Mechanism Of Targeting Metallothionein To Alleviate The Hepatic Toxicity Induced By Aflatoxin B1 To Duckling

Aflatoxin B₁（AFB₁）is known to contaminate a large portion of feed products and food supply,which seriously threatens the health of animals and the safety of food.Among poultry,ducks are the most susceptible species to AFB₁.In addition,liver is the main target organ of AFB₁.Recently,increased evidence shows that reactive oxygen species produced by AFB₁ cause toxicity through oxidative stress.Metallothionein（MT）is metal binding proteins,which is an antioxidant in the liver.Previous studies of our group found that AFB₁ inhibited the expression of MT in liver of duckling,which was related to the hepatic injury caused by AFB₁,but the mechanism was not clear.The objective of this study is to reveal the mechanism of AFB₁ regulating MT expression in hepatic damage by toxicity experiments in ducklings and cell experiments,and to explore the feasibility of ZnO NPs to alleviate AFB₁ hepatotoxicity.Part 1 The effect of AFB₁ on the hepatic injury of ducklings and the alleviating effect of ZnO NPs to AFB₁In this experiment,96 one-day-old Cherry Valley ducks were randomly divided into4 groups（24 ducks in each group）:control group（Basic diet,BD）,AFB₁ group（BD+40μg/kg BW AFB₁）,ZnO NPs group（BD+60 mg/kg ZnO NPs）and AFB₁+ZnO NPs group（BD+40μg/kg BW AFB₁+60 mg/kg ZnO NPs）.AFB₁ was administered by gavage.During the experiment,the body weight was recorded.Six ducks were randomly selected from each group for slaughter on the 7th day of continuous gavage and the 5th day after the end of gavage.Then,the serum biochemical indexes,organ index,histopathology,antioxidation,zinc content and the expression of related genes in liver were determined to explore the possible mechanism of hepatic damage induced by AFB₁ in ducklings and the alleviating effect of ZnO NPs to AFB₁.Results are as follows:1.Effects of AFB₁ and ZnO NPs on growth performance of ducklings:After 7 days of continuous intragastric administration of AFB₁,the body weight of the AFB₁ group was significantly decreased when compared to the control（P<0.05）;Besides,the body weight of the AFB₁+ZnO NPs group was significantly increased with respect to AFB₁group（P<0.05）,and there was no significant difference between the control and AFB₁+ZnO NPs group.On the 5th day after the end of intragastric administration of AFB₁,the body weight of ducklings in the AFB₁ group was still lower than the control group（P<0.05）;And there was no significant difference between the ZnO NPs group and the AFB₁+ZnO NPs group and that in the control group and AFB₁ group（P>0.05）.2.Effects of AFB₁ and ZnO NPs on liver:There were 6 livers of ducklings showed reticular lesions in AFB₁ group,and two ducklings in AFB₁+ZnO NPs group showed hepaic lesions,which were slighter than those in AFB₁ group.The pathology results showed that there were no pathological alterations in control and ZnO NPs group;Compared to the control group,hepatocyte necrosis and proliferation of bile duct epithelial cells were observed in AFB₁ group;Contrastingly,the injury induced by AFB₁were ameliorated and restored in AFB₁+ZnO NPs group.In addition,compared to the control group,the activities of ALT and AST were increased（P<0.05）,the contents of TP and Glu were decreased（P<0.05）in the AFB₁ group;The activities of AST and Glu in the AFB₁+ZnO NPs group were decreased（P<0.05）.3.Effects of AFB₁ and ZnO NPs on zinc content and antioxidant function in liver:Compared to the control group,the zinc content in the liver of AFB₁ group was significantly decreased（P<0.05）,and the zinc content in the liver of AFB₁+ZnO NPs group was significantly increased when compared to AFB₁ group（P<0.05）,and there was no significant difference between AFB₁+ZnO NPs group and control group.The activities of SOD,CAT and the content of GSH in the liver of AFB₁+ZnO NPs group were significantly increased than those of AFB₁ group（P<0.05）,and there was no significant difference between AFB₁+ZnO NPs group and control group.4.Effects of AFB₁ and ZnO NPs on hepatic antioxidation and apoptosis related genes:Compared to the control group,the m RNA expressions of MTF-1 and NQO1 in ZnO NPs group were significantly up-regulated（P<0.05）.Compared to the control group,the m RNA expressions of MT,SOD1,Nrf2 and p53 in AFB₁ group were significantly down regulated（P<0.05）,and the m RNA expressions of CAS3,Cas9,Bax and Bcl-2 were significantly up-regulated（P<0.05）.The related genes in AFB₁+ZnO NPs group were significantly different from those in AFB₁ group（P<0.05）,but had no significant difference from those in the control group.Part 2 The mechanism of ZnO NPs up-regulating MT expression to alleviate the toxicity of AFB₁ to hepatocytesHepG2 cells were cultured in MEM medium and divided into 4 different treatment.Except the control group,the other three treatment groups were treated with 20μg/m L AFB₁（AFB₁group）,20μg/m L ZnO NPs（ZnO NPs group）and 20μg/m L AFB₁+20μg/m L ZnO NPs（AFB₁+ZnO NPs group）for 24 hours.The main results are as follows:1.The effects of AFB₁ and ZnO NPs on cell viability,apoptosis and intracellular ROS:20μg/m L AFB₁ significantly reduced cell activity（P<0.05）,increased apoptosis rate and ROS positive rate（P<0.05）.And compared to the AFB₁group,the cell activity was significantly increased（P<0.05）,while the apoptosis rate and ROS positive rate were significantly decreased in AFB₁+ZnO NPs group（P<0.05）.2.Effects of AFB₁ and ZnO NPs on expression of related genes and nuclear translocation of MTF-1:Compared to the control group,the m RNA expressions of MT,MTF-1,NQO1 and SOD were significantly up-regulated（P<0.05）.After exposure to 20μg/m L AFB₁,the m RNA expressions of MT,MTF-1,Nrf2,NQO1 and SOD were significantly decreased（P<0.05）,while the m RNA expression of Bax was significantly increased（P<0.05）;The m RNA expressions in ZnO NPs group of MTF-1 and Nrf2 were significantly increased compared with the AFB₁ group（P<0.05）and has no differences with that in control group,but the m RNA expressions of MT,NQO1 and SOD were higher than those of the control group（P<0.05）.The expression of MTF-1 and nuclear translocation were detected by immunofluorescence,the result showed that MTF-1 was mainly located in the cytoplasm of HepG2 cells in normal state,and the expression of MTF-1 in the nucleus increased after treated with ZnO NPs.After AFB₁ exposure,MTF-1 was only expressed in the cytoplasm of a few cells.And when exposed to both AFB₁ and ZnO NPs,the fluorescence intensity of MTF-1 was increased when compared to AFB₁ group,while the expression of MTF-1 in nucleus was increased.In conclusion,our research suggested that 1)AFB₁ could reduce the zinc content in hepatocytes,and down regulated MTF-1 while inhibited the translocation of MTF-1 from cytoplasm to nucleus,and then MT expression was decreased.After that,the expression of antioxidant genes and mitochondrial apoptosis related genes in hepatocytes were down regulated,and the antioxidant capacity of hepatocytes was damaged.2)Furthermore,ZnO NPs could attenuate the hepatotoxicity of AFB₁ through the increase of Zn²⁺concentration in hepatocytes by ZnO NPs in diet.Aferwards,the expression of MTF-1and the nuclear translocation of MTF-1 were increased by Zn²⁺,then induce the expression of MT in hepatocytes,so as to alleviate the hepatotoxicity of AFB₁.