| In aquaculture,farming density exceeding the environmental capacity and deteriorating water environment lead to a large amount of stress in farmed animals,which in turn prone to large-scale disease outbreaks.The application of immune stimulants to improve the immunity and resistance to stress of aquatic animals is an effective way to safeguard the health of farmed animals and farming benefits.β-glucan,a common immune stimulant,is widely used in aquatic animals,but its mechanism of action has not been studied deeply enough.Therefore,the pearl gentian grouper was used as the object of study in this thesis,and we investigated the effects of long-term and short-term feeding strategies of β-glucan diets on the growth performance,intestinal health and disease resistance of grouper,and explored the roles of mitogen-activated protein kinase(MAPK)signaling pathway,nuclear factor-κB(NF-κB)signaling pathway and intestinal microbiota in the regulation of fish intestinal health by β-glucan,aiming to elucidate the specific pathways of action of β-glucan in regulating the intestinal health of pearl gentian grouper.Four main aspects were included in this thesis:1.Effects of long-term and short-term dietary yeast β-glucan on growth performance,intestinal health and disease resistance in pearl gentian grouperThree experimental groups were set up: the pearl gentian grouper in the control group(CD)was fed with basal feed(commercial feed)for 8 weeks,the grouper in the long-termβ-glucan administration group(LGD)was fed with β-glucan feed(basal feed + 0.1% β-glucan)for 8 weeks,while the short-term β-glucan administration group(SGD)was fed with basal feed for 4 weeks,followed by β-glucan feed for 4 weeks.At the end of the feeding,we found that LGD and SGD did not significantly affect the growth performance of grouper but reduced the cumulative mortality of grouper challenged with Vibrio harveyi.LGD and SGD significantly increased the intestinal muscle thickness,plica height and mucin secretion level of grouper,compared to CD group.Meanwhile,the catalase(CAT)enzyme activity and total antioxidant capacity(T-AOC),as well as the m RNA expression levels of sod1 and cat were significantly higher in the LGD group than in the CD group,while the malondialdehyde(MDA)content was significantly lower than that in the CD group.Superoxide dismutase(SOD)and CAT enzyme activities,as well as the m RNA expression levels of sod1 were significantly higher in the SGD group than in the CD group.Both LGD and SGD promoted the production of humoral immune factors and upregulated the expression of cytokines.Compared with the CD group,the m RNA expression levels of the pro-apoptotic genes caspase(casp)3 and casp9 were significantly lower in the LGD and SGD groups,while the m RNA expression levels of the anti-apoptotic gene bclxl were significantly higher in the LGD and SGD groups.In addition,the m RNA expression levels of key genes related to MAPK and NF-κB signaling pathways and the phosphorylation levels of key proteins were significantly upregulated in the LGD and SGD groups.2.Exploration of the role of p38 MAPK signaling pathway in the regulation of intestinal health by yeast β-glucan in pearl gentian grouperFour experimental groups were set up: the CD group was injected with PBS,the βG group was injected with 80 mg/kg of β-glucan,the SB203580(p38 MAPK inhibitor)group was injected with 1 mg/kg of SB203580,and the βG+SB203580 group was injected with 80mg/kg of β-glucan+1 mg/kg of SB203580.Sampling after 48 h of injection,the results showed that βG treatment promoted the secretion of mucin,increased intestinal SOD,CAT and T-AOC enzyme activities,decreased intestinal MDA content,promoted the production of complement(C)3,inducible nitric oxide synthase(i NOS),tumor necrosis factor α(TNFα)and interleukin 1β(IL1β)in the intestine,and promoted the m RNA expression of MAPK kinase(mkk)6,p38α,p38β,C-jun amino-terminal kinase(jnk)2,extracellular signalregulated protein kinase(erk)1,MAPK-activated protein kinase(mapkapk)2 and mapkapk3,and the phosphorylation of p38 MAPK,JNK and ERK proteins.After the intervention with SB203580,the intestinal muscle thickness increased significantly,while the level of mucin secretion decreased significantly;SOD enzyme activity and MDA content increased significantly,while CAT and T-AOC enzyme activities decreased significantly;AKP enzyme activity,and m RNA expression levels of il6 and il18 increased significantly,while C3,i NOS,TNFα and IL1β levels,and m RNA expression levels of il1β were significantly decreased;m RNA expression levels of casp8,casp9 and bcl2 were significantly increased,while m RNA expression levels of casp3 were significantly decreased.3.Exploration of the role of NF-κB signaling pathway in the regulation of intestinal health by yeast β-glucan in pearl gentian grouperFour experimental groups were set up: the CD group was injected with PBS,the βG group was injected with 80 mg/kg of β-glucan,the PDTC(NF-κB inhibitor)group was injected with 30 mg/kg of PDTC,and the βG+PDTC group was injected with 80 mg/kg ofβ-glucan+30 mg/kg of PDTC.Sampling after 48 h of injection,the results showed that the thickness of muscle layer in the βG group was significantly reduced,while the mucin secretion level was significantly increased;SOD,CAT and T-AOC enzyme activities were significantly increased,while MDA content was significantly decreased;ACP and AKP enzyme activities,C3,i NOS,TNFα and IL1β content,and m RNA expression levels of tnfα,il1β,il6 and il18 were significantly increased,while m RNA expression levels of il8 were significantly decreased;The m RNA expression levels of casp3,bcl2 and bclxl were all significantly elevated,whereas the m RNA expression level of casp8 was significantly decreased.The m RNA expression levels of myd88,ikkα2,iκbα and nf-κb p65,the protein levels of P-IκBα and P-NF-κB p65,and the ratios of P-IκBα/IκBα and P-NF-κB p65/NF-κB p65 were significantly increased,while the protein expression levels of IκBα were significantly decreased.After intervention with the NF-κB inhibitor PDTC,the intestinal muscle thickness and mucin secretion level were significantly reduced;CAT enzyme activity and MDA content were significantly reduced;AKP enzyme activity,C3,the content of i NOS and IL1β,and m RNA expression levels of tnfα,il1β,il6 and il18 were significantly reduced;casp3,casp8,casp9 and bcl2 m RNA expression levels were significantly increased.4.Exploration of the role of intestinal flora in the regulation of intestinal health by yeast β-glucan in pearl gentian grouperThree experimental groups were set up: pearl gentian grouper was fed basal diet(CD),β-glucan diet(LGD)and β-glucan diet supplemented with antibiotics [β-glucan diet + 0.2%oxytetracycline(OTC),GOD] for 8 weeks.At the end of the feeding,we found that GOD did not affect the growth performance and the cumulative mortality of grouper after challenged with V.harveyi compared to the LGD group,but significantly reduced the intestinal muscle thickness and plica height.GOD treatment significantly elevated the MDA content and significantly decreased the enzymatic activity of SOD as well as the m RNA expression level of cat.Meanwhile,the m RNA expression levels of acp,tnfα,il1β,il6 and il8 were significantly increased in the GOD group,while the enzymatic activity of AKP and the content of TNFα were significantly decreased;casp3,casp8,casp9 and m RNA expression levels of bclxl were significantly increased in the GOD group;the expression of genes and proteins related to JNK and ERK signaling pathways,as well as the phosphorylation levels of proteins related to JNK and ERK signaling pathways were significantly increased in the GOD group,while those of p38 MAPK signaling pathway were significantly decreased;the m RNA expression levels of myd88 and ikkα2 were significantly increased in the GOD group,while the protein expression levels of IκBα and P-IκBα were significantly decreased.Although there was no significant difference in the intestinal microbiota diversity indices between the GOD and LGD groups,the intestinal microbiota structure of these two groups could be significantly distinguished in PCo A and NMDS analyses.Metabolomics results showed that a total of 70 differential metabolites were identified in the LGD group compared to the CD group,of which 41 metabolites were significantly upregulated and 29 metabolites were significantly downregulated;a total of 91 differential metabolites were identified in the GOD group compared to the LGD group,of which 74 metabolites were significantly upregulated and 17 metabolites were significantly downregulated.Among them,LGD treatment significantly elevated the content of glutathione and significantly decreased the content of leukotrienes,creatinine and docosahexaenoic acid(DHA),while GOD treatment significantly elevated the content of leukotrienes and significantly decreased the content of DHA.In conclusion,long-term and short-term feeding strategies of β-glucan diets improved intestinal health,enhanced disease resistance,and regulated the expression of MAPK and NF-κB signaling pathways,although they did not affect the growth performance of pearl gentian grouper.It was further found that both p38 MAPK and NF-κB signaling pathways were involved in the regulation on intestinal histomorphology,antioxidant,humoral immune response and apoptosis by β-glucan in grouper.Additionally,the intestinal microbiota was also involved in the regulation on intestinal histomorphology,antioxidant,humoral immune response and apoptosis as well as the expression of p38 MAPK and NF-κB signaling pathways by β-glucan in grouper. |
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