The Researches On The Mechanisms Of Autophagy And Extracellular Traps Induced By Aflatoxin B1 In Macrophages

Autophagy is a conserved intracellular process that delivers the damaged,degenerated or aging proteins to the lysosome for degradation.According to the degradation of the substrate and formed in a different way will be divided into giant autophagy,micro-autophagy and molecular chaperone autophagy.They are different of the mechanism and function in the body.Autophagy occurs by the regulation of autophagy-related genes and related proteins.At present more than 36 autophagy-related genes(ATG)were defined in yeast,and the homologous gene was appeared in most of the mammalian cells.Autophagy-related proteins encoded by ATG constitute the core molecular mechanism of autophagy,and they are involved in the regulation of various autophagic processes.In recent years,a variety of autophagy related pathways have been found.Extracellular traps,a new cellular natural immune mechanism,which is the fibrous network composed of nuclei DNA can be used to limit the activity of pathogenic microorganisms and kill it.It is a unique form of cell death that is different from apoptosis and necrosis,and it is a unique way of self-protection to resist infection and poisoning.It is currently found that microbes that can induce ETs include bacteria,fungi and parasites,but whether mycotoxins can induce the mechanism has not been reported.Aflatoxin B1(AFB1)is the most toxic,and one of the most extensive contaminations,trace can cause great harm.In vivo,the mechanism of aflatoxin B1 has become a hot issue at home and abroad.Macrophages were used in this research to solve the following problems: whether aflatoxin B1 can induce autophagy and extracellular traps,what's the mechanisms involved in aflatoxin B1-induced autophagy and extracellular traps.And to explore the relationship between autophagy and extracellular traps induced by aflatoxin B1.Firstly,we investigated the production of macrophage autophagy that induced by aflatoxin B1.The fluorescence microscope was used for GFP-LC3 and ptf-LC3 punta observation in aflatoxin B1-treated RAW264.7 cells.The expressions of LC3,P62 or Beclin1,ERK,P-ERK,P-MEK in aflatoxin B1-treated RAW264.7 cells and THP-1cells were assayed by western blot.And by autophagy inhibitor chloroquine(CQ)to observe whether AFB1-induced autophagy is completely autophagy,the relationship between Beclin1,MEK,ERK and autophagy was observed by MEK inhibitor PD98059.The results showed that there was an increase in the number of GFP-LC3 punta during aflatoxin B1 treatment.The LC3 generally increased but P62 decreased in two cells,this results indicating that AFB1 can induce macrophage autophagy.RFP-LC3 puncta were more than GFP-LC3 puncta in aflatoxin B1-treated RAW264.7 cells.The CQ can inhibit the process of LC3 degradation.These indicating that the AFB1-induced autophagy is a complete autophagy.The phosphorylation of ERK,MEK could be induced by aflatoxin B1.Furthermore,PD98059 pre-treatment inhibited the phosphorylation of ERK and MEK and the expresstion of LC3 and Beclin1.These results suggest that AFB1-induced macrophage autophagy is achieved through MEK,ERK pathway and associated with Beclin1.In summary,AFB1 induced human and murine macrophage completed autophagy,mediated by MEK,ERK pathway and regulated by Beclin1.Secondly,we analyzed the AFB1-induced macrophage ROS and extracellular traps.we measured the production of Reactive oxygen species(ROS)and extracellular DNA in RAW264.7 cells and THP-1 cells treated with aflatoxin B1 by fluorescence microplate reader.The structure of extracellular traps produced by THP-1 cells was observed by fluorescence microscopy.The results showed that AFB1 was able to induce the production of ROS in RAW264.7 cells and THP-1 cells in a time-and dose-dependent manner and inhibited by NOX2 inhibitor DPI,and the expression level of extracellular DNA was increased in a dose-dependent manner.The morphology of extracellular traps changes with increasing concentrations of AFB1.Histone H3,elastase,and myeloperoxidase are components of macrophage extracellular traps.In summary,these results indicate that AFB1-induced production of NOX2-dependent ROS in macrophages.AFB1 is able to induce extracellular traps and its structure is similar to neutrophil extracellular traps(NETs),while histone H3,myeloperoxidase,elastase is the main constituent of extracellular traps.Finally,we explored the relationship between autophagy,ROS and extracellular traps induced by aflatoxin B1 at the molecular mechanism.The production of extracellular traps of THP-1 cells treated with AFB1 was observed by fluorescence microscopy,and the relationship between ROS,autophagy and extracellular traps was studied by using NOX2 oxidase inhibitor DPI and autophagy inhibitor wortmannin.The production of ROS in THP-1 cells treated with PMA was detected by fluorescence microplate reader to study the relationship between ROS and extracellular traps.The expression level of LC3 in THP-1 cells was detected by western blot.The relationship between ROS and autophagy was studied by using NOX2 oxidase inhibitor DPI and autophagy inhibitor wortmannin.Then,the degradation of aflatoxin B1 was detected by ELISA with extracellular traps of macrophages.The results showed that NOX2 oxidase inhibitor DPI and autophagy inhibitor wortmannin inhibited the generation of extracellular traps,indicating that the formation of extracellular traps depends on the production of ROS and autophagy.NOX2 oxidase inhibitor DPI can significantly inhibit the expression level of cell LC3,indicating that AFB1-induced macrophage autophagy is regulated by ROS.AFB1 has also been significantly degraded at the same time as induced extracellular traps.In summary,AFB1 induced NOX2-dependent ROS production to induce the formation of extracellular traps of macrophages,ROS by regulating autophagy to regulate the generation of extracellular traps.And the production of extracellular traps of macrophages has the effect of degrading AFB1.In conclusion,the results of this study show that AFB1 is can induce a complete autophagy of macrophages RAW264.7 and THP-1,which is achieved by activating the MEK/ERK pathway and upregulating Beclin1.And induce NOX2-dependent ROS production and autophagy to induce macrophage extracellular traps.And the production of ROS induced by aflatoxin B1 in the upstream of autophagy,had regulation of autophagy.And aflatoxin B1 induces extracellular traps to significantly degrade itself.These results will pave the way to exploring the strategy of Aflatoxin contorl.