝组织中MDA水平比对照组分别增加了38.6%和10.8%,GSH含量比对照组分别下降38.6%和70.5%,组织中CAT活性明显的下降(P＜0.05);姜黄素预处理减轻AN引起的作用,脑组织和肝组织MDA水平明显降低,GSH含量和CAT活性增加明显,在脑组织中,GSH水平恢复到正常组83.2%,而在肝组织中,仅恢复了46.6%;③AN组中CcOx活性与对照组相比有明显的下降,该作用可被100mg/kgCur预处理明显减轻,100mg/kgCur+AN组脑组织中的CcOx活性恢复要比肝组织恢复高,分别是75.7%和58.4%;④各组CYP2E1活性变化不明显。亚急性实验:①每次腹腔注射AN后20分钟,大鼠出现流泪,流涎,耳廓发红,腹泻,鼻孔渗血,1小时后症状基本消失,Cur+AN组以上症状有所改善;②AN组MDA在脑组织和肝组织中明显增加(P＜0.05),GSH含量显著减少(P＜0.05),CAT活性对照组分别下降54.3%和10.2%;SOD活性下降,仅脑组织有显著性差异(P＜0.05),姜黄素预处理明显降低脑组织和肝组织MDA水平,明显增加GSH含量、脑组织SOD和CAT活性,与AN组相比MDA分别下降33.9%和25.2%,CAT活性分别升高68.1%和17.2%;③脑组织中AN组与对照组相比CHE含量升高明显,Cur+AN组与AN组相比CHE含量明显下降;④在肝组织中,各组CYP2E1活性无明显的区别,各组CYP2E1蛋白,基因表达无差异。结论姜黄素降低组织脂质过氧化,升高酶性抗氧化酶SOD,CAT活性和非酶性抗氧化酶GSH活性,姜黄素对AN诱导的氧化性神经损伤具有明显的保护作用。姜黄素对AN诱导氧化性神经损伤的保护作用可能不是通过抑制CYP2E1水平来完成,是一种安全有效防治AN及相关毒物的神经毒性的植物。更多还原

【Abstract】 Objective: Acrylonitrile (Acrylonitril, AN) is an important raw material in organic synthesis industry, widely used in synthesis of acrylic fibers, nitrile rubber, ABS engineering plastics, synthetic resin, etc. In recent years, with rapid development and expansion of AN industry, occupational and daily life exposure significantly increased, Thus the protection of human beings exposed to AN, much attention has been paid to its toxicity, especially neurotoxicity. Therefore,it is necessary to explore the mechanism of neurotoxicity of AN and look for effective chemopreventive agents against AN toxicity. In this study, based on the acute and subacute experiments, by evaluation of biomarkers of oxidative stress, energy metabolism and inflammmation, the protection of Curcumin, a phytochemical extracted from Curcuma longa L. against AN neurotoxicity was explored. And it was hoped that the obtained findings will be useful to development of health food products for occupational protection.Methods: In acute experiments, twenty-eight adult healthy male Sprague-Dawley (SD) rats were randomly divided into four groups according to their body weights (seven rats in each)control group, AN group, AN following 50mg/kgCur pretreatment group and AN following 100mg/kgCur pretreatment group. The rats were orally administered curcumin at doses of 0 (olive oil control), 50 or 100 mg/kg bodyweight daily for 7 consecutive days. Two hours after the last dose of curcumin, rats received an intraperitoneal injection of 50mg AN/kg body weight. In subacute experiments, twenty-eight adult healthy male SD rats were randomly divided into four groups (seven rats in each). control group, Cur group,AN group and AN following 100mg/kgCur pretreatment group(Cur+AN). The rats were orally administered curcumin at doses of 0 (olive oil control), 100 mg/kg body weight daily, two hours after the dose of curcumin, rats received an intraperitoneal injection of 50mg AN/kg body weight for 14 consecutive days. Neurotoxicity was assessed by behavioral observations, the reduced glutathione levels (GSH), glutathione lipid peroxidation (MDA), catalase(CAT) and superoxide dismutase(SOD) activity in the brain and liver. The cytochrome c oxidase activity in brain and liver, CN~- content in rat brain, The activities of CYP2E1, CYP2E1 gene, CYP2E1 protein in the liver and histological changes were detected.Results: Acute experiment:①Behavioral observations showed that all AN-treated animals exhibited similar pattern of cholinomimetic signs.②A significant increase in levels of MDA was observed in the AN-treated group both in the brain and liver compared with the control group(38.6% and 10.8% respectively), These increases were accompanied by asignificant decrease in GSH content (38.6% and 70.5 %respectively)and a significant reduction in CAT activity in the same tissues. Pretreatment with curcumin reversed the AN-induced effects, reducing the levels of MDA and enhancing CAT activity and increasing reduced GSH content both in the brain and liver. At 100 mg/kg curcumin, the brain level of GSH returned to 83.2% of the control level, whereas hepatic GSH recovery was only 46.6%.③AN treatment significantly reduced thecytochrome c oxidase activity in the brain and liver compared with the control group, and these effects were significantly mitigated by 100 mg/kg curcumin pretreatment both in the brain and liver. The extents of recovery of activity of cytochrome c oxidase upon curcumin pretreatment in the brain was relatively higher compared with the liver, reaching approximately 75% and 58% of the control values, for brain and liver, respectively.④The hepatic CYP2E1 activity in the control and the various treatment groups. Neither the change between the AN alone and the control group, nor changes between the AN alone and the ANwith curcumin pretreatment groups were statistically significant.Subacute experiment:①Twenty minuntes later, the cholinomimetic signs, such as salivation or lacrimation and labored breathing, were observed for in AN group rats, and after about one hour, the previous symptoms disappeared gradually, the effects were significantly mitigated by curcumin pretreatment.②A significant increase in levels of MDA was observed in the AN-treated group both in the brain and liver compared with the control group, These increases were accompanied by asignificant decrease in GSH content and a significant reduction in CAT activity (54.3% and 10.2% respectively)in the same tissues. SOD activity in the brain and liver was reduced upon AN treatment, a significant decrease in SOD activity in the brain compared with controls. Pretreatment with curcumin reversed the AN-induced effects, reducing the levels of MDA and enhancing CAT activity and increasing reduced GSH content both in the brain and liver, enhancing SOD activity in the brain. Compared with the AN group, levels of MDA was significantly decreased both in brains and livers of rats in the Cur+AN group ( 33.9% and25.2% respectively). Compared with the AN group, CAT activity was significantly increased both in brains and livers of rats in the Cur+AN group (68.1% and 17.2% respectively).③A significant increase in levels of CHE was observed in the AN-treated group both in the brain compared with the control group, Pretreatment with curcumin reversed the AN-induced effects.④The activities of CYP2E1 in each group showed no significant difference by Western blot and RT-PCR detection.Conclusion:The present study provides direct evidence for the involvement of curcumin in neuroprotection against oxidative stress. Curcumin may also terminate lipid peroxidation by induction ofenzymatic and non-enzymatic antioxidants, such as GSH, SOD and CAT. curcumin is a safe and effective plants to prevent oxidative damage and related toxins, the way that curcumin pretreatment protects against acrylonitrile-induced oxidative damage, may not be the means by changing the level of CYP2E1.更多还原