| Wheat germ accounts for 3%of the whole wheat grain,and it is praised as“the natural treasure trove of human nutrition”by nutritionists.Wheat germ globulin(WGG)accounts for about 20%of the total protein in wheat germ,and has physiological activities of enhancing immunity,anti-inflammation and anti-oxidation.Proteomics and bioinformatics were used to analyze and predict the proteomics,functional domains,and secondary and tertiary structures of WGG.The mouse models were established and multimodality was used to examine the immunoregulatory effects and explore the mechanisms of WGG on model mice,and WGG's effects on intestinal microflora and metabolites.The main contents are as follows:Proteomics technology was used for the composition analysis,functional annotation,functional domain prediction,and secondary and tertiary structure simulations of WGG.The infrared chromatography,SEM and SDS-PAGE electrophoresis were used to characterize the different precipitation methods of WGG.The results showed that three-level ethanol precipitation was suitable for the purification of WGG.Proteome analysis of wheat germ protein using LC/MS showed that WGG contains 1575 proteins and 584 protein groups.It is mainly composed of the family of Wheat germ globulin,Heat shock proteins,Histones,Typical functional proteins and Uncharacterized proteins.The GO function annotation and KEGG Pathway analysis of the identified proteins revealed that WGG involved a total of 31molecular functions,47 cell components,and 87 biological processes.They participated in199 metabolic pathways,including Systemic lupus erythematosus(31,1.97%),Alcoholism(30,1.96%),Ribosome(26,1.65%),Viral carcinogenesis(20,1.27%),Spliceosome(18,1.14%),etc.55 immunoregulatory signaling pathways were involved,accounting for 22.5%of the total participating pathways.PPI analysis revealed that there were five interacting subnetworks of WGG,including protein splicing and folding network,biological enzyme modification and activation network,protein translation and modification network,carbohydrate metabolism network and protein molecular assembly network.The CBS database and tools were used to predict the functional domain of WGG,and it was found that WGG have immunologically active sites and nutrient supply function.Twenty-two kinds of the tertiary structure of five categories WGG model were visualized by Swiss-Model and Phyre2.The results showed that typical proteins such as Globulin 3A,Heat shock protein,Histone,and WHEAT 5a2 protein may be representative components of the immune function of WGG.Proteomic analysis and functional prediction of WGG provide reference for the development and evaluation of their functions in the next phase.Based on high-throughput sequencing and metabonomics,the effects of WGG on intestinal microflora and metabolic profiles were investigated.A specific dietary intervention mouse model was made with WGG,whey protein,egg white protein.Its results showed that WGG had no significant effect on the basic physiological indicators(body weight,blood routine,liver function,kidney function,blood lipids)of mice and it was eaten for long periods of time without toxic side effects.The consumption of WGG can significantly increase the secretion of IgG and IL-6 in peripheral blood of mice.The immunity of mice in WG group were significantly increased.The predominant microfloras in the intestine of the WG group were Lachnospiraceae nk4a 136 group,Norank bacteroidales s24-7 group,Norank lachnospiraceae,Roseburia,Unclassified lachnospiraceae,Lactobacillus,Alistipes,Lachnoclostridium,etc.At the level of the portal,the number of Firmicutes in the mouse model of wheat germin were increased and the abundance of Bacteroides and Actinobacteria were decreased.At the genus level,the number of Unclassified Lachnospiraceae,Blautia,Anaeroticuncus,and Anaeroplasma in the mice of WGG group were increased,while the abundances of the norank Bacteroidales S24-7 group,Ruminococcaceae UCG-010,Ruminococcaceae NK4A214 group,and Eubacterium nodatum group were decreased.WGG increased the number of beneficial bacteria in the gut microbiota and maintains homeostasis between the gut microbes.The established mathematical model of intestinal microbial diversity and the level of immune molecule secretion showed that intestinal flora diversity and the body's immunity promoted each other.The classification and comparison of intestinal metabolites in each group of model mice identified revealed that there were 53 differential metabolites in the WG-NC group,30 differential metabolites in the WG-EG group,and 66metabolites in the WG-EG group.These differential metabolites mainly involve pathways such as Biosynthesis of amino acids,Glycerophospholipid metabolism,Secondary bile acid biosynthesis,Bile secretion,Choline metabolism in cancer,Retrograde endocannabinoid signaling,and Primary bile acid biosynthesis.And multiple pathways are related to immune system regulation.Correlation analysis and Procrustes analysis were carried out on the intestinal microbial diversity and differential metabolites in each group model mice.Structural changes of intestinal flora and differential metabolites were biologically relevant.The above results showed that wheat germin globulin affected the dynamic changes of immune factors,intestinal microflora and metabolites of model mice,which affected the body's physiological state and immune level.Four aspects of immunological organs,immune cells,immune molecules and mRNA expression revealed the immunological activity and mechanism of action of WGG through experimental animal models.The mouse model of immunosuppression was established by cyclophosphamide.It was found that WGG could increase the body weight,spleen index,and thymus index of model mice.WGG can activate the activity of LDH and ACP in mice,activate the body's macrophages,and increase the total antioxidant capacity of model mice,which balance the redox state of model mice.WGG can increase the activity of lymphocyte ATP in mice,significantly increase the proliferation of mouse spleen T lymphocytes and B lymphocytes,and increase the ratio of CD3~+and CD4~+/CD8~+in peripheral blood.Thus,the cellular immunity of model mice were improved.The ratio of immune cytokines and immune lymphocyte subsets in peripheral blood of model mice increased the rate of differentiation of Th1 cells into Th2 cells in immunosuppressed mice,and this mutual transformation was unbalanced.WGG may regulate the transformation of Th1/Th2 subsets of immunosuppressed mice by increasing the expression of T-bet mRNA in model mice and decreasing the expression of GATA-3 mRNA.The effect of WGG on the proliferation of lymphocytes may be achieved through the mitochondrial-dependent apoptosis pathway that promotes the expression of Bcl-2 mRNA and increases the ratio of Bcl-2/Bax.The results showed that WGG can improve the immune level of immunosuppressed mice to a certain extent,and there is a linear correlation in dose,which has biological statistical significance.WGG is a functional protein related to immunity and nutrient supply and plays a key role in the regulation of biological processes.It can be isolated and purified as a special food and plant-derived immunomodulator. |
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