Study On Anti-inflammatory Activity And Mechanism Of Major Yolk Protein From Sea Cucumber

Sea cucumber is a kind of precious medicine and food aquatic products,with high economic value and nutritional value.The aquaculture areas are mainly distributed along the coasts of the Yellow Sea and Bohai Sea in China,and the environment may affect the nutritional quality and functional activity.In order to fully explore the biological active components of sea cucumber,the changes of nutrient contents in multi-region and multi-season sea cucumber were investigated.The main edible part of sea cucumber is the body wall,which contains a variety of active components,such as polysaccharides,essential amino acids,polyunsaturated fatty acids and other biological active substances.Studies have shown that edible sea cucumbers have biological functions such as hypoglycemia,hypolipemia and anti-nephritis,and these chronic diseases are often accompanied by inflammatory reactions.However,there are few reports on whether sea cucumbers play these functions by inhibiting inflammatory reactions and the specific functional components that can exert anti-inflammatory activities.In this study,the nutritional components of sea cucumbers in different regions and seasons were comprehensively evaluated,and the anti-inflammatory activities of sea cucumbers and their main components were evaluated through animal experiments.High abundance major yolk protein with anti-inflammatory activity was screened by TMT proteomics.The separation and purification method was established for major yolk protein.Further we analyzed its antiinflammatory activity and mechanism.The research contents are as follows:(1)Study on the main nutritional components of sea cucumber body wall: This paper selected sea cucumber caught in spring and autumn in four representative breeding areas as the research object,and analyzed the content of nutrients such as protein,polysaccharide,lipid,the amino acids,fatty acids and mineral elements in the body wall.The results show that the content of main nutrients was affected by the season and region,but the influence by season is more significant.When analyzing the nutritional components of sea cucumber,the cumulative contribution rate of principal components was 74.6%,and the main nutritional components were amino acids,fatty acids and polysaccharides.(2)Study on the anti-inflammatory activity of sea cucumber and its principal components:Sea cucumber and its principal components were administered to mice with colitis in different doses(50,100 and 300 mg/kg,respectively)to verify the anti-inflammatory activity of mice.The results showed that the clinical score of the sea cucumber group decreased,and the colonic mucosal tissue repair and anti-inflammatory factors IL-4 and IL-10 were increased by 39.7%and 33.1%,respectively.The results showed that sea cucumber had the activity of alleviating colitis.To further identify the functional components with anti-inflammatory activity in sea cucumber,the composition of sea cucumber was divided into three components: ethanol extract,polysaccharides and protein.The colitis mice were gavaged with the main composition.The results showed that low-dose sea cucumber polysaccharide and all protein groups had the function of alleviating colitis,and mainly manifested in reducing symptoms of loose stools and bloody stools,decreasing inflammatory cells,repairing the mucosal tissue,decreasing contents of pro-inflammatory factors(TNF-α,IL-6,TGF-β and IFN-γ)and increasing contents of antiinflammatory factors(IL-4 and IL-10),and especially the high-dose protein group had the best anti-inflammatory effect.(3)Major yolk protein isolation and purification and identification: The composition and content of sea cucumber protein in different regions and seasons were studied by TMT quantitative proteomics.The results showed that sea cucumber yolk protein was a high abundance protein.Major yolk protein was then extracted by three-step separation precipitation and the purity was 88.54% and the molecular weight was 2.0×106 k Da.The results showed that major yolk protein was composed of four subunits,and their molecular weights were 171 k Da,123 k Da,79 k Da and 59 k Da respectively.The electrophoretic bands were stained with Coomassie brilliant blue,periodate-Schiff base,methyl green-molybdate and Sudan black B,all of which were positive,indicating that major yolk protein is a high molecular weight glycophospholipid protein.In addition,combined with bioinformatics analysis,major yolk protein has the motif of transferrin,and transferrin has anti-inflammatory activity,so it is speculated that vitelloprotein is the main anti-inflammatory factor in the protein components of sea cucumber.(4)Study on anti-inflammatory activity and mechanism of major yolk protein: Colitis mice were gavaged with different doses of major yolk protein.The results showed that major yolk protein could alleviate colitis in mice,especially the medium dose(100 mg/m L)and high dose(300 mg/m L)major yolk protein could significantly improve the morphological characteristics,reduce the mucosal damage and reduce inflammatory cell infiltration.And the mechanism of major yolk protein alleviating colitis was discussed.Major yolk protein increased Firmicutes and Bacteroidetes,reduced microflora,and promoted the increase of short chain fatty acids.Furthermore,molecular docking method was used to explain the anti-inflammatory mechanism of major yolk protein.Further using the molecular docking,the results showed that major yolk protein could inhibit inflammatory response by binding anti-inflammatory peptide to colitis target,and short peptides FL and SYF could exert anti-inflammatory effect through multi-target,while AFPAL only acted on IL1 B target protein.In summary,this paper firstly screened major yolk protein by the proteomic technology,and established a method for the separation and purification of the yolk protein.Further,the DSS induced colitis model revealed that major yolk protein was the main component of the anti-inflammatory activity of sea cucumber,and its possible mechanism was preliminarily discussed.In conclusion,animal experiments and molecular docking has analyzed the sea cucumber major yolk protein can alleviate colitis,providing development ideas and data support for the future development of functional food.