【作者】 张彩英；

【导师】 胡国良；

【作者基本信息】 江西农业大学 ， 兽医学， 2022， 博士

【摘要】 钼（Mo）是动物机体必需的微量元素,但过量的钼会对机体产生毒性作用。镉（Cd）是毒性极强的累积性的工业和环境重金属污染物。工农业生产常造成钼、镉对环境的联合污染,最终通过食物链危害人畜健康。肾脏是钼、镉毒性作用的重要靶器官,肾小管是其致肾毒性损伤的靶部位。细胞自噬和焦亡是两种在形态和机制上完全不同的程序性细胞死亡,但它们又紧密联系、相互影响。目前,钼镉联合致肾损伤过程中自噬和焦亡的发生及其机制以及两者之间的调控关系的研究未见报道。因此,本研究以绍兴鸭为研究对象,旨在探讨钼镉联合诱导鸭肾脏细胞自噬和焦亡的作用机制。体内试验选用240羽8日龄健康绍兴鸭随机分为4组（60羽/组）,对照组（Control组,饲喂基础日粮）、Mo组[100 mg/kg Mo,（NH₄）₆Mo₇O₂₄·4H₂O]、Cd组（4 mg/kg Cd,3Cd SO₄·8H₂O）和Mo+Cd组（100 mg/kg Mo+4 mg/kg Cd）,在试验第4、8、12和16周,每组随机选取10羽鸭,采集血液和肾脏,应用透射电镜、实时荧光定量PCR和免疫印迹等方法,检测肾脏微量元素含量、氧化应激指标、自噬和焦亡水平等。体外试验以原代鸭肾小管上皮细胞为研究对象,分别用Mo或Cd处理细胞12 h的IC₅₀与一系列浓度的Cd或Mo联合处理细胞12 h,或用500μM Mo、4μM Cd和500μM Mo+4μM Cd处理细胞12 h,,最后分别用抗氧化剂100μM BHA、焦亡抑制剂（10μM MCC950和10μM Z-YVAD-FMK）和自噬抑制剂（2.5μM 3-MA和10μM CQ）干预Mo+Cd染毒试验12 h,收集细胞和上清样,应用流式细胞术、实时荧光定量PCR和免疫印迹等方法,检测细胞活力、Mo和Cd含量、氧化应激指标、自噬和焦亡水平等。结果表明:1.Mo或/和Cd显著增加其在肾脏中的含量,降低Cu、Fe、Zn、Se含量、CAT、T-SOD、GSH-Px活性和提高MDA、H₂O₂、UA和CREA含量;导致肾小管上皮细胞变性、坏死、肾间质充血、出血、炎性细胞浸润,线粒体肿胀、嵴断裂甚至空泡化、核变形和固缩等;显著增加自噬相关因子（AMPKα-1、Atg5、Beclin-1、LC3A、LC3B和LC3II/LC3I）表达、自噬体和LC3II聚点数量,降低自噬相关因子（m TOR、Dynein和p62）表达;显著上调焦亡相关因子（NLRP3、Caspase-1、ASC、NEK7、GSDMD、GSDME、GSDMA、IL-1β和IL-18）表达、IL-1β和IL-18含量,且呈时间-效应。以上结果表明,Mo或/和Cd能够导致肾脏微量元素稳态失衡、氧化应激和形态结构异常,以及发生自噬和焦亡。2.Mo或/和Cd导致细胞发生皱缩、部分细胞呈圆形、空泡化,显著降低细胞活力,钼镉非等毒暴露呈协同作用。3.Mo或/和Cd显著增加其在细胞内的含量、诱导细胞发生氧化应激和自噬,MCC950显著降低钼镉诱导的焦亡,说明Mo或/和Cd可诱导细胞发生自噬和焦亡,且钼镉联合能通过NLRP3/Caspase-1通路诱导细胞发生焦亡。4.BHA显著降低钼镉联合诱导的氧化应激、自噬和焦亡,说明氧化应激介导了钼镉联合诱导的自噬和焦亡的发生。5.Z-YVAD-FMK显著降低钼镉联合诱导的自噬,说明抑制焦亡能显著降低钼镉联合诱导的自噬。6.3-MA（或CQ）显著提高钼镉联合诱导的氧化损伤和焦亡,说明抑制自噬能显著提高钼镉联合诱导的焦亡。综上所述,钼镉及其联合能够干扰鸭肾脏微量元素稳态、引起氧化应激、从而导致肾功能异常和肾脏组织发生病理损伤;钼镉非等毒暴露呈协同作用;钼镉联合能诱导鸭肾脏发生氧化应激介导的细胞自噬和焦亡;抑制焦亡能显著降低钼镉联合诱导的鸭肾小管上皮细胞自噬;抑制自噬能显著提高钼镉联合诱导的鸭肾小管上皮细胞焦亡。更多还原

【Abstract】 Molybdenum（Mo）is an essential trace element for animals,but excess Mo has toxic effects on the body.Cadmium（Cd）is a highly toxic,cumulative industrial and environmental heavy metal pollutant.The combined pollution of Mo and Cd to the environment is often caused by industrial and agricultural production,and ultimately endangers animal and human health through the food chain.The kidney is a crucial target organ for the toxicity of Mo and Cd,and the renal tubule is the target site of nephrotoxic damage.Autophagy and pyroptosis are two kinds of programmed cell death that are completely different in morphology and mechanism,but they are closely related and affect each other.At present,there is no report on the occurrence mechanism of autophagy and pyroptosis in the process of kidney injury caused by the combination of Mo and Cd as well as the regulation relationship between the two metals.Therefore,in this study,duck was used as the experimental animal to explore the mechanism of autophagy and pyroptosis co-induced by Mo and Cd in duck kidneys.240 healthy 8-day-old Shaoxing ducks（Anas platyrhyncha）were randomly divided into 4 groups（60 birds/group）,and Cd or/and Mo were added to basic diet per kilogram（kg）in vivo:control group（0 mg Mo and 0 mg Cd）,Mo group[100 mg Mo,（NH₄）₆Mo₇O₂₄·4H₂O],Cd group（4 mg Cd,3Cd SO₄·8H₂O）,and Mo+Cd group（100 mg Mo and 4 mg Cd）.At the 4^th,8^th,12^th,and 16^thweeks of the experiment,10 ducks were randomly collected from each group,blood and kidneys of each duck were collected,respectively.Transmission electron microscopy,real-time fluorescent quantitative PCR and Western blotting and so on were used to detect the contents of trace elements,oxidative stress indexes,and the levels of autophagy and pyroptosis in kidneys.Additionally,duck primary renal tubular epithelial cells were used as the research object in vitro.The IC₅₀of cells treated with Mo or Cd alone for 12 h was combined with a series of concentrations of Cd or Mo for 12 h,or cells were treated with 500μM Mo,4μM Cd and500μM Mo+4μM Cd for 12 h,respectively.Finally,antioxidant 100μM BHA,pyroptosis inhibitors（10μM MCC950 and 10μM Z-YVAD-FMK）and autophagy inhibitors（2.5μM3-MA and 10μM CQ）were applied for the intervention of Mo and Cd co-exposure for 12h,respectively.Cells and supernatant samples were collected,respectively.The cell viability,Mo and Cd contents,oxidative stress indexes,the levels of autophagy and pyroptosis were detected by using flow cytometry,real-time fluorescent quantitative PCR,Western blotting and so on.The results were as following:1.Mo or/and Cd obviously increased their contents in kidneys,decreased Cu,Fe,Zn,Se contents and CAT,T-SOD,GSH-Px activities,and elevated MDA,H₂O₂,UA and CREA contents,resulting in renal tubular epithelial cells degeneration,necrosis,interstitial congestion,bleeding and inflammatory cell infiltration as well as mitochondrial swelling,cristae fragmentation,even vacuolization,nuclear deformation and pyknosis;increased the number of autophagosomes and LC3II puncta,evidently upregulated autophagy-related factors（AMPKα-1,Atg5,Beclin-1,LC3A,LC3B and LC3II/LC3I）expression levels,markedly downregulated autophagy-related factors（m TOR,Dynein and p62）expression levels;obviously elevated pyroptosis-related factors（NLRP3,Caspase-1,ASC,NEK7,GSDMD,GSDME,GSDMA,IL-1βand IL-18）expression levels and the contents of IL-1βand IL-18,and showed time-effects.The above results showed that Mo and/or Cd could interfere with the homeostasis of trace elements in duck kidneys,thereby causing oxidative stress,ultimately leading to pathological damage to renal tissue,and induced autophagy and pyroptosis.2.Mo or/and Cd caused shrink,round and vacuolation of cells,and significantly reduced cell viability.Toxicity of non-equitoxic binary mixtures of Mo and Cd presented synergic effects.3.Mo or/and Cd markedly increased the contents of Mo and Cd in cells,induced oxidative stress and autophagy,and MCC950 remarkably decreased pyroptosis caused by Mo or/and Cd,which demonstrated that Mo or/and Cd triggered autophagy and pyroptosis and the combination of Mo and Cd induced pyroptosis through the NLRP3/Caspase-1pathway..4.BHA markedly reduced oxidative stress,autophagy and pyroptosis co-induced by Mo and Cd,which indicated that autophagy and pyroptosis co-induced by Mo and Cd were mediated by oxidative stress.5.Z-YVAD-FMK dramatically alleviated autophagy co-induced by Mo and Cd,indicating that inhibiting pyroptosis notably reduced autophagy co-induced by Mo and Cd.6.3-MA（or CQ）distinctly enhanced oxidative damage and pyroptosis co-induced by Mo and Cd,illustrating that inhibiting autophagy obviously promoted pyroptosis co-induced by Mo and Cd.In conclusion,Mo or/and Cd could interfere with the homeostasis of trace elements in duck kidneys and cause oxidative stress,thereby leading to abnormal renal function and renal pathological damage.Toxicity of non-equitoxic binary mixtures of Mo and Cd presented synergic effects.Mo and Cd co-exposure induced autophagy and pyroptosis mediated by oxidative stress in duck kidneys.Inhibition of pyroptosis remarkably reduced autophagy co-induced by Mo and Cd in duck renal tubular epithelial cells,and inhibition of autophagy markedly increased pyroptosis co-induced by Mo and Cd.更多还原