Study On The Mechanism Of Bisphenol A Exposure And Selenium Deficiency Inducing Inflammatory Injury In Chicken Trachea By Regulating The MiR-155/TRAF3/NF-κB Pathway

Bisphenol A(BPA)is an organic synthetic compound used as an industrial raw material to prepare products containing polycarbonate and epoxy resin.With the widespread use of plastic products,BPA is widely present in the environment and poses a great threat to the health of organisms.BPA can enter human and animal bodies through the food chain,causing multiple tissue damage,including inflammation,cell apoptosis,and necrosis.Selenium,as an essential nutrient trace element,plays an important role in various physiological functions of the body.Selenium deficiency can cause inflammation in various tissues,and the NF-κB signaling pathway is one of the important inflammatory response regulatory pathways involved in the occurrence and development of various diseases.More and more studies have shown that selenium deficiency can cause damage to various tissues and organs in the body,and the trachea is also one of the target organs for selenium deficiency.MiRNAs are a class of endogenous non-coding RNA with regulatory functions.They participate in and regulate many biological processes by regulating the expression of a variety of genes,and MiRNAs are closely related to inflammation.However,the specific molecular mechanisms of BPA exposure and selenium deficiency damage to chicken tracheal tissue,as well as the role played by miRNAs,are still unclear.Therefore,this study further explored the mechanism of tracheal injury in broilers caused by BPA exposure and selenium deficiency and the role of miRNA in it,in order to clarify the toxicity of BPA and the molecular mechanism of the occurrence of selenium deficiency diseases in broilers.This study first replicated in vitro and in vivo BPA exposure or/and selenium deficient chicken models.In vivo experiments were divided into four groups: control group(Control group),BPA exposure group(BPA group),selenium deficient group(-Se group),selenium deficient+BPA exposure group(-Se+BPA group).The changes of histopathology and morphology of broiler trachea were analyzed by H&E staining,PAS staining and ultrastructural observation.The miRNAs with significant differential expression in broiler trachea were screened out,namely miR-155.The in vitro trial was divided into two parts,the first part was grouped in the same way as the in vivo trial,The second part was divided into Control group,selenium deficiency+BPA exposure group(-Se+BPA group),selenium deficiency+BPA exposure group+miR-155 inhibitor group(-Se+BPA+I group),selenium deficiency+BPA exposure group+NAC group(-Se+BPA+NAC group).The specific target genes of miR-155 were predicted and verified by bioinformatics website,double Luciferase reporter gene,q RTPCR and other methods.In broiler tracheal tissue,tracheal epithelial cells transfected with miRNA-155 inhibitor or added with NAC,the expression changes of antioxidant genes,the changes of ROS levels,inflammatory signaling pathways and cytokines,immune functionrelated β-defensin,heat shock protein and immunoglobulin were detected by q RT-PCR,Western Blot,ELISA and immunofluorescence staining.The main research results are as follows:(1)The histopathological results showed that BPA exposure and selenium deficiency caused cilia loss,detachment and mucus secretion in the trachea mucosal epithelium of chickens,and increased goblet cells in airway epithelium accompanied by inflammatory cell infiltration of varying degrees.-Se+BPA group had the most serious pathological changes,such as epithelial cell arrangement disorder,serious loss and loss of cilia,significant increase in airway epithelial goblet cells,and a large number of inflammatory cell infiltration and mucus secretion.Ultrastructural observation results showed that the concentration of nuclear chromatin and the vacuolation of mitochondria were observed in the-Se group and BPA group,while the concentration of nuclear chromatin and the vacuolation of mitochondria were observed in the-Se+BPA group,suggesting that BPA exposure and selenium deficiency could cause inflammatory damage of trachea tissue.(2)BPA exposure and selenium deficiency caused the decrease of antioxidant enzyme activities of CAT,SOD and GPX,and the content of GSH,while the content of MDA increased.The activity of antioxidant enzyme was further inhibited and the content of MDA increased significantly in the trachea of-Se+BPA group.The results indicated that the combination of BPA exposure and selenium deficiency could further aggravate airway oxidative stress in chickens.(3)BPA exposure and selenium deficiency can activate the NF-κB signaling pathway,significantly increase the expression levels of pro-inflammatory cytokines IL-1β,IL-2,IL-6,COX-2,i NOS,TNF-α and IFN-γ,and decrease the expression of anti-inflammatory cytokine IL-10.The expressions of heat shock protein(HSP60,HSP70,HSP90)were increased,and the levels of β-defensin(Av BD6,Av BD7,Av BD8,Av BD9,Av BD10)and immunoglobulin(Ig A,Ig Y,Ig M)were decreased.Compared with the single treatment group,the changes of BPA exposure and selenium deficiency combined group were more significant.The results indicated that BPA exposure and selenium deficiency induced trachea inflammation and immune damage in chickens,and the combination of BPA exposure and selenium deficiency aggravated trachea inflammation and immune damage in chickens.(4)MiR-155 was selected as one of the specific miRNAs associated with BPA exposure and selenium deficiency.The m RNA expression level of miR-155 was significantly increased in the tracheal tissues of broilers.The potential target gene TRAF3 was predicted by Targetscan,miRDB and other websites,and the interference model of miR-155 was successfully constructed on the basis of cultured tracheal epithelial cells in vitro.Dual luciferase reporter gene,q RT-PCR and Western Blot were used to verify the targeting relationship between miR-155 and TRAF3.(5)A model of BPA exposure and selenium deficiency was established in vitro.ROS levels were significantly increased in the-Se+BPA group,and the overexpression of miR-155 significantly increased the production of ROS.The addition of NAC significantly blocked the production of ROS,and silenced TRAF3 could reduce the production of ROS induced by the overexpression of miR-155.These results indicate that overexpression of miR-155 and silencing of TRAF3 can reverse ROS production,and the miR-155/TRAF3 axis stimulates ROS accumulation and leads to oxidative stress.In addition,after transfection with miR-155 inhibitor or NAC treatment,the expression of NF-κB signaling pathway was significantly down-regulated,and the levels of cytokines IL-1β,IL-2,IL-6,COX-2,i NOS,TNF-α and IFN-γ were significantly decreased,while the level of IL-10 was significantly increased,and the expression levels of heat shock proteins(HSP60,HSP70,HSP90)were also down-regulated.This suggests that miR-155 induces inflammatory response and immune damage in tracheal epithelial cells by targeting TRAF3 to stimulate ROS accumulation.In conclusion,BPA exposure and selenium deficiency can significantly increase the expression of miR-155 in tracheal tissues of broilers,increase ROS levels through targeted inhibition of TRAF3,activate the NF-κB signaling pathway mediating tracheal inflammation,and ultimately lead to tracheal tissue inflammation and immune damage of broilers.The results of this study not only enriched the research content of BPA and selenium deficiency on tracheal damage in poultry,but also provided a new theoretical basis for the prevention and treatment of BPA exposure and inflammatory damage in selenium deficient poultry.