Study On The Mechanism Of Liver Inflammation Induced By Bisphenol A Exposure Or/and Selenium Deficiency Through ROS-Mediated Pyroptosis-M1 Polarization Crosstalk In Chicken

Bisphenol A(BPA)is a common environmental endocrine disruptor which is abundant in the food chain.BPA causes damage to endocrine,reproductive,immune and liver tissues through oxidative stress.Selenium(Se)is one of the essential trace elements in the body,which plays an important role in anti-oxidation in life activities.Se deficiency reduces the antioxidant capacity of the body and causes tissue damage such as liver necrosis and skeletal muscle necrosis.Due to the natural environmental factors of the earth and man-made industrial pollution,BPA exposure and Se deficiency often occur at the same time.In addition,oxidative stress can cause pyroptosis and M1 polarization,which are closely related to the occurrence and development of liver inflammation.Both BPA exposure and Se deficiency can cause liver injury,but whether the two regulate the occurrence of chicken hepatitis induced by hepatocyte pyroptosis and M1 polarization crosstalk through oxidative stress is not clear,and whether the effect of joint exposure is stronger than that of single exposure is also unknown.Based on this,we established chicken,chicken hepatocyte(LMH),chicken macrophage(HD11),and NAC cell intervention models exposed to BPA and Se deficiency alone or in combination,observed the histomorphology and inflammatory cell infiltration of chicken liver by H&E staining,detected the oxidative stress index of liver tissue,and detected the ROS levels of HD11 and LMH cells by DCFH-DA method.Immunofluorescence double staining,real-time fluorescence quantitative PCR and western blot were used to detect the expression of chemokines,pyroptosis,and macrophage polarization-related genes.In addition,the co-culture system of LMH and HD11 was constructed in transwell chamber to explore the crosstalk between pyroptosis and M1 polarization.The results are as follows:(1)H&E staining showed that the interlobular space of liver became large r in the BPA or-Se group,and the hepatocytes were watery degeneration accompanied by a small amount of inflammatory cells infiltration between hepatic sinuses.In the-Se+BPA group,the interlobular space of liver was enlarged,the tissue structure was d isordered,the hepatocytes were watery and steatosis,some nuclei were dissolved,and there was obvious inflammatory infiltration.The results displayed that the combined exposure of Se deficiency and BPA aggravated the inflammatory damage of chicken liver.(2)The results of oxidative stress showed that,in BPA or-Se group,the contents of MDA and NO and the activity of i NOS enhanced(p<0.05),while the activities of SOD,CAT,T-AOC and GSH-Px and GSH content declined,especially in-Se+BPA group(p<0.05).In LMH and HD11 cells,ROS level in BPA or-Se group was significantly enhanced,and it was further enhanced in the combined group(p<0.05).NAC treatment counteracted the excessive production of ROS in-Se+BPA group.The results showed that BPA or/and l ow-Se exposure induced oxidative stress in liver,and the combined group was stronger than the single group.(3)The results of pyroptosis showed that,in BPA or-Se group,the fluorescence intensity of NLRP3 and GSDMD increased in chicken liver tissue and LMH.The expressions of NLRP3 inflammasome(ASC and pro-Caspase-1),Caspase-1,GSDMD,GSDMD-N,IL-1βand IL-18 were up-regulated.The expression of-Se+BPA was further up-regulated(p<0.05).In LMH,the expression of these genes was alleviated by adding NAC.The results showed that the combined exposure of BPA and low-Se aggravated the pyroptosis of chicken hepatocytes through oxidative stress.(4)The results of macrophage polarization showed that the fluorescence intensity of CD86 and CD163 enhanced in the BPA or-Se group,CD86,TNF-α and IL-1β expression enhanced,and CD163,IL-10 and Arg-1 expression declined in chicken liver and HD11 cells.The fluorescence intensity of-Se+BPA group enhanced further,while the expression of marker genes kept the same trend and changed further(p<0.05).In HD11,NAC treatment reversed the expression changes of these genes.The results showed that BPA exposure combined with low-Se exposure aggravated M1 polarization of chicken liver through oxidative stress.(5)The detection result of chemokines show ed that the expression levels of MIF,CCL4,CCL17 and CCL19 in liver tissue and hepatocytes of BPA or-Se group were all increased,and the expression levels in-Se+BPA group were further increased(p<0.05).In cell experiments,the addition of NAC to the culture medium reduce d the expression of some chemokines,indicating that the combined exposure of BPA and low-Se further increased the expression of chemokines.(6)The detection results of cell crosstalk show ed that co-culture with HD11 after treating LMH with low-Se or/and BPA caused the expression of M1 related genes(CD86 and TNF-α)to increase in HD11,while M2 decreased;However,co-culture with LMH after treating HD11 with low-Se or/and BPA induced the pyroptosis of LMH cells and increased the inflammatory factors.After NAC was added to the co-culture system,the above changes were alleviated,which confirmed that BPA and low-Se exposure were mediated by ROSmediated pyroptosis-M1 polarization crosstalk.To sum up,BPA exposure and Se deficiency induce chicken liver inflammation through ROS-mediated pyroptosis-M1 polarization crosstalk.This study clarifie s the new toxic mechanism of liver inflammation caused by BPA exposure or/and Se deficiency,which provides a new perspective for the research of animal toxicology and nutritional deficiency,and also provides a reference for comparative medicine.