| Choline is an essential vitamin for animals.Previous studies in our laboratory found that dietary choline can improve the growth performance of on-growing grass carp and improve the digestion and absorption capacity of juvenile Jian carp.However,there are no reports research the effects of choline on growth performance,digestive and absorptive capacity and intestinal immune function of juvenile grass carp(Ctenopharyngodon idella).Firstly,the effects of choline on growth performance,intestinal transport and metabolism of choline,lipid metabolism,digestion and absorption capacity,intestinal morphology and structure by growth test.Choline requirement of juvenile grass carp was also determined.Secondly,intestinal immune function and related signal molecules of juvenile grass carp were investigated by challenge test.Thirdly,in vitro experiments,we study the absorption and metabolic pathways of choline,the antimicrobial substances contents,the expression of inflammatory factors and JAK/STAT3 signaling molecule in the IECs of grass carp,which explained the regulation mechanism of choline on the intestinal immune function of juvenile grass carp.Fourthly,in vitro experiments,we conducted to study the absorption and metabolic pathways of choline,activity of complement system,expression of inflammatory factors,phagocytosis capacity,oxygen respiratory,nitrogen respiratory and mitochondrial morphology of intestinal macrophages of grass carp were studied through the JAK/STAT3signaling pathway.The regulation mechanism of choline on intestinal immune function of juvenile grass carp was further revealed.1.Effects of choline on growth,digestibility and absorption capacity of juvenile grass carpMethods:A total of 1440 healthy grass carps(9.29±0.02 g)were randomly divided into 6 treatments with 6 replicates per treatment and 40 fish per replicate.Six diets containing different levels of choline(0,400,800,1200,1600 and 2000 mg/kg)were fed,and the actual contents of choline in the six diets were 142.2,407.4,821.6,1215.8,1589.3 and 1996.6 mg/kg,respectively.The effects of choline on growth performance,choline transport and metabolism,digestion and absorption,and lipid metabolism of juvenile grass carp were investigated after feeding for 70 days.1.1 Effects of choline on performance and choline transport metabolism of juvenile grass carpThe results showed as follows:first,dietary choline can improve the growth performance of juvenile grass carps;Increased intestinal choline,phosphatidylcholine(PC),betaine,and acetylcholine(Ach,excluding hindgut)levels(P<0.05),the upregulation of intestinal choline content may be related to the choline transporter.Dietary choline only upregulates the expression of CHT1 in midgut(P<0.05),and increased the expression of CTL1,CTL2,CTL4(excluding hindgut)and CTL5 in three intestinal segments(P<0.05).Second,dietary choline significantly decreased the contents of TG and TC in hepatopancreas,but significantly increased the contents of TG and TC in serum(P<0.05).These results suggest that dietary choline may reduce lipid deposition in the liver.Thirdly,the choline requirements of juvenile grass carps were calculated as 1330.7 and 1283.4 mg/kg,using PWG and FE respectively.1.2 Effects of choline on digestion and absorption of juvenile grass carpsThe results showed that dietary choline could increase the activities of intestinal digestion and absorption enzymes(trypsin,lipase,amylase,AKP,Na~+/K~+-ATPase,CK andγ-GT)(P<0.05),alleviate goblet cell proliferation caused by choline deficiency,and enhance the absorption capacity of amino acids in the intestine of grass carp.Strikingly,choline influenced the absorptive capacity for various sorts of AAs in different intestinal segments by non-uniform mechanisms.Firstly,choline deficiency reduced the contents of free neutral AAs including Met,Trp,Thr,Ile,Leu,Val,Gly,Pro,Ser,Cys and Tau in all intestinal segments partly attributed to decrease the m RNA levels of the corresponding transporters including SLC7A7,SLC7A6,SLC1A5,SLC7A9(except in midgut),SLC7A8,SLC6A6,SLC6A19b,SLC38A2(except in foregut)and peptide transporter 1(SLC15A1)as well as the protein level of SLC1A5 in the intestine.Secondly,choline deficiency decreased the contents of free basic AAs including Lys,Arg,and His(except in hindgut)in three segments partly associated with downregulating cationic AAT SLC7A1 and SLC15A1 m RNA levels as well as the protein level of SLC7A1 in gut segments.Thirdly,choline deficiency decreased the contents of free acidic AAs including Glu and Asp in three gut segments partly associated with the decreasing m RNA levels of the corresponding transporters including SLC1A2a and SLC15A1 in the intestine.Choline insufficiency depressed the TOR(target of rapamycin)signalling molecule m RNA levels and TOR protein abundance in the fish gut.2.Effect of choline on intestinal immune function of juvenile grass carpIt was found that choline could improve the growth performance and digestion and absorption capacity of juvenile grass carp,the digestion and absorption capacity of fish was related to intestinal immune barrier in experiment 1.Therefore,in order to explore the effect of choline on intestinal inflammation,Aeromonas hydrophila challenge test was carried out6 days after the end of the growth test.42 grass carp with close to average body weight were selected from each treatment,and 1ml of Aeromonas hydrophila was intraperitoneal injected.Through the apparent score of grass carp,pathological section observation,the content and activity of antibacterial substances.To investigate the effect of choline on intestinal immune function of juvenile grass carp,the gene expression of antimicrobial peptides and inflammatory factors as well as JAK/STAT3 gene and protein levels were determined.The results showed that:First,dietary choline significantly reduced the incidence of enteritis and intestinal pathological symptoms(bleeding,goblet cell proliferation,inflammatory cell infiltration).Second,dietary choline significantly increased the activities of lysozyme(LZ)and acid phosphatase(ACP),as well as the contents of Ig M,complement C3 and C4(except foregut)(P<0.05).Dietary choline increased the gene expression levels ofβ-defedin-1(except foregut),Hepcidin,LEAP2B,Musin2 and CSF-1R in three segments(P<0.05).Dietary choline decreased the m RNA levels of pro-inflammatory factors IL-12p40(excluding foregut),IL-8(excluding foregut),IL-6,TNF-α(excluding foregut),and IL-1β(P<0.05),increased the m RNA levels of anti-inflammatory factors TFG-β1,IL-4/13B,and IL-10(P<0.05).These results indicated that choline can improve the intestinal immune function of juvenile grass carp.Third,choline increased m RNA levels of JAK1(excluding foregut and midgut),JAK2,TYK2(excluding foregut),STAT3B1,and STAT3B2 in all three segments(P<0.05).Dietary choline increased the p-STAT3S727(except foregut)and p-STAT3Y705 levels(P<0.05).These results indicate that dietary choline increases intestinal immune function,which may be related to the JAK/STAT3 signaling pathway.3.Effect of choline on immune function in IECs of grass carpExperiment 2 showed that choline could enhance the intestinal immune function of grass carp,which was related to the JAK/STAT3 signaling pathway.In order to further explore the role of choline in regulating the intestinal immune function of grass carp,experiment 4 was designed.IECs were used as the research model,and four cell experiments were carried out:3.1 Effects of choline on immune function of grass carp IECsExperiment 3.1,A total of 3 treatments were designed,including without choline,LPS and LPS+choline,with 6 replicates per treatment.The addition of choline to LPS model significantly up-regulated JAK1 and JAK2 m RNA levels of in IECs(P<0.05),had no effect on m RNA levels of JAK3,TYK2,STAT3A,STAT3B1 and STAT3B2 as well as P-JAK3Y1002 and P-TYK2 Y1054 protien level(P>0.05);Significantly increased the protein phosphorylation of P-JAK1Y1022/1023 and P-JAK2Y1002(P<0.05);Choline can increase p-STAT3Y705 phosphorylation level in the IECs nucleus,but has no effect on the phosphorylation level of p-STAT3S727 in the nucleus and mitochondria.These results suggest that choline may improve the immune function of IECs through the regulation of inflammatory cytokines by JAK1 and JAK2/STAT3.3.2 Choline improves the immune function of grass carp IECs through JAK signaling moleculeResults of Experiment 3.1 showed that the immune function of IECs may be regulated by JAK1 and JAK2/STAT3 signaling pathways.In order to further investigate whether the immune function of IECs is regulated by JAK1 and JAK2,experiment 3.2 was carried out on the basis of the LPS inflammatory model.Gene silencing of JAK1 and JAK2 was performed,and 5 treatments were designed,including LPS,LPS+Si JAK1,LPS+Si JAK2,LPS+Si JAK1+Choline and LPS+Si JAK2+Choline,with 6 replicates for each treatment.The results showed that there were differences in the regulation of different inflammatory factors by adding choline in LPS models.Choline down-regulated the m RNA levels of pro-inflammatory factors(IL-8,IL-6,TNF-α,and IL-1β),and up-regulated anti-inflammatory factors IL-10 and TGF-β1 m RNA levels(P<0.05),through JAK1 and JAK2(P<0.05),choline down-regulated IL-12p40 m RNA level through JAK1,but not through JAK2.Choline up-regulated the phosphorylation of p-STAT3Y705 in the nucleus by JAK1and JAK2(P<0.01).These results suggest that choline regulates the m RNA levels of inflammatory factors in grass carp IECs through JAK1 and JAK2/STAT3 in the LPS model.3.3 Transport and utilization of choline in the grass carp IECsResults of Experiment 3.2 showed that the immune function of IECs was regulated by the JAK1 and JAK2/STAT3 signaling pathways.In order to further explore the regulatory mechanism of choline on the immune function in IECs,a study on the transport and metabolism of choline in IECs was carried out(Experiment 3.3).A total of 3 treatments were designed in the experiment.The results showed that choline can up-regulate m RNA levels of CTL1,CTL2,and CTL5(excluding CTL4 and CHT1)and protein levels of CTL1 and CTL2 in IECs in LPS-induced inflammation model(P<0.01).Choline up-regulated the content of PC and the m RNA level of Ckαin IECs(P<0.05),but did not affect the contents of Ach and betaine,as well as the m RNA levels of CHDH and CHA(P<0.05).These results suggest that choline absorption in intestinal epithelial cells is mediated by choline transporters(CTL1,CTL2 and CTL5),and may play a more important role in immune regulation through phosphorylation and metabolism after entering cells.3.4 Choline can improve the immune function of IECs of grass carp through phosphorylation metabolic pathwayTest of 3.3 the results showed that the choline in IECs of the immune function regulation may through phosphorylation metabolic pathways,in order to further proven choline on IECs immune function regulation is phosphorylation,by the inflammation model induced by LPS and Ckαgene silence,to carry out the test of 3.4,test a total of four processing design,they were LPS,LPS+si CKα,LPS+Choline and LPS+si CKα+Choline,and each treatment had 6 replicates.The results showed that most inflammatory cytokines(TGF-β1,IL12p40,IL-1β,TNF-α,IL-6 and IL10)were regulated by choline through CKα(P<0.01),choline regulates the m RNA levels of JAK1 and JAK2 by CKα,and choline regulates the regulating P-JAK2Y1002 and P-STAT3Y705 phosphorylation by CKα(P<0.01),and the phosphorylation level of p-JAK1Y1022/1023 was not regulated by Ckα(P>0.05).These results suggest that choline regulates most inflammatory cytokines(TGF-β1,IL12p40,IL-1β,TNF-α,IL-6,and IL10)and JAK2/STAT3 signaling pathway through the phosphorylated metabolic pathway.4.Effect of choline on immune function in intestinal macrophages of grass carpThe results of Experiment 2 and Experiment 3 showed that choline could improve the intestinal immune function and thus improve the resistance to enteritis.The improvement of the intestinal immune function of grass carp was related to the regulation of the immune function of intestinal epithelial cells by choline.Macrophages are important immune cells.In order to further explore the specific mechanism,experiment 4 was designed on this basis.A total of 6 cell experiments were carried out:4.1 Culture and identification of intestinal macrophages of grass carp The cells grew well and were identified as macrophages.4.2 Effects of choline on immune function of intestinal macrophages of grass carpExperiment 4.2 Three treatments were designed with 6 replicates per treatment Without choline,LPS and LPS+choline.The results showed as follows:1)choline could significantly improve the phagocytosis capacity of intestinal macrophages,stabilize the morphology of mitochondrial crest,and increase the contents of C3 and C4(P<0.05),significantly down-regulated the contents of NO and ROS(P<0.01);The m RNA levels of TNF-α,IL-6,IL-1β,IL-12p40 and IL-8 in intestinal macrophages were down-regulated(P<0.01);The m RNA levels of IL-4/3B,TGF-β1 and IL-10 in intestinal macrophages were up-regulated(P<0.05);2)upregulated the activity of Complex 1,2 and 4(P<0.05)and the m RNA levels and protein expressions of mitochondrial fusion proteins(OPA1,Mfn1 and Mfn2)(P<0.01);3) m RNA levels of JAK2 and TYK2 in intestinal macrophages were significantly up-regulated(P<0.05),P-JAK2Y1002 and P-TYK2 Y1054,and phosphorylation of P-STAT3S727 in mitochondria(P<0.01).These results suggest that choline regulates the expression of inflammatory factors and the phosphorylation of mitochondrial STAT3S727 by activating JAK2 and Tyk2 in intestinal macrophages of grass carp,further regulating mitochondrial morphology,up-regulating the activity of mitochondrial respiratory complex(1,2 and 4),phagocytosis,and reducing the contents of NO and ROS.4.3 Choline improves the immune function of intestinal macrophages of grass carp through JAK signaling moleculeExperiment 4.2 The results showed that choline could improve the immune function of macrophages,which was related to the regulation of the expression of inflammatory factors and mitochondrial function by JAK2 and TYK2/STAT3.In order to further explore the regulatory mechanisms of JAK2 and TYK2/STAT3,JAK2 and TYK2 were silenced on the basis of LPS challenge model using intestinal macrophages as the model.Experiment 4.3 A total of 5 treatments were designed,including LPS,LPS+si JAK2,LPS+si TYK2,LPS+si JAK2+Choline,and LPS+si TYK2+Choline,with 6 replicates per treatment.The results showed that in LPS model,choline silenced JAK2 and TYK2,respectively.1)Choline down-regulated the m RNA levels of anti-inflammatory factors(IL-6 and IL-1β)in JAK2and TYK2(P<0.01).Choline up-regulated the m RNA levels of anti-inflammatory factors(IL-10,TGF-β1,and IL-4/13B)by JAK2 and TYK2(P<0.01),while the m RNA levels of TNF-αand IL-8 were regulated by choline through JAK2 but not through TYK2(P<0.05);2)Choline down-regulated the contents of NO and ROS in macrophages by JAK2 and TYK2(P<0.01),choline up-regulated mitochondrial fusion protein expression(OPA1,Mfn1 and Mfn2)m RNA levels and OPA1 protein levels by JAK2 and TYK2(P<0.01),while Mfn1protein level was up-regulated only by JAK2 but not by TYK2;The activity of complex 1and 4 was regulated by choline through JAK2 and TYK2,while the activity of complex 2was up-regulated only by JAK2 but not TYK2(P<0.01);3)Choline cooperates with JAK2and TYK2 to up-regulate phosphorylation of P-STAT3S727 in mitochondria(P<0.01).These results suggest that choline regulates the immune function of intestinal macrophages through TYK2 and JAK2 signaling molecules.4.4 Transport and utilization of choline in intestinal macrophages of grass carpAccording to the results of Experiment 4.3,the immune function of intestinal macrophages was regulated by the JAK2 and TYK2/STAT3 signaling pathways.To further explore the mechanism,3 treatments were designed in Experiment 4.4,which were Without choline,LPS and LPS+choline,with 6 replicates for each treatment.The results showed that choline could up-regulate the m RNA and protein levels of CTL1 and CTL2 in intestinal macrophages(P<0.01).Choline can up-regulate the contents of PC and betaine in intestinal macrophages(P<0.01),but had no effect on the content of Ach(P<0.01).Meanwhile,choline can up-regulate the m RNA levels of Ckαin intestinal epithelial cells,but not CHDH and CHA(P<0.05).These results suggest that the absorption of choline in intestinal macrophages is mediated by choline transporters(CTL1 and CTL2),and that choline entering intestinal macrophages may play a role in the regulation of immune function through phosphorylation and oxidative metabolism.4.5 Choline improves the immune function of intestinal macrophages of grass carp through phosphorylation metabolizationAccording to the results of Experiment 4.4,the regulation of immune function of intestinal macrophages by choline may play a role through the phosphorylation metabolic pathway.In order to further explore the mechanism,CKαwas silence in the LPS-induced inflammation model.A total of 4 treatments were designed in Experiment 4.5.They were LPS,LPS+si CKα,LPS+Choline and LPS+si CKα+Choline,and each treatment had 6 replicates.The results showed as follows:1)choline down-regulated the m RNA levels of pro-inflammatory cytokines TNF-α,IL-6,IL-1βand IL-8 by CK-α(P<0.01),the m RNA levels of anti-inflammatory factors TGF-β1 and IL-4/13B were up-regulated by CK-α(P<0.01),while choline did not up-regulate IL-10 m RNA by CKα(P<0.01);2)Choline decreased the contents of NO and ROS by Ckα(P<0.01),choline increased the activity of complex 2 and4 by Ckα(P<0.01),while Complex 2 does not pass through Ckα(P<0.05).In addition,choline up-regulated the m RNA and protein levels of OPA1 and Mfn1 by CKα(P<0.01);3)Choline up-regulated m RNA levels of JAK2,TYK2,STAT3B1 and STAT3B2 by Ckα(P<0.01)choline up-regulated p-JAK2 Y1002 and mitochondrial p-STAT3S727phosphorylation by Ckα(P<0.01).These results suggest that the regulation of intestinal macrophage inflammatory cytokines IL-6,IL-1β,TGF-β1,IL-8,IL-4/13B,and TNF-α(excluding IL-10)is related to the activation of JAK2(but not TYK2)by the phosphorylation metabolic pathway.Choline activates JAK2 through the phosphorylation metabolic pathway to regulate ROS,NO,OPA1,Mfn1,complex 2 and 4,which may be related to macrophage mitochondrial p-STAT3S7274.6 Choline improves the immune function of intestinal macrophages of grass carp through oxidative metabolismThe results of Experiment 4.4 showed that choline played a role in the regulation of immune function of intestinal macrophages through oxidative metabolism.To further explore the mechanism,CHDH was silence in the LPS-induced inflammation model.Experiment 4.6 a total of 4 treatments were designed,including LPS,LPS+si CHCD,LPS+Choline,and LPS+si CHCD+Choline with 6 replicates per treatment.The results showed as follows:1)choline down-regulated the levels of IL-6,IL-1βand IL-8 m RNA by CHCD(P<0.01),the m RNA levels of IL-10 and IL-4/13B were up-regulated by CHCD(P<0.01).The m RNA levels of TNF-αand TGF-β1 were not regulated by CHCD.2)Choline down-regulated ROS content by CHCD(P<0.01),and the down-regulation of NO content did not pass CHCD(P<0.05);Choline up-regulated the activity of complex 2 and 4 by CHCD(P<0.01);The up-regulation of m RNA and protein levels of OPA1 and MFN1 by ch CD(P<0.01);3)Choline up-regulated the levels of TYK2 and STAT3A m RNA,p-JAK2 Y1002and p-TYK2Y1052 and phosphorylation of p-STAT3S727 in mitochondria by CHCD(P<0.01).These results suggest that choline regulates the inflammatory cytokines IL-6,IL-1β,IL-10,IL-8,and IL-4/13B(excluding TGF-β1 and TNF-α)in intestinal macrophages by activating JAK2 and TYK2 through oxidative metabolic pathways.Choline regulates ROS(excluding NO),OPA1,MFN1,and Complex1,2,and 4 by activating JAK2 and TYK2through oxidative metabolic pathways,which may be related to macrophage mitochondrial p-STAT3S727.In conclusion,choline can improve the growth and intestinal digestion and absorption capacity of juvenile grass carp,which may be related to that choline can promote the amino acid content and up-regulation amino acid transporter gene expression by TOR signaling pathway.Dietary choline can enhance the resistance of juvenile grass carp to enteritis,which is related to the enhancement of intestinal immune function through JAK/STAT3 signaling pathway.However,there are differences in the immune regulation of IECs and macrophages by choline.The regulation of the immune function of IECs by choline only activates JAK2through the phosphorylation metabolic pathway and regulates the expression of inflammatory factors,which is related to the nuclear translocation of P-STAT3Y705.The immune regulation of intestinal macrophages by choline is mediated by phosphorylation and oxidative metabolism pathways.The phosphorylation pathway only regulates the expression of inflammatory factors(excluding IL-10 m RNA level)by activating JAK2,but does not affect the nuclear translocation of p-STAT3Y705.The regulation of mitochondrial function(excluding complex 1)is related to the phosphorylation by mitochondrial P-STAT3S727.The oxidative pathway regulates the expression of inflammatory factors through JAK2 and TYK2(without TGF-β1 m RNA levels)but does not affect the nuclear translocation of p-STAT3Y705.Its regulation of mitochondrial function(without NO content)is related to phosphorylation of mitochondrial P-STAT3S727. |
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