Bisphenol A Exposure And Selenium Deficiency Induces Apoptosis And Necroptosis In Chicken Spleen Tissue Via INOS/TNF-α Pathway

Bisphenol A(BPA),a typical endocrine disruptor,is widely used in the production of polycarbonate,epoxy resin,thermal paper and food containers.In recent years,the market demand and production of BPA products have surged,which has increased the exposure risk of humans and animals.It has been confirmed that BPA exposure can lead to the accumulation of NO in the body,interfere with the regulation of signaling pathways,and cause tissue damage.Selenium is one of the essential trace elements to maintain the physiological functions of organisms.Selenium deficiency also induces excessive production of NO,interferes with the function of the immune system,and causes cell death and tissue damage.Due to the uneven distribution of Se in Earth’s surface soils,the co-occurrence of Se deficiency and BPA exposure becomes the norm.The combined occurrence of BPA exposure and selenium deficiency can aggravate tissue damage and the mechanism of injury has not been reported.Based on this,this study proposed that BPA exposure and selenium deficiency caused apoptosis and necroptosis via iNOS/TNF-α axis in chicken spleen tissue,leading to spleen tissue damage,and the combination of BPA exposure and selenium deficiency could aggravate this process.The in vivo model of broilers and the in vitro model of MSB-1 cells treated with combined BPA exposure and selenium deficiency were established.In vivo experiments were divided into four groups,a control group(NC group),a BPA exposure group(BPA group),a selenium deficiency group(-Se group),and a selenium deficiency+BPA exposure group(-Se+BPA group).In vitro experiments were divided into five groups: control group(NC group),BPA exposure group(BPA group),selenium deficiency group(-Se group),selenium deficiency+BPA exposure group(-Se+BPA group)and selenium deficiency+BPA exposure+TNF-αinhibitor QNZ group(QNZ group).H&E staining,transmission electron microscopy,fluorescence microscopy,TUNEL assay,flow cytometry,AO/EB staining,Western Blot,and q RT-PCR were used to detect the morphological changes of spleen cells,apoptosis and necrosis,iNOS/TNF-α pathway,mitochondrial apoptosis related pathway,and necroptosis related pathway.NO content and iNOS activity were also measured.The results of the study are as follows:(1)The models of selenium deficiency,BPA exposure and combined injury of selenium deficiency and BPA exposure were successfully constructed.The results of H&E staining showed that the area of white pulp was decreased,the area of red pulp was increased,and the wall of central artery was thickened in BPA group.In the-Se group,the boundary between red pulp and white pulp was blurred,the area of white pulp decreased,and the area of red pulp increased.The number of lymphocytes decreased and the arrangement of cells was sparse.The number of red blood cells between the red pulp increased,and the wall of the central artery thickened.In-Se+BPA group,the boundary between red pulp and white pulp was blurred,and the area of white pulp was reduced.The number of lymphocytes decreased and the arrangement of cells was sparse.The red pulp was hyperemic and the wall of the central artery was thickened.Cell lysis and fragmentation were also observed.The results of transmission electron microscopy showed nuclear pyknosis,perinuclear space enlargement,chromatin edge aggregation,mitochondrial cristae fracture and swelling,and cell membrane rupture in lymphocytes of BPA group.The lymphocytes in the-Se group were deformed,with nuclear pyknosis,enlarged peripheral space,chromatin condensation,mitochondrial cristae fragmentation,swelling and cell membrane fragmentation.The lymphocytes in-Se+BPA group showed obvious deformation,nuclear pyknosis,enlarged perinuclear space,chromatin condensation and edge polymerization,mitochondrial cristae rupture,swelling and vacuolization,cell shrinkage,cell membrane lysis and rupture.These results suggest that BPA exposure and selenium deficiency induce spleen tissue damage in chickens,and the combined treatment is more severe.(2)The content of NO and the activity of iNOS in spleen tissues and MSB-1 cells were significantly higher in the experimental groups than in the control group in vitro and in vivo,and the content of NO and the activity of iNOS in-Se+BPA group were significantly higher than those in BPA group and-Se group;in vitro addition of QNZ inhibited NO content and iNOS activity in the-Se+BPA group(p<0.05).The above results confirmed that BPA exposure and Se deficiency caused an increase in NO content and iNOS activity in chicken spleen tissue,and the combined treatment had more significant effects than the single treatment group.(3)TNF-α pathway analysis showed that the expression levels of iNOS,TNF-α,TNFR1 and FADD were significantly increased in BPA group,-Se group and-Se+BPA group;the expression levels of iNOS,TNF-α,TNFR1 and FADD in-Se+BPA group were higher than those in BPA group and-Se group;in the in vitro experiment,the expression levels of iNOS,TNF-α,TNFR1 and FADD in the-Se+BPA group were inhibited after the addition of QNZ(p<0.05).These results demonstrated that BPA exposure and selenium deficiency promoted the activation of the TNF-α pathway,and their combined effects promoted the activation of the TNF-α pathway.(4)TUNEL staining,AO&EB staining and flow cytometry showed that the levels of apoptosis and necrosis in the experimental group were significantly higher than those in the control group in vitro and in vivo,and the levels of apoptosis and necrosis in the-Se+BPA group were significantly higher than those in the BPA group and-Se group;in vitro,the addition of QNZ alleviated the occurrence of apoptosis and necrosis in the-Se+BPA group(p<0.05).The results of q RT-PCR and Western Blot showed that the expression levels of apoptosis related factors Bax,Caspase9,Caspase3,Cyt C and necroptosis related cytokines RIPK1,RIPK3 and MLKL were increased,and the expression level of Bcl2 was decreased in the experimental groups in vitro and in vivo;the expressions of apoptosis related factors Bax,Caspase9,Caspase3,Cyt C and necroptosis related cytokines RIPK1,RIPK3 and MLKL in the-Se+BPA group were significantly higher than those in the BPA group and-Se group,and the expression of Bcl2 was significantly lower than that in the BPA group;in vitro,QNZ inhibited the expression of Bax,Caspase9,Caspase3,Cyt C,RIPK1,RIPK3,and MLKL and upregulated the expression of Bcl2(p<0.05).These results suggested that BPA exposure and selenium deficiency promoted apoptosis and necroptosis in chicken spleen tissue,and the promoting effect was aggravated by the combined treatment of BPA exposure and selenium deficiency.(5)The results of heat shock protein detection showed that the expression levels of HSP60,HSP70 and HSP90 in the experimental groups were significantly higher than those in the control group,and the expression levels of HSP60,HSP70 and HSP90 in the-Se+BPA group were significantly higher than those in the BPA group and-Se group;in vitro,it was observed that QNZ inhibited the expression levels of HSP60,HSP70 and HSP90 in-Se+BPA group(p<0.05).These results suggest that heat shock proteins are involved in regulating the necroptosis of chicken spleen tissues induced by BPA exposure and selenium deficiency.In conclusion,BPA exposure and Se deficiency activate iNOS/TNF-α signaling pathway to induce apoptosis and necroptosis in chicken spleen tissue.This study enriches the toxicology of BPA and the immunotoxic mechanism of selenium deficiency,provides theoretical basis for the prevention and treatment of BPA exposure and selenium deficiency in poultry,and provides reference for comparative medicine.