Study On The Inhibition Of NETs Release By Chlorpyrifos Via Promoting Necroptosis And Inhibiting The PKC/MAPK-respiratory Burst Pathway In Carp

Chlorpyrifos(CPF),an organophosphorus pesticide,is widely used in the world which can effectively kill a variety of chewing and piercing and sucking mouthparts.However,it can accumulate in water through surface runoff or other ways to increase the risk of damage to aquatic organisms.Neutrophils play a major role in innate immune.At the same time,neutrophils are also a key component of blood nonspecific cellular immunity.The latest research shows that in addition of releasing immune factors and other forms of immune function,neutrophils can also prevent the harmful factors by releasing Neutrophil extracellular traps(NETs).To explore a relationship between NETs and CPF and its mechanism,this experiment was based on the establishment of a CPF-exposed neutrophil model,and neutrophils was stimulated with 4 ?M PMA to establish a NETs release model.At the same time,scanning electron microscopy,fluorescence microscopy,quantitative Real-Time PCR,and Western blotting etc.were used in the experiment.Meanwhile,in the perspective of necroptosis caused by oxidative stress,heat shock protein,and inflammation,Nec-1 was used to explore the PKC/MAPK-respiratory burst pathway in the process of CPF to NETs,further clarified the effect of CPF exposed on neutrophil immune disorders in carp.The results are shown as follows:(1)During acute exposed of CPF,the release of ROS was increased in a dose-dependent manner,damaging the carp neutrophil antioxidant system,promoting the expression levels of heat shock proteins and inflammatory factors,further promoting MLKL,RIP3 and other expressions,and decreasing caspase8 expression.Nec-1 could significantly increase the expression level of caspase8,while the expressions of RIP1,RIP3 and MLKL were down-regulated.The results showed that acute exposure of CPF could lead to necroptosis in neutrophils.(2)The exposure of CPF could cause damage to carp neutrophils,increase cell folds and reduce fibrils.The CPF+PMA group showed that CPF inhibited PMA-induced release of NETs and shortened the nets of NETs;after the addition of Nec-1,the release of NETs increased significantly,but the amount of NETs was still less than PMA group.In addition,under the co-treatment of CPF and PMA,the expression and release of MPO were significantly reduced.These results indicated that CPF could significantly inhibit PMA-induced release of NETs,while Nec-1 could alleviate the inhibition.(3)After the stimulation of PMA,the PKC/MAPK pathway was activated,and respiratory burst occurred.CPF pretreatment could inhibit the activation of PKC/MAPK-respiratory burst pathway caused by PMA.In addition,the ROS level was increased in a time-dependent manner in CPF group.The results indicated that CPF could negatively regulate the PKC/MAPK pathway,which promoted the increase of ROS release and induced oxidative stress,but it could inhibit the respiratory burst caused by PMA,thereby inhibiting the generation of NETs.In summary,acute CPF exposure caused oxidative stress in neutrophils of carp,caused inflammatory damage and heat shock protein accumulation,thereby promoted necroptosis and further hindered the release of NETs;In addition,CPF also negatively regulated PKC/MAPK pathway,reduced the level of respiratory burst and inhibited the generation of NETs.This study provides a reference for further research on the toxicological mechanism of CPF exposure,which is a new theoretical basis for environmental protection and biological innate immune regulation.