| Selenium(Se)is an essential trace element that is very important to human health.It participates in the regulation of the immune system,digestive system,respiratory system,urinary system,endocrine system,reproductive system and cardiovascular system.Se deficiency can induce damage to various tissues and cells,including brain,pancreas,breast,liver,arteries,and intestinal tissues.Bisphenol A(BPA)is an environmental endocrine disruptor chemicals(EDCs)commonly used in chemical products and food packaging materials.The degradation of these materials can release BPA into the environment and interfere with the endocrine system of humans or animals,causing numerous damage to the body.Meanwhile,numerous studies have shown that the cardiovascular system is also one of the targets for BPA and Se deficiency.BPA exposure and Se deficiency can induce oxidative stress,and some studies have proved that BPA exposure and Se deficiency can cause apoptosis of arterial endothelial cells by reducing the antioxidant capacity of the body,thus causing damage to arteries.Mi RNAs are a class of endogenous short chain non-coding RNA that can specifically regulate their target m RNA to regulate a series of biological reactions.More and more studies have revealed that miRNA is an important regulator of a series of pathophysiological cellular effects and molecular signaling pathways.However,it is currently unclear whether the mechanism of Se deficiency and BPA damage to the arterial tissue of broilers and whether miRNA participates in arterial damage through the ROS/PI3K/AKT signaling pathway.In order to further investigate the mechanism of miRNA mediated by BPA exposure and Se deficiency in regulating apoptosis of broiler arterial endothelial cells,this experiment used models of BPA exposure/Se deficiency in chicken arteries and endothelial cells,and established knockdown/overexpression patterns of miR-215-3p and Dio1 in vitro.In vivo experiments were divided into four groups: control group(group C),Se deficiency group(-Se group),BPA exposure group(BPA group),Se deficiency+BPA exposure group(-Se+BPA group).In vitro experiments were divided into four groups(C group,-Se group,BPA group,-Se+BPA group)and five groups(C group,-Se+BPA group,-Se+BPA+Inhibitor group,-Se+BPA+NAC group,-Se+BPA+LY294002 group).The detection of 25 selenoprotein in chicken artery tissue by q RT-PCR revealed that Dio1 was significantly differentially expressed.The bioinformatics targeting prediction website predicted the miRNAs targeting Dio1.The targeting relationship between miR-215-3p and Dio1 was analyzed and verified by double Luciferase gene reporting analysis,q RT-PCR and western blot.The morphological changes of chicken arterial tissue were observed by H&E staining,and the expression of caspase-3 was observed by transmission electron microscopy,TUNEL detection,flow cytometry,AO/EB staining and immunofluorescence to evaluate the apoptotic signal of chicken arterial endothelial cells.The expression of PI3K/AKT pathway,mitochondrial dynamics genes(Drp1,Mfn1,Mfn2,OPA1),and mitochondrial apoptosis pathway related genes(Bcl-2,Bax,cytc,APAF-1,caspase-9,caspase-3)in chicken arterial tissue and PAEC cells were detected using real-time fluorescence quantitative PCR,western blot,and other techniques.In addition,this experiment also measured the content or activity of CAT,GSH,GPX,SOD,MDA,e NOS,i NOS,ATPase,and ATP in different groups of chicken arterial tissue and PAEC cells.ROS staining was performed on different groups of PAEC cells.The research results are as follows:(1)Pathological observation revealed that exposure to BPA and Se deficiency led to damage such as collagen fiber hyperplasia,intimal plaque protrusion,scar tissue hyperplasia,and elastic fiber rupture in arterial tissue.Ultrastructural observation showed that BPA exposure and Se deficiency led to obvious apoptosis characteristics of vascular endothelial cells,such as karyopyknosis,chromatin aggregation and marginalization.The TUNEL staining and immunofluorescence staining results of arterial tissue showed that the number of apoptotic positive cells in chicken arterial endothelial cells significantly increased in the BPA exposure and Se deficiency groups,and the expression of the key apoptotic factor caspase-3 significantly increased.The combined effects of BPA exposure and Se deficiency were particularly evident in cell apoptosis.This indicated that BPA exposure and Se deficiency induced apoptosis of vascular endothelial cells,and showed a trend that BPA exposure aggravated Se deficiency induced apoptosis.(2)Under BPA exposure and Se deficiency conditions,the activity of antioxidant enzymes CAT,GSH,GPX,and SOD in the arterial tissue of broilers decreased,MDA levels increased,and ROS release significantly increased in PAEC cells.Under BPA exposure and Se deficiency conditions,the e NOS enzyme activity in arterial tissue decreases,while the i NOS enzyme activity increases.The results indicate that exposure to BPA and Se deficiency can lead to a decrease in the antioxidant capacity of arterial tissue.(3)Under BPA and Se deficiency conditions,the protein and m RNA levels of mitochondrial fusion proteins Mfn1,Mfn2,OPA1 in arterial tissues and PAEC cells were significantly reduced,and the protein and m RNA levels of mitochondrial fission protein Drp1 were significantly increased.BPA and Se deficiency lead to a significant decrease in ATPase activity and ATP levels.These results suggest that BPA exposure and Se deficiency may promote cell apoptosis by enhancing mitochondrial fission and inhibiting mitochondrial ATP synthesis.(4)Through AO/EB,flow cytometry,western blot,and q RT PCR detection,it was found that the PI3K/AKT pathway genes were significantly inhibited under BPA and Se deficiency conditions,and cell apoptosis signals were significantly enhanced.The expression of mitochondrial apoptosis pathway related factors cytc,APAF-1,Bax,caspase-9,and caspase-3was significantly enhanced,and the data showed that apoptosis was particularly significant under the combined effects of BPA exposure and Se deficiency.This indicates that BPA exposure and Se deficiency may induce cell apoptosis by inhibiting the PI3K/AKT pathway,and exhibit a trend of BPA exacerbating cell apoptosis under Se deficiency conditions.(5)After screening the selenoprotein with significant differential expression between Dio1 as BPA and Se deficiency,the miR-215-3p specifically targeting Dio1 was predicted through Targetscan,miRDB and other websites.The knockdown/overexpression model of miR-215-3p/Dio1 was successfully constructed in PAEC cells.The targeting relationship between miR-215-3p and Dio1 was verified by double Luciferase gene reporting analysis,q RT-PCR and western blot.(6)When miR-215-3p overexpression and Dio1 expression were reduced in PAEC cells,ROS release was significantly increased,PI3K/AKT pathway genes were significantly inhibited,mitochondrial fission protein expression was significantly increased,mitochondrial fusion protein expression was significantly reduced,ATPase activity and ATP level were significantly reduced,apoptosis signal was significantly enhanced,and expression of genes related to mitochondrial apoptosis pathway was significantly enhanced.In addition,miR-215-3p-Inhibitor and NAC blocked cell apoptosis caused by BPA exposure and Se deficiency,while LY294002 exacerbated apoptosis.These results indicate that BPA exposure exacerbates Se deficient arterial endothelial apoptosis in chickens by regulating the activation of ROS/PI3K/AKT pathway by miR-215-3p/Dio1.In summary,BPA exposure and Se deficiency can cause significant damage to the arterial tissue of broiler chickens.BPA exposure and Se deficiency cause overexpression of miR-215-3p,inhibit the expression of Dio1,produce excessive ROS,inhibit PI3K/AKT pathway,promote mitochondrial fission,and induce apoptosis through mitochondrial apoptosis.This study confirms that BPA exposure regulates the activation of ROS/PI3K/AKT signaling pathway by the miR-215-3p/Dio1 axis,exacerbating Se deficiency induced apoptosis in chicken arterial endothelial cells. |
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