Bisphenol A Exposure And Selenium Deficiency Induce Apoptosis And Cell Cycle Arrest In Broiler Jejunum Via ROS/p53 Pathway

Bisphenol A(BPA)is one of the industrial organic compounds with high yield and wide use.It is mainly used in the production of food packaging bags and containers.BPA is released into the atmosphere,surface water,wastewater and dust during production and use,and enters the organism in different ways,resulting in excessive ROS production,induction of oxidative stress,mitochondrial dysfunction and apoptosis to damage the heart,kidney,liver and intestine.Selenium(Se)is an essential trace element for the organism.It has the functions of anti-oxidation,improving immunity,preventing diseases and detoxification,which plays an important biological function.Se deficiency can cause oxidative stress and apoptosis in the kidney,heart,and liver.In addition,the intestine is not only affected by BPA exposure but also a target organ for Se deficiency.As a highly organized cell death process,apoptosis plays a regulatory role in organ development,tissue homeostasis,and immune response,and is related to the pathological processes of many organisms.A number of studies have shown that changes in the expression of some factors can cause apoptosis.Among them,tumor suppressor protein p53 is a redox-active transcription factor.When the organism lacks trace elements or is exposed to environmental pollutants,p53 organizes and guides cells to respond,including inducing apoptosis.With the deepening of p53 research in recent years,researchers have found that p53 is also involved in the regulation of the cell cycle.At present,there is no report on the damage of BPA exposure and Se deficiency to the intestine of broilers.In order to further study the damage of BPA exposure and Se deficiency alone or in combination with the jejunum of broilers and its specific molecular mechanism,this experiment first replicated the broiler models of Se deficiency(-Se),BPA exposure(BPA group),and BPA exposure and Se deficiency combined treatment(-Se+BPA).The pathological changes or ultrastructural changes of jejunum in broilers were observed by H&E staining or transmission electron microscopy.The apoptosis of broiler jejunum or CSIEC cells was evaluated and detected by TUNNEL staining,acridine orange/ethidium bromide(AO/EB)staining,and flow cytometry.The expression of Caspase3 and Bcl2 was detected by immunofluorescence staining.The kit was used to detect the oxidative stress indicators(MDA,GSH,CAT,GSH-Px)of broiler jejunum and ROS staining of CSIEC cells to detect the antioxidant capacity of broiler intestinal epithelial cells.The expression of p53,mitochondrial pathway apoptosis-related genes,cell cycle-related genes and mitochondrial dynamics-related genes were detected by q RT-PCR and Western blot.In addition,p53 inhibitor Pifithrin-α(PFTα)or ROS inhibitor N-Acetyl-L-cysteine(NAC)was used to pretreat CSIEC cells in vitro.The main research results are as follows:(1)The results of pathological structure observation showed that the intestinal villi and crypt structure were normal in the jejunum of broilers in the Control group.In the-Se group and BPA group,the villi were broken and disordered,and the crypt structure was abnormal.In the-Se+BPA group,the intestinal villi were atrophic and sparse,the crypt structure was abnormal and branched.The above results indicated that BPA exposure and Se deficiency damaged broiler jejunum and the combined treatment caused more serious damage to broiler jejunum.(2)The results of ultrastructural observation showed that the jejunum cells of broilers had obvious apoptotic characteristics in the-Se group and BPA group,including nuclear shrinkage;chromatin condensation,marginalization;mitochondrial structural abnormalities.The jejunum cells of broilers in the-Se+BPA group also showed obvious apoptotic characteristics,including nuclear shrinkage and unclear nuclear membrane boundaries;chromatin agglutination,marginalization;mitochondrial swelling,mitochondrial cristae rupture,and even vacuolization.It is indicated that BPA exposure or/and Se deficiency caused apoptosis in broiler jejunum.The results of TUNEL staining in vivo,AO/EB staining and flow cytometry in vitro were consistent with the above results.And the results of immunofluorescence staining showed that the expression level of Caspase3 in the experimental group was higher than that in the control group,and the expression level of Caspase3 in the-Se+BPA group was higher than that in the BPA group and-Se group.The expression level of Bcl2 in the experimental group was lower than that in the control group,and the expression level of Bcl2 in the-Se + BPA group was lower than that in the BPA group and-Se group.The above results indicated that BPA exposure and Se deficiency caused apoptosis in broiler jejunum,and the combined treatment of BPA exposure and Se deficiency induced more obvious apoptosis in broiler jejunum.(3)In the-Se group and BPA group,the activity of CAT and GSH-Px,the content of GSH in the broiler jejunum was significantly lower than those in the control group,and MDA content was significantly higher than the Control group.Compared with the-Se group and BPA group,the activity of CAT and GSH-Px,the content of GSH decreased significantly,and the content of MDA increased significantly in the-Se + BPA group.The results of ROS staining in vitro showed that the ROS production in the BPA group and-Se group was higher than that in the control group.The ROS release of CSIEC cells in the-Se+BPA group was significantly higher than that in the BPA group and the-Se group,and the production of ROS decreased after the use of ROS inhibitor NAC.The above results indicated that BPA exposure and Se deficiency caused oxidative stress in broiler jejunum,and the combined treatment induced more obvious oxidative stress in broiler jejunum.(4)Compared with the Control group,the expression levels of apoptosis-related genes p53,BAX,Caspase3,Caspase9,Cytc and Apaf1 in the jejunum or CSIEC cells of the BPA group and the-Se group were increased,and the expression level of Bcl2 was decreased.The expression level of mitochondrial dynamics related gene Drp1 increased,and the expression levels of OPA1,Mfn1 and Mfn2 decreased;ATP content and ATPase activity decreased.Compared with the BPA group and the-Se group,the expression levels of apoptosis-related genes and mitochondrial dynamics-related genes were changed in the-Se+BPA group,and the ATP content and ATPase activity were decreased.It is indicated that BPA exposure and Se deficiency induced mitochondrial dysfunction in broiler jejunum,caused apoptosis of mitochondrial pathway in broiler jejunum,and combined treatment caused more obvious apoptosis in the mitochondrial pathway of broiler jejunum.(5)Compared with the control group,the expression level of cell cycle-related genes in the jejunum or CSIEC cells of BPA group and-Se group decreased,and the expression level of p21 increased.Compared with BPA group and-Se group,the expression level of cell cycle related genes in jejunum or CSIEC cells of-Se+BPA group decreased,and the expression level of p21 increased.These results indicated that BPA exposure and Se deficiency induced cell cycle arrest in jejunal epithelial cells of broilers,and the combined treatment of BPA exposure and Se deficiency induced more obvious cell cycle arrest in jejunal epithelial cells of broilers.(6)In vitro experiments,after the application of inhibitors NAC or PFTα,the cell cycle arrest and apoptosis of CSIEC caused by BPA exposure and Se deficiency were significantly alleviated,indicating that BPA exposure and Se deficiency induced cell cycle arrest and apoptosis of chicken jejunum through ROS and p53,confirming the importance of ROS and p53 in this process.In summary,BPA exposure and Se deficiency can induce oxidative stress in the jejunum of broilers,activate ROS/p53 pathway to induce cell cycle arrest and apoptosis,and combined treatment increase cell cycle arrest and apoptosis in broiler jejunum through the ROS/p53 pathway.The results of this study further enriched the content of the molecular mechanism of BPA exposure and Se deficiency intestinal injury and provided a new direction for the prevention and treatment of intestinal injury caused by BPA exposure and Se deficiency in production practice.