Study On The Prevention Mechanism Of Chicken Water-soluble Selenium-containing Proteins Against Colitis And Its Purification,Identification And Antioxidant Activity

Selenium（Se）is an essential trace element in the human body.It is often inserted into proteins in the form of selenomethionine（Se Met）and selenocysteine（Sec）to form Se-containing proteins.Chicken is rich in protein,which provides natural insertion sites for Se and is a good source of nutrition as well as Se for the human body.Some Se-containing proteins perform specific anti-inflammatory,antioxidant and other defense functions in the human body.Therefore,we speculate that the intake of Se-containing proteins from chicken may have a preventive effect on inflammatory bowel disease（IBD）.The purification,identification and antioxidant activity of Se-containing proteins in chicken are of great scientific significance for understanding the types of selenoproteins in poultry,and of great practical significance for human health.In this study,water-soluble proteins with 1.78μg Se/g protein,salt-soluble proteins with1.66μg Se/g protein and alkali-soluble proteins with 1.64μg Se/g protein were extracted from chicken breast with high Se content,respectively.The water-soluble Se-containing protein（WSSeP）with 1.78μg Se/g protein was used as the target material,the prevention mechanism of them on ulcerative colitis（UC）induced by dextran sulfate sodium（DSS）in mice were studied.The selenoproteins in WSSeP was further isolated,purified and and characterized,its antioxidant activity in vitro was preliminarily analyzed.The results of the study are as following:1.The prevention of UC in mice by WSSeP showed that the higher the dose and Se content of WSSeP,the lower the disease activity index（DAI）and inflammatory response,the higher the expression levels of three tight junction（TJ）proteins in the crypt tissues and glands of the colon（including the membrane）,which are claudin-1,occludin and Zonula Occludens-1（ZO-1）,the lower the expression levels of Toll like receptor 4/Myeloid differentiation factor88（TLR4/My D88）in the inflammatory signaling pathway and the lower the levels of interleukin（IL）-1β,IL-6,tumor necrosis factor（TNF）-αand interferon（IFN）-γin the colon,the lower the levels of the lipopolysaccharide（LPS）and malondialdehyde（MDA）in the serum,the higher the activity of superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）in serum.The therapeutic effect of WSSeP at 1500 mg/kg BW/d（Se content 1.78μg Se/g protein）is similar to that of 5-aminosalicylic acid（5-ASA）,there are no side effects as 5-ASA in preventing and improving UC,suggesting that WSSeP is a potential natural anti-inflammatory activity substance.Based on previous findings and combined with the evidences of this study,it is clarified that the classical intervention mechanism of WSSeP on inflammation is to protect the crypts in the mucosal layer and the goblet cells in the glands of the colon through the high expression of three TJ proteins,claudin-1,occludin and ZO-1,the mucus secreted by the goblet cells normally relieves or inhibits the peroxidation of the pro-inflammatory substance LPS formed by pathogenic microorganisms in the colon,thus inhibiting the activation of the inflammatory signaling pathway TLR4/My D88,and reducing or eliminating the inflammation on mucosal layer caused by the damage of DSS.2.WSSeP（1.78μg Se/g protein）was preliminarily purified by anion exchange chromatography,to obtain three fractions F₁,F₂ and F₃.The Se content analysis results showed that the Se contents of the three fractions were 283.5μg Se/g protein,85.2μg Se/g protein and44.7μg Se/g protein,respectively.The F₁ fraction was further purified by gel filtration chromatography,and a purified selenoprotein monomer SePP was obtained,which was identified by HPLC-MS/MS to be highly homologous with glucose-6-phosphate isomerase（GPI）,with molecular weight of 62,091 Da,there is only one Sec insertion site,locating in the403rd amino acid in the domain SIS of the molecular,a highly conserved domain with glycometabolic function.The theoretical ratio of the Se relative atomic mass to molecular weight of the enzyme is 0.0013,and the metrological analysis shows that the ratio of Se content to protein content is 0.0011,and the agreement reaches 84.6%.3.The secondary structure of SePP was analyzed by Fourier transform infrared spectroscopy（FT-IR）and circular dichroism,it can be seen that it contains C-Se and theα-helix is the main secondary structure.The free radical scavenging ability of SePP was determined in vitro,the Fe²⁺reducing antioxidant power,DPPH radical and hydroxyl radical scavenging activity of SePP are concentration-dependent,and the hydroxyl radical（Reactive oxygen species,ROS）scavenging activity is the strongest among them,when SePP concentration reach 100 g/L,the scavenging rate is 99.27%,which was comparable to vitamin C.In conclusion,WSSeP in chicken has a good effect on prevention and improvement of acute UC induced by DSS in mice.It is a natural and excellent anti-inflammatory substance from food,and its anti-inflammatory activity is related to protein dose and Se content.The GPI in chicken WSSeP is an unreported selenoprotein with strong hydroxyl radical scavenging activity,Sec may improve the antioxidant activity of GPI to maintain the stability of this enzyme.Our results provide a theoretical basis for the study of the types and functions of poultry selenoproteins.