Preliminary Study On FB₁ Induced Chicken Heterophil Releasing ETs And Its Mechanism

Fumonisin B₁ is a water-soluble mycotoxin produced by fusarium which is ubiquitous in the world and mainly pollutes corn and its coproduct.After being ingested by the body,FB₁can cause multiple organ damage,immunosuppression,and even cause cancer,resulting in serious harm to animal health.ETs,as a new type of immune defense mechanism,play an important role in resisting pathogens.But it is also a double-edged sword.ETs can not only capture and kill pathogens but also is related to many diseases of the body.At present,whether FB₁can induce chicken heterophils to release heterophil extracellular traps（HETs）and its mechanism have not yet been reported.In this paper,the formation of HETs and its mechanism induced by FB₁in chicken heterophils were studied,and it is hoped that through this research,the immunotoxicity of FB₁will be further understood.The experimental methods and corresponding results are as follows:FB₁ induces the formation of HETs in chicken heterophils.Firstly,heterophils were isolated and purified.Chicken heterophils were treated with different concentration of FB₁（10μM,20μM,40μM）,and then the HETs induced by FB₁were observed by immunofluorescence staining.In order to further confirm whether FB₁induces the formation of HETs of chicken heterophils,zymosan was used as a positive control,and the release of HETs were detected by DNA quantitative experiment.The results showed that FB₁can induce the release of HETs in chicken heterophils,which are composed of DNA,H3 and NE.DNA quantitative detection results showed that FB₁-induced HETs peaked at 1.5h after FB₁exposure.Besides,FB₁-induced HETs increased in a dose-dependent manner among 10-40μM FB₁exposure.The role of reactive oxygen species（ROS）in HETs formation induced by FB₁.In order to clarify whether HETs induced by FB₁are dependent on ROS,DCFH-DA（ROS fluorescent probe）was used to detect intracellular ROS.To explain the cause of ROS generation,antioxidant enzymes SOD,CAT,GSH-Px activity and GSH content were detected by enzymatic activity experiments.Moreover,western blotting was used to detect the effect of FB₁on the p38 and ERK pathways.In parallel experiments,we used SB202190（p38 inhibitor）,U0126（ERK inhibitor）,DPI（NADPH oxidase inhibitor）to verify the relationship between the generation of ROS and p38,ERK pathway and NADPH oxidase.Finally,pharmacological inhibition experiments were conducted to verify the role of p38,ERK pathway and NADPH oxidase in the release of HETs induced by FB₁.The results showed that FB₁can increase the ROS production,and affect the antioxidant capacity of chicken heterophils by reducing the activity of antioxidant enzymes SOD,CAT,GSH-Px and GSH content.Furthermore,FB₁increased the phosphorylation level of p38 and ERK.However,the results of parallel experiments showed that the generation of ROS induced by FB₁was not dependent on p38,ERK pathway but NADPH oxidase.SB202190,U0126,and DPI could not reduce the release of HETs induced by FB₁,indicating that HETs formation induced by FB₁was not dependent on p38,ERK pathway and NADPH oxidase.The role of autophagy in the release of HETs induced by FB₁.In order to determine whether FB₁can induce autophagy in chicken heterophils,the autophagy marker LC3B was labeled with specific antibody,and observed by fluorescence microscope.Western blotting was used to detect the activation of PI3K-Akt-m TOR and AMPK-ULK1 signaling pathways and the expression of PI3K classⅢto verify the mechanism of FB₁-induced autophagy in chicken heterophils.The autophagy inhibitor 3-Methyladenine（PI3K classⅢinhibitor）and the autophagy promoter rapamycin（m TOR inhibitor）were used to prove the role of autophagy in the formation of HETs induced by FB₁.The results showed that the content of autophagy marker LC3B was increased after FB₁stimulation.Besides,FB₁reduced the phosphorylation level of PI3K,Akt and m TOR,increased the phosphorylation level of AMPK and ULK1,and the protein expression level of PI3K classⅢand LC3B,which further verified that FB₁can induce autophagy and the mechanism of autophagy induced by FB₁.Finally,inhibition experiments showed that3-Methyladenine significantly reduced the release of HETs induced by FB₁,while rapamycin significantly increased it,indicating that the release of HETs induced by FB₁depends on autophagy in chicken heterophils.In summary,this study proved for the first time that FB₁can induce the release of HETs in chicken heterophils,and the formation of HETs induced by FB₁depends on ROS and autophagy,while it is independent of NADPH oxidase,p38 and ERK signaling pathways.Autophagy-related PI3K classⅢ,PI3K-Akt-m TOR signaling pathway and AMPK-ULK1 signaling pathway play an important role in the formation of HETs induced by FB₁.This study can enrich the content of immunotoxic effects of FB₁on poultry,and provide an important theoretical basis and experimental reference for the prevention and treatment of diseases caused by FB₁.