Lead Inhibited The Formation Of Neutrophil Extracellular Traps And The Antagonism Of Selenium In Chicken

With the development of modern industry,lead(Pb)as a widespread chemical pollutant has seriously endangered human health and safety.Exposure to lead caused immune dysfunction and lead to neutrophil damage.Selenium(Se),as one of the essential trace elements in the body,has various functions such as anti-oxidation,anti-aging,and thallium heavy metals.Neutrophil extracellular traps(NETs)are one of the ways in which the body exerts its immune function.Its formation is mainly affected by various factors such as myeloperoxidase(MPO),neutrophil elastase(NE),respiratory burst,calcium ions,etc.And NETs are also related to other cell death methods such as apoptosis and autophagy.Many studies have shown that the formation of NETs is inhibited when apoptosis increases.Heavy metal Pb can cause apoptosis in hepatocytes,lymphocytes,and macrophages.However,whether Pb cause neutrophil apoptosis and inhibit the formation of NETs,and whether Se antagonize the toxicity of Pb and the specific mechanism is unknown.In this experiment,neutrophils(heterotrophic leukocytes)were isolated from Hyland Brown Layer Chicken peripheral blood,and an in vitro lead and selenium exposure model was established based on the results of pre-experiments,and were divided into NC group(blank group),Pb group(12.5 ?M),and Se group(1 ?M),Pb + Se group(12.5 ?M/1 ?M).In this experiment,phorbol 12-myristic acid 13-acetate(PMA)was used as an inducer for the formation of NETs.Scanning electron microscope and Hoechst 33258 fluorescence staining were used to observe the morphological changes of neutrophils.AO/EB dual fluorescence staining and Annexin V-FITC-PI flow cytometry to detect apoptosis,quantitative quantitative PCR(q RT-PCR)and western blot(WB)to detect mitochondrial apoptotic pathway,death receptor pathway m RNA and protein expression.Fluorescent staining was used to detect the level of reactive oxygen species(ROS),respiratory burst,endoplasmic reticulum calcium ion release,and q RT-PCR and WB to detect the expression of related genes m RNA and protein.The test results showed that:1.Lead inhibit the increase of m RNA expression of RAF-MEK-ERK-NOX2 pathway,phosphorylation of ERK protein and respiratory burst caused by PMA,and then inhibit the release of proteins such as MPO,NE,thereby inhibiting the NETs formation.Selenium antagonizes the toxic effects of lead and restores the expression of the above pathways and respiratory bursts.2.Lead inhibits the expression of PLC-IP3 R pathway m RNA and protein caused by PMA,impedes the outflow of calcium ions in the endoplasmic reticulum,and thus hinders the formation of NETs.Selenium antagonize the toxic effects of lead,restore the expression of PLC-IP3 R pathway m RNA and protein,and the outflow of calcium ions in the endoplasmic reticulum,and restore the formation of the external trapping network.3.Lead exposure causes an increase in neutrophil apoptosis.Lead cause oxidative stress,disrupt the balance of oxidative and antioxidant systems,and inhibit the expression of Bcl-2,Mcl-1 m RNA and protein,and increase the mitochondrial apoptosis pathway(P53,Bax,Bak,Cyt-c,Apaf-1,Caspase 9,Caspase 3)and death receptor pathway(Fas,Fadd,Caspase 8)m RNA and protein expression,lead changes the way of cell death through the above pathways,destroys the balance between apoptosis and NETs,and causes neutrophils apoptosis,thereby inhibiting the formation of NETs.Selenium reverse the above-mentioned changes in m RNA and protein expression caused by lead,hinder the occurrence of apoptosis,and then restore the release of the NETs.Based on the above results,Pb hinder the activation of the RAF-MEK-ERK-NOX2 pathway and subsequent respiratory bursts caused by PMA;it also hinder the PLC-IP3 R pathway upregulated by PMA and the outflow of calcium ions in the endoplasmic reticulum.Se antagonizes the toxic effects of Pb.In addition,exposure to Pb cause neutrophil oxidative stress,activate the death receptor pathway and mitochondrial apoptotic pathway,and subsequently cause apoptosis.Se can prevent Pb-induced apoptosis.Our results show that Pb inhibit the formation of NETs by causing neutrophil apoptosis,inhibiting respiratory bursts and the outflow of calcium ions in the endoplasmic reticulum.Se antagonizes the toxic effects of Pb and restores the formation of NETs.Our data enrich the study of Pb immunotoxicity and provide a certain reference for the study of the mechanism of immune damage caused by Pb exposure in humans.