Study On The Protective Mechanism Of Glutamine Against β-Conglycinin And Glycinin Induced Enteritis In Pearl Gentian Grouper (Epinephelus Fuscoguttatus♀ × Epinephelus Lanceolatus♂)

Soybean is a widely used protein source in fish feeds.However,a certain level of β-conglycinin and glycinin in soybean induce intestinal inflammation and inhibit fish growth.Glutamine（Gln）,a conditionally essential amino acid,is a major energy source for enterocytes and immune cells and can be involved in the regulation of the mucosal immune system.It has been widely used in the treatment of inflammatory bowel disease in mammals.The fish intestine is an important barrier to maintain the stability of the organism’s internal environment,which has great significance for organism’s defense system.Therefore,this study was conducted to reveal the role and potential mechanism of Gln in alleviating enteritis caused by soybean antigenic protein in pearl gentian grouper（Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂）from nutritional and molecular biological perspectives.The results of the study provide a reference for the rational use of soybean meals and the scientific configuration of grouper feed.The main results of the study are as follows:1.Establishment of a soybean meal-enteritis model and its immunoregulatory network for pearl gentian grouperFour isonitrogenous（48% crude protein）and isolipidic（12% crude lipid）diets were formulated by replacing 0%,10%,30%,and 50% fish meal protein with soybean meal（namely FM,SBM10,SBM30,and SBM50,respectively）.Each feed was fed to peral gentian grouper after 10 weeks.There were no significant differences in weight gain rate（WGR）and feed coefficient rate（FCR）among FM,SBM10,and SBM30 groups（P > 0.05）.However,WGR of the SBM50 group was significantly lower compared with other groups（P < 0.05）.In addition,FCR of the SBM50 group was significantly higher compared with other groups（P < 0.05）.Plica height,muscular layer thickness,and goblet cells of the distal intestine in the SBM50 group were extremely lower than those in the FM group（P < 0.01）.The expression levels of pro-inflammatory cytokines il-6 and tnf-α genes in the distal intestine of fish in the SBM50 group were significantly higher than those in the FM group（P < 0.05）,whereas the expression levels of anti-inflammatory cytokines tgf-β and il-10 genes were significantly lower than those in the FM group（P < 0.05）.The intestinal transcriptomic data,including transcriptome and miRNAome,showed that a total of 6,390 differentially expressed genes（DEGs）and 92 DEmiRNAs were identified in the SBM50-vs-FM comparison group.Differentially upregulated m RNAs were significantly enriched in immune-related pathways such as phagosomes,natural killer cell-mediated cytotoxicity,FcγR-mediated phagocytosis,intestinal immune network for Ig A-production,as well as inflammatory NF-κB signaling pathway（P < 0.05）.DEmiRNAs（10 known and 1 novel miRNAs）and their DE target genes are involved in the immune-related phagosome,natural killer cell-mediated cytotoxicity,ECM-receptor interaction pathways.2.Study on the protective mechanism of glutamine against β-conglycinin-induced enteritis in pearl gentian grouperFour isonitrogenous（48% crude protein）and isolipidic（12% crude lipid）trial diets were formulated: a basic diet（FM）,a diet containing 7% β-conglycinin（7S）,and 7S diets supplemented with 1% or 2% alanine-glutamine（1% and 2% Ala-Gln）.Compared with Group 7S,fish fed the 2% Ala-Gln diet had increased weight gain and specific growth rates（P < 0.05）;enhanced intestinal muscular layer thickness,plica height,and plica width（P < 0.05）;promoting intestinal cell proliferation（P < 0.05）;enhanced the content of each free essential and non-essential amino acids（P < 0.05）;increased intestinal glutamine,glutaminase,alpha-ketoglutarate,malate dehydrogenase,and ATP contents（P < 0.05）;increased intestinal lysozyme and acid phosphatase activities and complement（C3 and C4）and immunoglobulin M contents（P < 0.05）;downregulated expression levels of myd88,ikkβ,nf-κb p65,tnf-α,il-1β,ifn-α,and hsp70 genes;enhanced relative abundances of potentially beneficial bacterial genera Akkermansia,Allobaculum,Ileibacterium,Bifidobacterium,Blautia,Lactobacillus,and Sharpea（P < 0.05）.Integration of transcriptomic and proteomic showed that 117 differentially expressed genes were consistent at the transcriptional and translational levels in the 2% Ala-Gln-vs-7S comparison group.These differentially expressed genes were significantly enriched in intestinal physical barrier-related pathways（P < 0.05）,including extracellular matrix receptor interactions,focal adhesion,and tight junctions.Further,α-actin,collagen alpha-1（I）,collagen alpha-2（I）,collagen alpha-1（VI）,integrin-linked protein kinase,and caveolin-1 were significantly upregulated at transcriptional and translational expression levels（P < 0.05）.q PCR results of miRNA showed that miR-192-3p₂ and miR-210-5p（target gene cadherin-5）,and miR-24（target gene perforin-1）expression levels in fish fed Gln group were significantly lower compared to the 7S group fish（P < 0.05）.3.Study on the protective mechanism of glutamine against glycinin-induced enteritis in pearl gentian grouperFour isonitrogenous（48% crude protein）and isolipid（12% crude lipid）experimental diets were formulated: a basal diet（FM,40% fish meal）,a diet containing 10% glycinin（11S）,and an 11 S diet supplemented with 1% or 2% alanine-glutamine（1% Ala-Gln and 2% Ala-Gln）.Compared to the 11 S group,fish in the 2% Ala-Gln group had increased weight gain and specific growth rates（P < 0.05）;increased intestinal muscle thickness,fold height and fold width（P < 0.05）;promoting intestinal cell proliferation（P < 0.05）;increased intestinal free amino acids such as glutamine,glutamate,alanine,and glycine contents（P < 0.05）;increased intestinal glutaminase,α-ketoglutarate,malate dehydrogenase,and ATP contents（P < 0.05）;increased intestinal lysozyme and acid phosphatase activities and complement 3,antimicrobial peptide and immunoglobulin M（P < 0.05）;decreased intestinal caspase-9 activity;downregulated intestinal myd88,ikkβ,nf-κb p65,tnf-α,il-1β,ifn-α,and hsp70 gene expression levels（P < 0.05）;increased the relative abundance of potentially beneficial bacterial Brevibacterium,Rhodanobacter,and Arthrobacter（P < 0.05）.Integration of transcriptomic and proteomic showed that 117 differentially expressed genes were consistent at the transcriptional and translational levels in the Gln-vs-11 S comparison group.These differentially expressed genes were significantly enriched in intestinal physical barrier-related pathways（P < 0.05）,including ECM-receptor interaction,focal adhesion,tight junction,and cell adhesion molecules.Further,myosin,cortactin,tenascin,major histocompatibility complex classes I and II（MHC-I and II）,lysozyme,and NF-κB inhibitor were significantly upregulated at transcriptional and translational expression levels（P < 0.05）.q PCR results of miRNA showed that miR-212（target genes colla1 and colla2）and miR-18a-5p（target gene colla2）expression levels in fish fed Gln group were significantly lower compared to the 7S group fish（P < 0.05）.In summary,soybean meal replacing 50% fish meal protein significantly reduced the growth and induced enteritis in pearl gentian grouper.High levels of soybean meal in feed can mediate the development of grouper enteritis through activation of NF-κB signaling pathway,phagosome,and natural killer cell-mediated cytotoxicity.Dietary Gln can alleviate grouper enteritis caused by β-conglycinin or glycinin,enhance intestinal immunity and physical barrier function,and promote Gln metabolism by inhibiting the My D88/NF-κB pathway,ultimately improving healthy fish growth.Upregulation of gene/protein levels of α-actin,myosin,cortactin,tenascin,collagen,caveolin-1,integrin-linked kinase,NF-κB inhibitor,MHC-I,MHC-II,and lysozyme plays an important role in Gln alleviation of grouper enteritis.Some miRNAs such as miR-192b-3p₂,miR-210-5p,miR-24,miR-212,and miR-18a-5p may directly target intestinal physical and immune-related genes involved in the mechanism of Gln alleviation of grouper enteritis.Therefore,when groupers are fed diets containing >7% β-conglycinin or >10% glycinin（equivalent to 400 g/kg soybean meal）,2% Ala-Gln supplementation is recommended.