Bisphenol A Exposure And Selenium Deficiency Induces Pyroptosis In Chicken Brain Via NNOS/NF-κB Pathway

Bisphenol A(BPA)is a serious environmental endocrine disruptor that damages not only the human endocrine system,but also the nervous system,which is one of its target organs.Selenium(Se)is an essential micronutrient,necessary for the synthesis of glutathione peroxidase in the body,and acts as an antioxidant.If the body is deficient in selenium,it will not only lead to damage to tissues and organs such as the liver,pancreas,skeletal muscle and kidneys,but also cause brain softening in birds.With seventy-two percent of the country’s selenium-deficient provinces and the ubiquity of BPA in the environment,BPA exposure and selenium deficiency often occur together.Based on this,the hypothesis that the combined treatment of BPA exposure and selenium deficiency exacerbates pyroptosis in chicken brain tissue is put forward in this project.Pyroptosis is involved in a variety of neurologically damaging diseases,such as stroke and Alzheimer’s disease.In addition,activation of NF-κB inflammatory vesicles is an important step in pyroptosis.nNOS activates NF-κB inflammatory vesicles.Based on this,the hypothesis that the combined treatment of BPA exposure and selenium deficiency activates the nNOS/NF-κB pathway to induce pyroptosis in chicken brain tissue and chicken embryonic primary neuronal cells is proposed in this work.To prove this hypothesis,we developed in vivo and in vitro models of BPA exposure and selenium deficiency.The in vivo test was divided into four groups,control group(NC group),BPA exposed group(BPA group),selenium deficient group(-Se group),and BPA exposed and selenium deficient group(BPA+-Se group).In vitro tests were divided into six groups,control group(NC group),BPA exposed group(BPA group),selenium deficient group(-Se group),BPA exposed and selenium deficient group(BPA+-Se group),nNOS inhibitor spermidine(Spermidine)group(BPA+-Se+nNOS group),and NF-κB inhibitor QNZ group(BPA+-Se+NF-κB group).The expression of nNOS/NF-κB pathway and genes related to pyroptosis pathway(GSDMD,NLRP3,Caspase-1 and ASC)were measured by quantitative real-time fluorescence PCR(q RT-PCR)and Western blot(WB).In addition,we also tested the levels of nNOS and NO.The results of the study are as follows:(1)The results of H&E staining showed that the brain tissue of the NC group was structurally intact and closely and regularly arranged.The cells in the BPA group were disorganized,with swollen lysed nuclei and abnormal morphology of neurons.In-Se group,the nuclei were swollen and heavily stained,with inflammatory cells infiltrating.In the BPA+-Se group,the cells were structurally disorganized,with an increase in morphologically abnormal neurons and inflammatory cells.These results demonstrate that BPA exposure or selenium deficiency can cause inflammation and damage to chicken brain tissue,and that the combination of BPA and selenium deficiency can cause more severe damage to brain tissue cells than treatment alone.(2)Transmission electron microscopy showed that the nuclei of the NC group were intact and smooth,the chromatin was evenly distributed,the mitochondria were intact and the cristae were clearly visible.The BPA group showed the typical features of cell death,such as swelling of the cells,many bubbles protruding from the nucleus,forming a "fried egg" shape,and chromatin agglutination.Cell swelling and membrane rupture were observed in the-Se group.The BPA+-Se group observed swollen nuclei with bubble-like protrusions and swollen mitochondria and loss of cristae.Immunofluorescence staining of chicken brain tissues for NLRP3 and GSDMD showed that the fluorescence intensity of NLRP3 and GSDMD was significantly enhanced in the BPA and-Se groups compared to the NC group(p < 0.05).In addition,the enhanced fluorescence intensity of brain tissue was more significant in the BPA and-Se co-treated group compared to BPA or-Se alone(p < 0.05).Immunofluorescence staining of primary neuronal cells from chicken embryos showed results consistent with the in vivo results.These results demonstrate that BPA or selenium deficiency leads to pyroptosis in chicken brain tissue and chicken embryo primary neuronal cells,with more severe pyroptosis in the combined treatment group.(3)The results of the nNOS/NF-κB pathway assay showed that the mRNA and protein levels of nNOS and NF-κB were significantly up-regulated in the BPA or-Se alone group compared to the NC group(p < 0.05),and the mRNA and protein levels of the above genes were more significantly up-regulated in the BPA+-Se group compared to the BPA or-Se alone group(p < 0.05).In addition,the results of NO content and nNOS activity in brain tissue and primary neuronal cells of chicken embryos showed that NO content and nNOS activity were elevated in the BPA and-Se groups compared to the NC group(p < 0.05),and more significantly in the BPA+-Se group compared to the BPA or-Se treatment alone(p < 0.05).Spermidine and QNZ effectively inhibited NO content,nNOS activity and nNOS/NF-κB expression.The above results demonstrate that BPA or selenium deficiency leads to activation of the nNOS/NF-κB pathway in chicken brain tissue and chicken embryo primary neuronal cells.Activation of the nNOS/NF-κB pathway was higher in the combination treatment group than in the treatment group alone.(4)The results of the expression assay of genes of the pyroptosis-related pathway showed that the mRNA and protein levels of pyroptosis-related genes(GSDMD,NLRP3,Caspase1,ASC,IL-1β and IL-18)were significantly higher in the BPA or-Se alone treated group compared to the NC group(p < 0.05).Upregulation of mRNA and protein expression of pyroptosis-related genes was more significant in the BPA+-Se group(p < 0.05).Spermidine and QNZ effectively inhibited the expression of pyroptosis-related genes.The above results demonstrated that BPA or selenium deficiency induced pyroptosis,with higher levels of pyroptosis in the combined treatment group than in the separate treatment group.In summary,BPA exposure and selenium deficiency activated the nNOS/NF-κB pathway and induced pyroptosis in chicken brain tissue and chick embryo primary neuronal cells,and the combined treatment of BPA exposure and selenium deficiency aggravated the level of pyroptosis.Our results provide a theoretical basis for the study of the combined toxicity mechanism of BPA exposure and selenium deficiency on chicken brain,and further provide a new perspective for the prevention and control of BPA exposure and selenium deficiency in livestock.