| The replacement of fish oil(FO)by plant oil(PO)sources has been a focus of research in the aquatic feed industry.In studies on the screening of PO sources,it was found that the complete replacement of fish oil by corn oil and olive oil did not affect the growth of grouper,but corn oil caused liver lipid deposition and inflammatory reactions,while olive oil improved the antioxidant and anti-inflammatory properties of grouper.As the highest content of active substances in olive oil,squalene has excellent hypolipidemic,antioxidant and anti-inflammatory functions.Therefore,the experiments were designed to construct corn oil diet to induce inflammation and utilize squalene to prevent and repair it,and to verify the regulatory effect of squalene on the immunity of grouper and the response of lipid metabolism to squalene-regulated immunity through classical nutritional experiments.The potential mechanism of squalene-mediated immunity enhancement in hybrid grouper(♀ Epinephelus fuscoguttatus × ♂ E.lanceolatu)was revealed by combining modern macrotranscription technology with screening of squalene-regulated immune-related differential metabolic pathways and validating the function of squalene-regulated immune differential signalling pathways through intraperitoneal injection of activators or inhibitors.The detailed results of the study are as follows:1.Screening of suitable plant oil sources for hybrid grouperIn order to select suitable PO sources to replace FO in pearl grouper diets,7 groups of iso-nitrogenous(crude protein 50.35%)and iso-lipidic(crude lipid 9.70%)diets were formulated with 5% FO,corn oil(CO),sunflower oil(SO),tea seed oil(TO),olive oil(OO),rice oil(RO),and mixed oil(MO,the first 6 oils were mixed in equal proportions)as the main oil sources,respectively.A random selection of 840 juvenile hybrid grouper15.09 ± 0.01 g was distributed to 21 fiberglass buckets(1000 L)for an 8-week breeding test.The results showed that the substitution of PO for FO did not affect the growth performance of grouper(P > 0.05),with the highest weight gain rate(WGR)and specific growth rate(SGR)in the CO group and the second highest in the OO group.Substitution of PO for FO significantly altered serum biochemical parameters,liver lipid metabolism enzyme activity and m RNA expression levels of lipid metabolismrelated genes in grouper(P < 0.05),and increased serum total cholesterol(TC)and triglyceride(TG)levels and triggered liver cell damage in the CO group compared with the FO group.There was no significant difference in cumulative mortality among the groups after challenge(P > 0.05),and the CO group had significantly lower antioxidant capacity accompanied by significant inflammatory response(P < 0.05),while the OO group had higher antioxidant capacity and reduced inflammatory response(P < 0.05),thus suggesting that OO could be a suitable lipid source to replace FO.2.Functional validation of suitable oil sourcesTo further verify the efficacy of OO in promoting lipid metabolism and improving immunity,3 groups of isonitrogenous(50.70%)and isolipidic(10.31%)test diets were formulated with FO,CO and OO as the main oil sources,respectively,feeding grouper with an initial weight of 8.76 ± 0.01 g for 8 weeks.The results showed that there was no significant effect(P > 0.05)on the growth performance of grouper by ingesting diets with three different oil sources,but the survival rate(SR)of the CO group was significantly lower than that of the FO and OO groups after the challenged(P < 0.0001).Compared with the FO group,CO caused hepatic lipid deposition abnormally and led to lipid metabolism disorder;while OO showed a positive effect in promoting lipid metabolism.Meanwhile,CO group of grouper showed significantly lower antioxidant performance(P < 0.05),significantly up-regulated m RNA expression of proinflammatory factors(P < 0.05),and significantly down-regulated m RNA expression of anti-inflammatory factors(P < 0.05);while OO significantly improved antioxidant performance and reduced inflammatory response of tissues in grouper(P < 0.05).In addition,CO altered the structural homeostasis of grouper gut microorganisms,disrupting the structural integrity of the intestine,which in turn reduced the digestive capacity of the intestine,while OO had a protective effect on the structural integrity and flora homeostasis of the grouper gut.In a comprehensive evaluation,under the present experimental conditions,CO reduced the antioxidant performance and immunity of grouper and the utilization of diets,while OO had a positive effect in improving immunity and promoting lipid metabolism in grouper.3.Modulation of growth,lipid metabolism and immunity by squalene in the hybrid grouperSqualene,as one of the most abundant active substances in OO,we speculate that OO to promote lipid metabolism,improve the antioxidant performance and disease resistance of grouper may be related to squalene.To investigate the modulating effect of squalene on the growth,lipid metabolism and immunity of hybrid grouper,four groups of iso-nitrogenous(50.70%)and iso-lipidic(10.31%)test diets(noted as CO,COJ1,COJ2,COJ3)were prepared by adding 0,0.05%,0.1%,and 0.2% squalene to the diets with CO as the main lipid source,and fed to juvenile hybrid grouper with initial body weight of 8.76 ± 0.01 g for 8 weeks,respectively.The test results showed that the addition of squalene to CO did not affect the growth performance and morphological indices of grouper(P > 0.05),but significantly increased the SR of grouper after challenge Vibrio harveyi(P < 0.0001).The addition of squalene to CO did not affect the whole fish composition of grouper(P > 0.05),significantly reduced the muscle crude lipid content and significantly increased the muscle crude protein content(P < 0.05).The addition of squalene to CO significantly promoted lipid metabolism and reduced abnormal liver lipid deposition in grouper(P < 0.05).The addition of squalene significantly down-regulated the m RNA expression of proinflammatory factors in the liver and distal intestinal of grouper(P < 0.05)and significantly up-regulated the m RNA expression of anti-inflammatory factors and antioxidant-related genes(P < 0.05).Meanwhile,the addition of squalene to CO also significantly increased the intestinal digestive enzyme activity of grouper(P < 0.05),increased the relative abundance of probiotic bacterial and decreased the relative abundance of harmful bacterial in the intestine,thus playing a regulatory role in intestinal flora homeostasis.In conclusion,the presence of squalene in OO may be a major factor in the enhanced lipid metabolism,antioxidant capacity and disease resistance of grouper,and squalene may modulate the antioxidant status and intestinal flora structure of grouper by reducing lipid peroxide damage,which in turn reduces inflammation counter and has a positive effect on the improvement of immunity and disease resistance in grouper.4.Screening for differential metabolic pathways in the regulation of immunity by squalene in the hybrid grouperWe selected samples from CO and COJ2 groups for transcriptomic and metabolomic and interaction analyses to screen for squalene-regulated differential metabolic pathways in grouper.The results showed that the transcriptome analysis screened a total of 461 pairs of differentially expressed genes(P < 0.05),of which 253 pairs were up-regulated(P < 0.05)and 208 pairs were down-regulated(P < 0.05);GO enrichment analysis obtained a total of 3 ontologies(Ontology),which describe the molecular functions,cellular components;KEGG enrichment analysis of 461 differential genes obtained 284 pathways,mainly involving 6 primary entries,and the top 30 significantly differential pathways analyzed by KEGG enrichment to immunerelated pathways were immune system-related hematopoietic cell lineage,Erb B signaling pathway,Jak-STAT signaling pathway,TGF-β signaling pathway,and also involved several cancer-related pathways,all of these pathways involved significantly downregulated pi3 k gene and significantly upregulated akt gene and il21 r in PI3K-Akt signaling pathway(P < 0.05),whereas these pathways all involved significantly downregulated m RNA expression of frizzled and axin(P < 0.05).A total of 978 metabolites were identified in the non-targeted metabolome,including 58 differential metabolites,30 of which were significantly up-regulated and 28 of which were significantly down-regulated(P < 0.05);the 58 differential metabolites were enriched to 27 differential signaling pathways,and the top 20 pathways of KEGG enrichment analysis were mainly associated with metabolic,digestive,nervous system,sensory system,endocrine system,and signal transduction pathways.Association analysis of differential genes and differential metabolites,load diagram,correlation network diagram and heat map results showed strong association between differential metabolites and transcriptomic differential genes,and KEGG enrichment analysis revealed that PI3K-Akt signaling pathway is common differential pathway to transcriptomic and metabolomic.Collectively,we concluded that squalene may regulate the Wnt/β-Catenin signaling pathway by activating the PI3K-Akt signaling pathway,and the activation of the Wnt/β-Catenin signaling pathway can reduce the inflammatory response through NF-κB mediated inflammatory factors as a way to improve the immunity of grouper.5.Function of Wnt/β-Catenin signalling pathways and modulation of Wnt/β-Catenin signalling pathways by squaleneBased on the results of transcriptomic analysis,we hypothesized that squalene may reduce the inflammatory response by activating the Wnt/β-Catenin signaling pathway.To further investigate the function of the Wnt/β-Catenin signaling pathway,this chapter of the study used the Western blot technique to determine the expression of key proteins in the Wnt/β-Catenin signaling pathway based on the previous experiments,and squalene,TWS119 inhibitor,and IWR-1 inhibitor were injected to interfere with RNA,and the expression of key genes in Wnt/β-Catenin signaling pathway was measured by q-PCR technique to explore the function of differential signaling pathway regulated by squalene,respectively;Squalene,squalene + TWS119,squalene + IWR-1 were injected into grouper(24.1 ± 0.03 g)after identifying the optimal working concentration and optimal working time of squalene,TWS119,and IWR-1,while PBS injection was set as the control group,the test was performed to investigate the regulation of Wnt/β-Catenin signaling pathway by squalene.The results showed that the addition of squalene to CO significantly decreased the expression of Axin2 protein and significantly up-regulated the expression of β-Catenin protein in grouper liver(P <0.05).Injection of squalene,TWS119 could activate the m RNA expression of β-catenin by inhibiting the m RNA expression of gsk3β,while injection of IWR-1 could inhibit the β-catenin signaling pathway by activating the m RNA expression of gsk3β.Therefore,the activity and function of squalene-regulated β-Catenin signaling pathway can be achieved by activating/inhibiting the expression of gsk3β.In consideration of the cost and effects,the recommended levels of squalene,TWS119,and IWR-1 for grouper injection were 0.5 mg/kg,10 μg/kg,and 10 μg/kg,respectively.Further injection results showed that the injection test did not cause changes in liver histology of grouper,but both could significantly elevate antioxidant enzyme activities in grouper liver(P < 0.05);the test groups injected with squalene and squalene + TWS119 both significantly activated the liver PI3K-Akt and Wnt/β-Catenin signaling pathways in grouper(P < 0.05)and significantly increased the m RNA expression of antiinflammatory factors in grouper liver and SR after challenged Vibrio harveyi(P <0.001);in contrast,the test group injected with squalene + IWR-1 inhibited the liver PI3K-Akt and Wnt/β-Catenin signaling pathways in grouper,significantly up-regulated the m RNA expression of liver pro-inflammatory factors(P < 0.05),while significantly down-regulated the m RNA expression of anti-inflammatory factors(P < 0.05)and significantly reduced SR after challenged Vibrio harveyi(P < 0.001).In conclusion,squalene can activate the PI3K/Akt regulated Wnt/β-Catenin signaling pathway by combining to the il21 r,and the activation of the Wnt/β-Catenin signaling pathway can inhibit the NF-κB mediated inflammatory response,which will then enhance the immunity and disease resistance of grouper.Comprehensive,we concluded that PO would induce inflammatory response by triggering abnormal hepatic lipid deposition,lipid metabolism disorder and disrupting intestinal flora homeostasis,thus decreasing grouper immunity and disease resistance;while OO has high immunity and disease resistance because it enriched in squalene,which can activate PI3K-Akt signaling pathway-regulated Wnt/β-Catenin by binding to il21 r,the activation of β-Catenin can reduce the inflammatory response,improve the immunity and disease resistance by inhibiting NF-κB-mediated inflammatory factors,and thus improve the lipid utilization of grouper. |
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