The Mechanism Of Bisphenol A Exposure And Selenium Deficiency Induce Necroptosis In Broiler Vein Cells Through MiR-26a-5p Targeting ADAM17

Selenium(Selenium,Se)is an essential nutritional trace element,which has anti-inflammatory and anti-oxidative functions,and plays a vital role in maintaining the homeostasis of humans and animals.When dietary selenium intake is insufficient,the redox system in the body is disturbed,leading to adverse reactions such as oxidative stress,lipid metabolism disorder,and inflammation.Bisphenol A(Bisphenol,BPA)is a widely used plasticizer.It enters the body through diet,breathing and other ways,causing liver necrosis,myocardial necrosis and other diseases,and affecting the normal life activities of organisms.Necroptosis is involved in the pathological process of cardiovascular diseases,in which cells undergo cell membrane rupture and cell death,thereby releasing cell contents into surrounding tissues and causing tissue inflammatory damage.Micro RNA(miRNA)regulates gene expression by changing the level of target m RNA by causing m RNA degradation or translation inhibition,directly or indirectly regulating cell cycle progression,necroptosis and other reactions.In recent years,people have focused on the damage of BPA exposure and selenium deficiency to human and animal tissues,but the research on the combined damage to tissue and its damage mechanism,as well as the role of micro RNA is still very limited.In this experiment,on the basis of establishing BPA-exposed and selenium-deficient broiler vein models and chicken arterial endothelial cell(PAEC)models,the control group(Control group),BPA-exposed group(BPA group),and selenium-deficient group(-Se group)were respectively set up,Se deficiency + BPA exposure group(-Se + BPA group).By observing the pathological changes and ultrastructure of vein tissue,the damage of BPA exposure and selenium deficiency to broiler vein tissue was evaluated;combined with the results of bioinformatics and dual luciferase reporter genes,miR-26a-5p and target gene ADAM17 were screened out.To determine its possible signaling pathways in BPA exposure and selenium deficiency-induced venous injury,established miR-26a-5p mimic/inhibitor PAEC model in vitro,and detected oxidative stress levels,ADAM17,TNFR1 pathways,necroptosis,inflammation and heat shock proteins and other genes.To further elucidate the regulatory mechanism of miRNA in vein injury induced by BPA exposure and selenium deficiency,we use NAC,Nec-1,miR-26a-5p mimic-treatment PAEC exposed to BPA and selenium deficient,the expression of genes such as oxidative stress level,ADAM17,TNR1 pathway,necroptosis,inflammation and heat shock proteins were detected to verify the combined effect of BPA exposure and selenium deficiency on broiler vein endothelial cells Injury mechanism of necroptosis.The test results are as follows:(1)Vein histopathological and ultrastructural observations showed that in the broiler veins of-Se group,the cytoplasm of venous endothelial cells condensed,the nuclear membrane ruptured,a small number of endothelial cells fell off,and the vascular smooth muscle cells in the medial layer were edematous and cytoplasmic Loose light staining,thickening of blood vessel walls.In the BPA group,the nuclei of vascular cells were irregular,the nuclear membrane was broken,the fibers of the smooth muscle layer were irregular,the endothelial cells were slightly exfoliated,and a small number of foamy macrophages infiltrated.In the-Se+BPA group,the cell membrane of venous endothelial cells ruptured,and the contents overflowed.The number of vascular endothelial cells decreased and some of them fell off.The structure of the elastic membrane is disordered,a small amount of lymphocyte infiltration can be seen,and the blood vessel wall is thickened.It shows that BPA exposure and selenium deficiency can cause vein injury in broiler chickens,and the combined treatment group is more serious than the single treatment group.(2)In broiler veins,the expression of miR-26a-5p was significantly increased.Combining the predictions of Targetscan,miRDB and other websites and the results of dual luciferase reporter genes,the target gene ADAM17 of miR-26a-5p was screened out,and the miR-26a-5p mimic/inhibitor model in vitro was constructed to detect the expression of ADAM17.The results showed that the expression of ADAM17 in the miR-26a-5p mimc group was significantly reduced,while the expression of ADAM17 in the miR-26a-5p inhibitor group was significantly increased,which verified the relationship between miR-26a-5p and ADAM17 target relationship.(3)In the vein tissue,compared with the control group,the GSH content and GSH-PX,CAT,SOD activities in the-Se group,BPA group,and-Se+BPA group were significantly decreased,and the MDA content was significantly increased;in PAEC,The ROS content of-Se+BPA group was significantly higher than that of-Se group,BPA group and control group;after miR-26a-5p inhibitor treatment in vitro,the release of ROS was significantly increased.It shows that BPA exposure and selenium deficiency can reduce the antioxidant capacity of broiler veins by regulating miR-26a-5p.(4)The detection results of TNFR1 pathway and necroptosis-related indicators in broiler vein tissue and PAEC showed that compared with the control group,the immunofluorescence of the necroptosis marker gene Caspase8 in the-Se group,the BPA group,and the-Se+BPA group The m RNA and protein expressions of TNFR1 pathway and necroptosis-related indicators(RIPK1,RIPK3,MLKL)increased,and the expression of Caspase8 decreased significantly in the-Se group,BPA group,and-Se+BPA group.It indicated that BPA exposure and selenium deficiency could activate the TNFR1 pathway and cause venous necroptosis in broilers.(5)The detection results of inflammation-related genes and heat shock protein-related genes in broiler veins and PAEC showed that the inflammation-related genes IL-6,COX2,IL-1β and heat shock protein in-Se group,BPA group,-Se+BPA group The m RNA and protein expressions of protein-related genes HSP60 and HSP90 were significantly increased,and the-Se+BPA group was significantly higher than that of the BPA group and the-Se group.It shows that BPA exposure and selenium deficiency can cause venous inflammatory response in broiler chickens.(6)On the basis of establishing an in vitro model of co-treatment with BPA and selenium deficiency,cells were treated with NAC,Nec-1 and miR-26a-5p Mimic,and the results showed that after NAC and miR-26a-5p mimic treatment,ROS in cells The content of necroptosis-related genes,inflammation-related genes,and heat shock protein-related gene expression levels were all decreased.After adding Nec-1,the expression levels of necroptosis-related genes,inflammationrelated genes,and heat shock protein-related genes were all reduced.The results indicated that BPA exposure and selenium deficiency promoted oxidative stress by regulating miR-26a-5p targeting ADAM17,and induced necroptosis and inflammatory response in PAEC.In summary,BPA exposure and selenium deficiency promote the expression of ADAM17 by inhibiting the expression of miR-26a-5p,activate the TNFR1 pathway,induce oxidative stress,necroropsis and inflammation,and thus lead to vein injury in broiler chickens.This study enriches the biological functions of miRNAs in regulating selenium deficiency and BPA exposure-induced vein injury in broiler chickens,and provides a theoretical basis for future research on trace element selenium deficiency,BPA ecological pollution and health risks.