【作者】 徐敏；

【导师】 李英伦；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2018， 硕士

【摘要】 纳米铜（Cu NPs）已逐渐作为饲料添加剂使用。Cu NPs的主要毒性靶器官是肝脏、肾脏和脾脏,进入机体可引起应激反应、炎症反应和组织损伤。但有关Cu NPs亚慢性中毒引起组织中细胞色素P450酶（CYP450s）的变化及其变化机制鲜有报道。本课题重点关注Cu NPs-肾CYP450s相互作用及机制。通过研究不同粒径Cu NPs的动力学特征,发现肾脏是Cu NPs的蓄积器官和毒作用靶器官。为进一步探讨Cu NPs是否通过其毒性效应影响肾CYP450s的表达和活力,我们建立大鼠Cu NPs（80-100nm）28 d亚慢性染毒模型,研究Cu NPs对大鼠肾脏的功能、组织病理、氧化/氮化因子和细胞因子的影响,最后通过荧光定量PCR（RT-PCR）、Western Blot、“鸡尾酒”探针药物法及液相芯片技术分别检测肾脏中CYP450s的mRNA表达、蛋白表达和体外酶活力及信号通路相关蛋白的表达。进一步分析Cu NPs影响肾CYP450s表达和活力的机制。结果如下:1.Cu NPs体内分布研究Cu NPs与Cu²⁺在组织的分布无明显差异,Cu NPs达到各脏器无明显延迟效应,肝脏和肾脏是Cu NPs分布最多且可能积累的主要器官。2.Cu NPs对大鼠肾脏的毒性研究（1）对肾脏的影响:Cu NPs 200 mg/kg亚慢性染毒可显著升高尿素（UREA）和肌酐（CREA）水平（p<0.05）;体重极显著下降（p<0.01）,肾脏质量及肾脏器指数显著升高（p<0.05）;且对肾具有病理损伤作用。（2）对应激因子的影响:Cu NPs 200 mg/kg可极显著升氧化应激因子高谷胱甘肽过氧化酶（GSH-Px）、超氧化物歧化酶（SOD）、总抗氧化力（T-AOC）和丙二醛（MDA）的水平（p<0.01）,也极显著升高了氮化应激因子一氧化氮（NO）和诱导型一氧化氮合酶（iNOS）的水平（p<0.01）,且具有剂量依赖性。（3）对细胞因子的影响:Cu NPs 200 mg/kg可极显著升高白介素（IL）-2、干扰素（IFN）-γ、肿瘤坏死因子（TNF）-α和趋化因子（MIP）-1α等炎性细胞因子的表达水平（p<0.01）。3.Cu NPs对肾CYP450s的影响Cu NPs可极显著下调CYP2C11、CYP2D6和CYP3A1的mRNA和蛋白表达（p<0.01）,并极显著抑制CYP450s各亚型的酶活力（p<0.01）。4.Cu NPs对肾CYP450s的作用机制研究Cu NPs可显著下调芳香烃受体（AhR）、组成型雄烷受体（CAR）和孕烷X受体（PXR）的蛋白表达水平（p<0.05）;可显著上调CREB、p70S6K、JNK、Akt和STAT3的总蛋白与磷酸化水平的表达（p<0.05）,及NF-κB、P38和STAT5的磷酸化水平（p<0.05）,表明Cu NPs可激活与炎症相关的信号通路。综上,Cu NPs对大鼠肾脏有较强的毒性作用,可显著影响肾CYP450s的表达和活力,推测Cu NPs进入机体后,引起应激及炎症反应,通过信号通路的级联作用与胞内活性分子的网络调节,引起下游靶因子的变化,从而影响CYP450s的正常表达和功能。更多还原

【Abstract】 Cu Nanoparticles（Cu NPs）has gradually been used as a feed additive.The toxic target organs of Cu NPs are mainly liver,kidney and spleen,and Cu NPs enters into the body,resulting in stress reaction,inflammatory reaction and tissue damage.However,there are few reports on the changes and mechanisms of cytochrome P450 enzymes（CYP450s）in tissues caused by chronic Cu NPs poisoning.This topic focused on the interaction and mechanism of Cu NPs-renal CYP450s.By studying the dynamic characteristics of Cu NPs with different particle sizes,it was found that the kidney was the accumulating organ and the target organ of toxic action of Cu NPs.In order to further explore whether Cu NPs affects the expression and activity of renal CYP450s through its toxic effects.The 28 d subchronic exposure model was established by continuous perfusion of 80-100 nm Cu NPs,study on the effect of Cu NPs subchronic exposure on rat kidney function,tissue pathology,oxidation/nitriding factors and cytokines,Finally,the mRNA expression,protein expression,in vitro activity of CYP450s and signal pathway related protein expression in kidney were detected by quantitative real-time PCR（RT-PCR）,Western Blot,“Cocktail”probe drug and luminex technology multiplex assay,respectively.To further analyze the mechanism of Cu NPs affecting the expression and activity of CYP450s in the kidney.The results are as follows:1.The pharmacokinetics of Cu NPs in ratsThere was no significant difference in the distribution of Cu NPs and Cu²⁺in the organs,organs of Cu NPs reached no delayed effect,and determine the liver and kidney are the main organs of Cu NPs that is most distributed and may accumulate.2.Study on the toxicity of Cu NPs on rat kidney（1）Effects on kidney:Cu NPs 200 mg/kg subchronic exposure could significantly increase urea（UREA）and creatinine（CREA）level（p<0.05）,weight significantly decreased,kidney and kidney index increased significantly（p<0.01）,and had significant pathological damage effect（p<0.05）.（2）Effect of stress responses:Cu NPs 200 mg/kg could increase the glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）,total antioxidant capacity（T-AOC）and malondialdehyde（MDA）levels（p<0.01）,also significantly increased nitric oxide（NO）and inducible nitric oxide synthase（iNOS）expressions,and there are dose dependent（p<0.01）.（3）Effects of cytokines:Cu NPs 200 mg/kg could significantly increase the expression levels of inflammatory cytokines such as IL-2,interferon（IFN）-gamma,tumor necrosis factor（TNF）-A and chemokines（MIP）-1 alpha（p<0.01）.3.The effect of Cu NPs on renal CYP450sCu NPs could significantly reduce the mRNA expression and protein expression of CYP2C11,CYP2D6 and CYP3A1（p<0.01）,and inhibit the activity of all the subtypes of renal CYP450s in vitro（p<0.01）.4.The study of the mechanism of Cu NPs on renal CYP450sCu NPs could significantly decrease the aryl hydrocarbon receptor（AhR）,constitutive androstane receptor（CAR）and pregnane X receptor（PXR）protein expression levels（p<0.05）;Cu NPs significantly increased total protein and phosphorylation levels of CREB,p70S6K,JNK,Akt and STAT3（p<0.05）,and significantly increased NF-κB,P38,and STAT5 phosphorylation levels（p<0.05）.Showed that Cu NPs can activate the signaling pathways associated with inflammation.In conclusion,Cu NPs had strong toxic effects on rat kidney,could significantly affect the expression and activity of renal CYP450s.It is speculated that Cu NPs can cause stress and inflammatory reaction after entering the body,through the cascading of signal pathways and the network of intracellular active molecules,thereby affecting the normal expression and function of CYP450s.更多还原