| Selenium(Se)is an essential nutrient trace element in the orgnism,which is very important for the health of the organism.Se\ deficiency can cause diarrhea and even death in chickens,thus affecting the economic development of poultry production.Diarrhea is related to intestinal health.The small intestine is an important place for digestion and absorption of the organism,as well as an important part for absorption of Se.Intestinal damage caused by Se deficiency will seriously harm the health and production performance of animals.Studies have found that Se deficiency can cause intestinal damage,including intestinal mucosal bleeding,intestinal integrity damage,oxidative stress,inflammatory infiltration,epithelial cell necrosis and so on.In the orgnism,Se plays antioxidant function in the form of selenocysteine.Oxidative stress damage caused by Se deficiency is related to the inactivation of selenoproteins.GPX3 in glutathione peroxidases(GPXs)family is one of the selenoproteins with antioxidant function.GPX3 plays a key role in reducing oxidative stress and inflammation in the intestine.Competitive endogenous RNAs(ce RNA)are a novel regulatory network discovered in recent years,involving a novel mode of post-transcriptional molecular regulation.long non coding RNA(Lnc RNA)can be used as ce RNA to combine competitively with Micro RNA(miRNA)and participate in the regulation of necroptosis.More and more studies have found that there is an interaction between selenoprotein and ce RNA regulatory network,but the correlation between changes in GPX3 expression induced by Se deficiency and ce RNA regulatory network and the mechanism of action of non-coding RNA/GPX3 axis in Se-deficient broiler intestinal inflammation remains unclear.Our previous studies have found that Lnc RNAWSF27,miR1696 and GPX3 have ce RNA regulatory relationships.In order to further determine the role and regulatory mechanism of Lnc RNAWSF27/miR-1696/GPX3 in the occurrence of Se-deficient broiler intestinal inflammation,the intestinal inflammation model of Se-deficient broiler was replicated in this study.The histopathological changes of duodenum and jejunum were observed by transmission electron microscopy and H&E staining.The expressions of 25 selenoproteins,oxidative stress,necroptosis,inflammatory factors and tight junction related genes and proteins were detected by immunohistochemistry,western blot and q RT-PCR.The small intestinal epithelial cell model,Lnc RNAWSF27 knockdown model,miR-1696 knockdown/overexpression model and GPX3 knockdown/overexpression model of chicken small intestinal epithelial cells were established in vitro.WB,q RT-PCR,AO/EB staining,ROS staining,fluorescence glucan labeling,HRP tracer labeling and other techniques were used to detect the level of intestinal oxidative stress,activation of necroptosis,inflammation and expression of tight-junction related factors,and cell bypass permeability,.The objective was to determine the regulatory relationship between changes in intestinal GPX3 expression induced by Se deficiency and Lnc RNAWSF27/miR1696,and to reveal the mechanism of Lnc RNAWSF27/miR-1696/GPX3 axis in regulating the occurrence of Se deficient broiler intestinal inflammation.(1)The small intestine damage model of Se-deficient broilers was successfully replicated in broilers fed with low Se diet.H&E staining showed that the muscle layer of duodenum and jejunum of Se deficient broilers was broken,intestinal glands and villi were seriously deformed,inflammatory cell infiltration occurred in the muscle layer and mucous lamina propria,and nuclear contraction of mucous lamina propria cells in the intestinal tissue of Se deficient broilers was observed.Ultrastructure of duodenum and jejunum villi of Se deficient broilers were broken and dissolved by transmission electron microscopy.The cell structure was deformed,cell membrane was broken,nucleolysis and disappearance,endoplasmic reticulum was swollen and broken,mitochondria were enlarged and ridge disappeared,and the tight intercellular connection structure was destroyed,showing obvious necroptosis characteristics,indicating that Se deficiency induced necroptosis of small intestine of broilers.(2)Se deficiency can affect the expression of selenium protein in broilers small intestine.The m RNA levels of 25 selenoproteins showed that GPX1,GPX3,Dio1,Dio3 decreased significantly in jejunum,and GPX3,Dio1,Dio2,Sel I,Sel K,Sel M and Sel W decreased significantly in duodenum.GPX3 has the most significant reduction.According to previous studies,in order to further verify the regulatory relationship between intestinal GPX3 and Lnc RNAWSF27 and miR-1696,the expression of Lnc RNAWSF27 was significantly decreased and the expression of miR-1696 was significantly increased in the small intestine of Se deficient broiler.At the same time,the expression of Lnc RNAWSF27 and GPX3 was decreased and the expression of miR-1696 was increased in chicken small intestinal epithelial cells due to Se deficiency.In chicken small intestinal epithelial cells,knockdown of Lnc RNAWSF27 increased the expression of miR-1696 and thus decreased the m RNA and protein levels of GPX3.Overexpression or knockdown of miR-1696 can decrease or increase the expression of Lnc RNAWSF27 and GPX3.This suggests that Lnc RNAWSF27 can act as a molecular sponge of miR-1696 in bioiler small intestine,bind miR-1696 competitively and regulate the expression of miR-1696 and GPX3.(3)Further study on the oxidative damage of small intestine caused by Se deficiency in chickens showed that CAT activity,GSH-PX,GSH,SOD content and MDA content were significantly decreased in jejunum and duodenum.In addition,m RNA levels of CAT,GST,HO-1,SOD1 and SOD2 were significantly decreased.The m RNA expression level of selenoprotein was decreased in 60% of small intestinal epithelial cells of Se-deficient chickens,and the decrease of GPX3 was most significant.The expression levels of oxidative stress related indicators showed that the expressions of CAT,GST,HO-1,SOD1 and SOD2 were significantly down-regulated,while ROS levels were significantly increased.NAC treatment could reduce ROS levels and cell death,indicating that Se deficiency led to the activation of oxidative stress in small intestine of broilers.In chicken small intestinal epithelial cells,after knockdown Lnc RNAWSF27,knockdown GPX3 and overexpression of miR-1696,ROS levels increased and expression levels of oxidative stress-related indicators were significantly down-regulated,accompanied by the aggravation of necroptosis,and eventually led to inflammation.These results indicated that low expression of Lnc RNAWSF27,overexpression of miR-1696 and low expression of GPX3 could activate oxidative stress in chicken small intestinal epithelial cells.After Lnc RNAWSF27 and miR-1696 knockdown at the same time,the low expression of miR-1696 can save the occurrence of oxidative stress in chicken small intestinal epithelial cells caused by Lnc RNAWSF27 knockdown,inhibit the pathway of necroptosis,and prevent inflammation.The results indicated that the change of Lnc RNAWSF27/miR-1696 expression in chicken small intestinal epithelial cells can reduce the expression of GPX3 and cause oxidative stress response,aggravate necroptosis,and ultimately lead to cell inflammation.(4)According to the necrotic features presented by morphology,immunohistochemical results of jejunum and duodenum showed that the content of RIPK1 in tissues increased due to Se deficiency.The m RNA and protein levels of necroptosis signaling pathway genes in jejunum and duodenum were detected,and the expressions of TNF-α,RIPK1,RIPK3 and MLKL were increased,while the expressions of Caspase-8 and FADD were decreased.The expressions of TNF-α,RIPK1,RIPK3 and MLKL were increased and the expressions of Caspase-8 and FADD were decreased in small intestinal epithelial cells of Se deficient broilers,indicating that Se deficiency caused the occurrence of small intestinal necroptosis of broilers.In chicken small intestinal epithelial cells,the level of necroptosis increased after Lnc RNAWSF27 knockdown,GPX3 knockdown and overexpression of miR-1696,and was relieved after treatment with Nec-1.These results indicated that low expression of Lnc RNAWSF27,overexpression of miR-1696 and low expression of GPX3 could cause necroptosis of chicken small intestinal epithelial cells.Lnc RNAWSF27 and miR-1696 were simultaneously knocked down,and it was found that low expression of miR-1696 could alleviate necroptosis of chicken small intestinal epithelial cells caused by Lnc RNAWSF27 knockdown,suggesting that Lnc RNAWSF27 could competitively bind to miR-1696 and reduce GPX3 expression.Involved in necroptosis of chicken small intestine cells caused by Se deficiency.(5)As inflammatory cell infiltration was observed in H&E staining,the detection results of inflammatory factors in tissues showed that the expressions of IL-1β,IL-2,IL-6,NF-κB,i NOS,COX-2and PTGE were significantly up-regulated,while the expression of IL-10 was significantly down-regulated.At the same time,Se deficiency led to the increase of inflammatory response in chicken small intestinal epithelial cells.The above results suggest that Se deficiency induced intestinal inflammatory response in broilers.In chicken small intestinal epithelial cells,the inflammation level was increased after Lnc RNAWSF27 knockdown,GPX3 knockdown and miR-1696 overexpression,and the expression of pro-inflammatory factors was significantly decreased after Nec-1 treatment.These results indicated that low expression of Lnc RNAWSF27,overexpression of miR-1696 and low expression of GPX3 would aggravate the inflammatory response of small intestinal epithelial cells in chickens.Lnc RNAWSF27 and miR-1696 were simultaneously knocked down,and it was found that the low expression of miR-1696 could save the necroptosis and inflammatory reaction of chicken small intestinal epithelial cells caused by Lnc RNAWSF27 knockdown,suggesting that Lnc RNAWSF27/miR-1696 could reduce GPX3 expression.Aggravate the inflammatory reaction of chicken small intestinal epithelial cells.(6)Se deficiency can significantly increase the expression of ERK,JNK and p38 in jejunum and duodenum,and significantly decrease the expression of ZO-1,Occludin-1,RAPGEF2,Rap2 c and Claudin-1,while significantly increase the expression of Rho A and Rock.The level of MAPK pathway genes in small intestinal epithelial cells of Se deficient chickens was increased,the expression of genes related to tight junction integrity was decreased,and the cell bypass permeability was increased,suggesting that Se deficiency could lead to the damage of small intestinal tight junction.In chicken small intestinal epithelial cells,after Lnc RNAWSF27 knockdown,GPX3 knockdown and miR-1696 overexpression,MAPK pathway was activated and intensified tight junction damage,which was relieved after treatment with Nec-1.These results indicated that low expression of Lnc RNAWSF27,overexpression of miR-1696 and low expression of GPX3 would lead to tight junction damage of chicken small intestinal epithelial cells and increase cell bypass permeability.Lnc RNAWSF27 and miR-1696 were knocked down at the same time,and it was found that the low expression of miR-1696 could save the tight junction damage of chicken small intestinal epithelial cells caused by Lnc RNAWSF27 knockdown and restore the cell bypass permeability,suggesting that Lnc RNAWSF27/miR-1696 could reduce GPX3 expression.Breaking the tight connections of chicken small intestinal epithelial cells.In conclusion,Se deficiency can inhibit the expression of Lnc RNAWSF27,lead to increased expression of miR-1696,and thus reduce the expression of GPX3.Low expression of GPX3 will activate oxidative stress in broiler small intestine,leading to necroptosis and then lead to intestinal inflammation.Our results suggest that Lnc RNAWSF27,miR-1696 and GPX3 are involved in regulating necroptosis and inflammatory damage of chicken intestinal induced by Se deficiency.Lnc RNAWSF27/miR-1696 can down-regulate GPX3 expression which participate in necroptosis mediated by Se deficiency in broiler intestinal inflammation.At the same time,oxidative stress can also activate MAPK pathway and induce intestinal tight junction injury.This conclusion enriched the understanding of the mechanism of intestinal injury caused by Se deficiency,further enriched the biological function of Se,and provided reference for comparative medicine.At the same time,this study will contribute to the diagnosis and drug development of Se-deficient intestinal inflammation. |
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