| Mannan oligosaccharides(MOS),a common prebiotic,are widely used as functional feed additives in livestock,poultry and aquaculture due to their excellent antioxidant properties and immune stimulatory effects.In this study,firstly,the effects of MOS on intestinal growth and development,intestinal flora,short-chain fatty acids(SCFAS)and intestinal digestion and absorption function of grass carp(Ctenopharyngodon idella)and the possible mechanism were investigated through growth trial.Secondly,the effects of MOS on intestinal structure and immune barrier function and the possible mechanisms were investigated by establishing a challenge model.Thirdly,primary intestinal epithelial cells(IECs)and primary intestinal macrophages(MΦ)of grass carp were used as the research subjects to establish an immune stress model in vitro,and the effects of MOS on the proliferation and immune regulation of intestinal cells were investigated,which further revealed the mechanism of MOS regulating the intestinal immune barrier function of grass carp.The main research contents and results are as follows:1.Effects of MOS supplementation on intestinal growth and development,digestive and absorption function,and health index of grass carpA total of 540 healthy grass carp(215.85 ± 0.30 g)was selected for 60-day growth test.Six treatment groups(MOS supplemental levels were 0,200,400,600,800 and 1000 mg/kg,respectively)were set up with 3 replicates in each treatment group and 30 grass carp in each replicate.The results showed that dietary MOS supplementation at different levels significantly increased intestinal length,fold height,hepatopancreas,intestine,head kidney and spleen weights and related health indexes of grass carp(P < 0.05),promote the growth and development of functional organs of grass carp;The number of Bifidobacterium and Lactobacillus and the contents of propionic acid and butyric acid were increased(P < 0.05),decreased the number of Escherichia coli and Aeromonas hydrophila and the content of acetic acid(P < 0.05)to optimize the intestinal flora and short-chain fatty acid production of grass carp,among which 400 mg/kg MOS group had the best effect.Dietary MOS supplementation at different levels significantly increased the activities of chymotrypsin,lipase and amylase in hepatopancreas and intestine(P < 0.05),increased the activity of brush border enzymes in different intestinal segments(P < 0.05),promote intestinal digestion and absorption capacity of grass carp;it up-regulated the m RNA levels of anionic amino acid transporters(SLCA2α and SLC1A3),cationic amino acid transporters(SLC7A1),neutral amino acid transporters(SLC1A5,SLC7A5,SLC7A8,etc.)and cationic and neutral amino acid transporters(SLC7A6,SLC7A7,and SLC7A)in the proximal(PI),middle(MI)and distal intestine(DI)(P < 0.05),promote the intestinal amino acid transport of grass carp;up-regulated TOR m RNA and protein levels in the PI,MI and DI(P < 0.05),the results indicated that the promotion of amino acid transport in intestinal tract of grass carp by MOS may be related to the activation of TOR signaling pathway.2.Effects of MOS on intestinal structure and immune barrier function of grass carp under challenge conditionIn experiment 2,the influence and possible mechanism of MOS on intestinal structure and immune barrier function under challenge condition was discussed by establishing a challenge model.Based on experiment 1,15 healthy grass carp was selected from each treatment group and Aeromonas hydrophila was injected intraperitoneally for 14 days.2.1 Effects of MOS on intestinal antioxidant capacity of grass carp under challenge conditionThe results showed that diets supplemented with different levels of MOS significantly decreased the reactive oxygen species levels,malondialdehyde and protein carbonyl contents in the PI,MI and DI of grass carp(P < 0.05),increased the enzyme activities and m RNA levels of Cu Zn SOD,CAT,GST,GR and GPx in the PI,MI and DI of grass carp(P < 0.05),and improved the intestinal antioxidant damage capacity of grass carp.Most of them,400 mg/kg MOS group had the best effect.Further studies showed that dietary MOS supplementation significantly up-regulated PKCδ and Nrf2 m RNA levels and protein levels(P < 0.05),suggesting that MOS may regulate intestinal antioxidant capacity of grass carp through MR/PKCδ/Nrf2/Keap1 signaling pathway.2.2 Effects of MOS on intestinal antioxidant capacity of grass carp under challenge conditionDietary MOS supplementation at different levels decreased the contents of diamine oxidase and D-lactic acid(P < 0.05),and protected the intestinal structural integrity of grass carp;up-regulated m RNA levels of tight junction proteins such as ZO-1,Occludin and Claudinb and adhesive junction proteins such as JAM-A,E-cadherin and α-catenin,ZO-1 and Occludin(P < 0.05),these results indicated that MOS could enhance the structural integrity of the intestinal apical junction complex of grass carp,of which 400 mg/kg MOS group had the best effect on most indexes in three intestinal segments.The m RNA levels of MLCK,Rho A,ROCK and NMII and the m RNA and protein levels of Rho A were significantly down-regulated by different levels of MOS supplementation(P < 0.05)in three intestinal segments,indicating that MOS may regulate the protein expression of apical junction complex by inhibiting MLCK and Rho A/ROCK signal to protect the structural integrity of IECs.2.3 Effects of MOS on intestinal immune barrier function of grass carp under challenge conditionIn addition,dietary MOS supplementation at different levels significantly decreased the incidence of enteritis(P < 0.05),increased the activities of enteric lysozyme and ACP and the contents of complement C3,C4 and Ig M of grass carp in three intestinal segments(P < 0.05),and up-regulated the m RNA levels of complement molecules such as C1,C2 and C3(P < 0.05).The m RNA levels of β-defensi-1,Hepcidin and LEAP-2A were up-regulated(P < 0.05),indicating that MOS could improve intestinal non-specific immunity of grass carp,and 600mg/kg MOS group had the best effect on most indexes;further studies showed that different dietary levels of MOS can down-regulate the m RNA levels of pro-inflammatory cytokines such as TNFα,IL-1β and IL-6,and up-regulate the mrna levels of anti-inflammatory cytokines such as IL-10,TGF-β1 and IL-4/13B(P < 0.05)in three intestinal segments,indicating that MOS can inhibit intestinal inflammatory response.For most of them,600 mg/kg MOS group had the best effect;in addition,different levels of dietary MOS can significantly down-regulate m RNA levels of receptors and related signaling molecules,such as TLR2,TLR5,NF-κBp65,c-Rel,IKKβ and IKKγ,in three intestinal segments of grass carp,the m RNA and protein levels of My D88,TRAF6,IRAK1 and NF-κBp65 were down-regulated(P < 0.05),but the m RNA levels of TLR1,TLR4,TRIF,NF-κBp52 and IKKα were not affected(P > 0.05)in three intestinal segments.These results indicate that MOS may regulate TLR2/5/NF-κBp65 signal rather than TLR1 and TLR4 to alleviate intestinal inflammatory response of grass carp.3.Effects of MOS on intestinal cell immunity of grass carp and its possible mechanismIn order to establish immune stress model with primary IECs and primary intestinal MΦof grass carp as research objects through in vitro experiments,explore and verify the effects of MOS on intestinal cell proliferation and immune regulation,and reveal the mechanism of MOS regulating intestinal immune barrier function of grass carp.Two in vitro experiments were designed: 1)The effect of different levels MOS on the immune stress of primary intestinal IECs and MΦ under LPS induction;2)Possible pathways for the effects of MOS on primary intestinal immune stress of IECs and MΦ.3.1 Effects of MOS on immune stress of primary intestinal IECs and MΦ under LPS inductionIn order to study the effects of MOS on the proliferation of IECs and MΦ of grass carp,CCK-8 method was used.The results showed that compared with the control group,the proliferation ability of IECs and MΦ was significantly enhanced at the MOS supplemental concentrations of 0.4,0.6,0.8 and 1.0 mg/m L(P < 0.05),and the best effect was in the 0.6mg/m L MOS group.These results indicated that 0.4-1.0 mg/m L MOS was effective on IECs and macrophage proliferation.In order to investigate the effects of MOS on the immune function of primary IECs and MΦ of grass carp and its possible pathways,a screening method based on LPS(epithelial cell induced concentration: 20 μg/m L,macrophage induced concentration: 40 μg/m L)and the optimal MOS concentration under induction conditions.A total of 7 treatment groups was designed.0(Ctrl),0(LPS),0.2(LPS+MOS),0.4(LPS+MOS),0.6(LPS+MOS),0.8(LPS+MOS)and 1.0 mg/m L(LPS+MOS),respectively.The results showed that under LPS immune stress,0.6-1.0 mg/m L MOS could significantly reduce the inflammatory response of X IECs and MΦ,and m RNA levels of TNFα,IL-1β,IL-6 and IL-8 were significantly downregulated(P < 0.05).IL-10 was significantly up-regulated(P < 0.05),and 0.6 mg/m L MOS group had the best effect.3.2 The effect of MOS on immune stress of primary intestinal IECs and MΦ and its possible mechanism under LPS inductionIn order to further investigate the effect of MOS on primary IECs and MΦ TLRs signals,two experiment were set up in vitro experiments 3.2.In the first experiment,a total of 4treatments were designed,namely Ctrl,MOS(0.6 mg/m L),LPS(20 μg/m L or 40 μg/m L),LPS+MOS(LPS: 20 μg/m L or 40 μg/m L,MOS: 0.6 mg/m L),to investigate the effects of MOS on the immune response of primary IECs and MΦ of grass carp under physiological conditions and LPS induction and the possible pathways.The results of IECs(LPS: 20 μg/m L)showed that compared with LPS group,the m RNA levels of TGF-β1 and TGF-β2 in LPS+MOS group were significantly up-regulated(P < 0.05),and the m RNA levels of TNFα,IL-1β,IL-6 and IL-8 were significantly down-regulated(P <0.05),the m RNA levels of TLR5 and NF-κBp65 and protein levels of My D88,IRAK1,TRAF6 and NF-κBp65 were significantly down-regulated(P < 0.05).These results suggest that MOS may alleviate the inflammatory response of IECs of grass carp by inhibiting TLR5/My D88/NF-κBp65 signaling pathway(instead of TLR1,TLR2 and TLR4);the results of MΦ(LPS:40 μg/m L)showed that m RNA levels of IL-10,TGF-β1 and TGF-β2 in LPS+MOS group were significantly up-regulated compared with LPS group(P < 0.05);the m RNA levels of TNF-α,IL-1β,IL-6,IL-8,IL-12p35 and IL-12p40 were significantly down-regulated(P < 0.05).m RNA levels of TLR2,TLR5 and NF-κBp65,and protein levels of TLR2,My D88,IRAK1,TRAF6 and NF-κBp65 were significantly down-regulated(P < 0.05).These results indicate that MOS may relieve intestinal macrophage inflammation of grass carp by inhibiting TLR2/5/ NF-κBp65signaling pathway(instead of TLR1 and TLR4).In the second experiment,six treatment groups were designed,which were Ctrl,LPS(20μg/m L or 40 μg/m L),LPS+MOS(LPS: 20 μg/m L or 40 μg/m L,MOS: 0.6 mg/m L),LPS+TH1020(LPS: 20 μg/m L or 40 μg/m L,TH1020:0.5 μM or 3 μM),LPS+C29(LPS: 20μg/m L or 40 μg/m L,C29:50 μM),LPS+TH1020+C29(LPS: 20 μg/m L or 40 μg/m L,TH1020:0.5 μM or 3 μM,C29:50 μM),to explore the inhibition effect of MOS and TLR2/5inhibitors on inflammation mediated by TLRs/NFκB in IECs and MΦ of grass carp under LPS induction.The results of IECs(LPS: 20 μg/m L,TH1020:0.5 μM,C29: 50 Μm)showed that compared with LPS group,LPS+MOS group up-regulated the m RNA levels of TGF-β2(P <0.05),and down-regulated the m RNA levels of TNFα,IL-1β,IL-6 and IL-8(P < 0.05).The m RNA levels and protein levels of My D88,IRAK1,TRAF6 and NF-κBp65 were downregulated(P < 0.05),and the m RNA levels of TNF-α,IL-1β,IL-6 and IL-8 in LPS+MOS group were significantly lower than those in LPS+C29 group(P < 0.05).The m RNA levels of IL-1βand IL-6 in LPS+TH1020 group were significantly lower than those in LPS+TH1020+C29group(P < 0.05),and the m RNA levels of TNF-α,IL-1β,IL-6 and NF-κBp65 protein were significantly lower than those in LPS+TH1020+C29 group(P < 0.05).These results indicated that 0.6 mg/m L MOS could relieve the inflammatory response of IECs of grass carp,and the effect was better than that of single or combined inhibitor.The results of intestinal macrophage(LPS: 40 μg/m L,TH1020:3 μM,C29:50 μM)showed that compared with LPS group,LPS+MOS group up-regulated m RNA levels of IL-10 and TGF-β1,and down-regulated m RNA levels of TNFα,IL-1β,IL-6,IL-12p35 and IL-12p40;down-regulated m RNA and protein levels of My D88,IRAK4,IRAK1,TRAF6 and NF-κBp65(P < 0.05),the m RNA levels of TNF-α,IL-1β,IL-6,IL-8 and IL-12p35 in LPS+MOS group were significantly higher than those in LPS+TH1020 group(P < 0.05);the m RNA levels of IL-1β,IL-6 and IL-12p35 were significantly higher than those of LPS+C29(P < 0.05).The m RNA levels and NF-κBp65protein levels of TNF-α,IL-1β,IL-6,IL-8 and IL-12p35 were significantly higher than those of LPS+TH1020+C29 group(P < 0.05),indicating that 0.6 mg/m L MOS could alleviate the inflammatory reaction of intestinal MΦ of carp.The effect was weaker than that of single or combined inhibitors.In conclusion,MOS can promote intestinal growth and development and disease resistance of on-growing grass carp,which is closely related to enhancing intestinal digestion and absorption capacity,structural integrity and immune function.MOS may enhance intestinal antioxidant capacity and inhibit MLCK and Rho A signaling pathways by activating Nrf2 signaling pathway,enhance intestinal apical junction complex,and maintain intestinal cellular structure and intercellular structural integrity.It is also possible to regulate intestinal immune barrier function of grass carp by down-regulating TLR2 and TLR5(but not TLR1 and TLR4)to inhibit NF-κBp65 signaling pathway.Based on the incidence of enteritis,the recommended supplemental level of MOS in the diet of grass carp with the body weight of 200-800 g is 499.1mg/kg. |
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