Study On The Inhibition Of NETs Release By Avermectin Via Activating PTEN Demethlation To Negatively Regulate The PI3k-ERK Pathway And Reducing Respiratory Burst In Carp

Avermectin(AVM)is a kind of broad-spectrum and highly effective insecticides,which is widely used in animal husbandry and agricultural production.AVM will eventually enter the water environment with precipitation,surface runoff and groundwater migration because of its heavily used.AVM has been reported to cause genomic demethylation,which is associated with diseases of immune system.The release of neutrophil extracellular traps(NETs)is considered to be a novel response mode of neutrophils for participating in immune defense.NETs formation is regulated by cellular respiratory burst and the PI3K-ERK pathway.The enriched AVM has toxic effects on non-target organisms such as mammals,poultry and invertebrates,there are few studies about the toxicity of AVM on aquatic organisms.Toxicology exposure tests of aquatic animal(mainly fish)cells are important detection methods in toxicology stud ies,and are often used to assess the toxicity of environmental pollutants such as pesticides.Therefore,in this study,peripheral blood neutrophils of carp were used as the research object,the acute toxity of AVM to carp neutrophils was first evaluted,and then 5 ?g/L AVM or 4 ?M DNA demethylation inhibitor(ATA)was added into the cell culture to pretreated cells for grouping,and 4 ?M PMA was used as the inducer for stimulating NETs release.Aotodock docking simulation,scanning electron microscopy,fluorescence staining,quantitative real-time PCR,western blot,Mass Array methylation detection and other experimental techniques were used.We simulated the docking method of AVM-MBD2,and detect the neutrophil and NETs morphology,NETs release and MPO expression,the expression of DNMTs and MBD2,the degree of demethylation in PTEN promoter region and the expression of PTEN and indicators in PI3K-ERK pathway,which regulates NETs release.Also,the level of cellular respiratory burst was tested.The study aim to explore the effects of AVM acute exposure on NETs release and the possible mechanism,as well as whether DNA methylation plays a role in this process.The results shows as follows:(1)AVM acute exposure changed the morphology of neutrophils in carps,NETs re leased from AVM-pretreated neutrophils was shortened and thinned and the amount of NETs was significantly reduced,while the quantity of PMA-stimulated NETs from neutrophils co-pretreated with AVM-ATA was significantly increased,but did not reach the leve l in PMA group.In addition,AVM significantly reduced the expression of MPO in neutrophils during the formation of NETs,and the expression of MPO was increased significantly after the addition of ATA.The results showed that AVM significantly inhibited PMA-induced NETs release,and ATA significantly attenuated this inhibition.(2)AVM acute exposure significantly reduced the m RNA expression of DNMTs in carp neutrophils,and significantly increased the m RNA and protein expression of MBD2.After the addition of ATA,the m RNA expression of DNMTs was significantly up-regulated,while the m RNA and protein expression of MBD2 was significantly down-regulated.In addition,the molecular docking model of AVM-MBD2 was successfully constructed using Autodock software.These indicated that AVM acute exposure may change the expression level of DNA methylation regulatory enzymes in carp neutrophils,and MBD2 may be a targeted regulatory protein of AVM.(3)AVM acute exposure significantly up-regulated the expression of PTEN in neutrophils and increased the degree of demethylation in the PTEN promoter region.After adding ATA,the expression of PTEN was significantly down-regulated and the degree of demethylation of promoter was significantly decreased.Compared with PMA stimulation alone,AVM pretreatment significantly down-regulated the expression of the PI3K-ERK pathway involved in the release of NETs negatively regulated by PTEN in neutrophils.The addition of ATA significantly increased the m RNA expression levels of AKT,Raf and ERK and the proteins expression levels of PI3 K,AKT,MEK and ERK1/2.However,the results above were not elevated to the expression level of PMA alone.(4)PMA could stimulate the respiratory bursts in neutrophils,wheres AVM could block this response,and ATA could increase ROS production during respiratory bursts after AVM exposure.In addition,ROS content in neutrophils treated by AVM alon was increased in a time-dependent manner.These indicated that AVM caused oxidative stress in carp neutroph ils and increased ROS release,but inhibited PMA-stimulated cellular respiratory burst.In summary,acute AVM exposure altered neutrophil morphology,suppressed NETs release via increasing MBD2 expression,PTEN demethylation and expression to incativate th e PI3K-ERK pathway and attenuating respiratory burst levels.This study enriched the mechanism of AVM's immunotoxicity to fish,and provided a reference for further research on the toxicological mechanism of AVM chronic exposure,which is of great signific ance for the protection of ecological environment and human health.