| In recent years,with the continuous development of large-scale and intensive farming models,China’s aquaculture has made considerable progress and rapid development.However,a series of diseases caused by over-depositing of fish fat will follow,which will reduce its immunity in severe cases,resulting in a large number of deaths.This has become a common problem faced by the aquaculture industry.The intestinal microbes and their encoded components play an important role in animal nutrition metabolism,defense against infection and immune response.In order to effectively alleviate the problem of fatty liver in fish,this research paper is intended to excavate effective probiotic elements by Akkermansia muciniphila which are used to regulate lipid metabolism in fish and reduce liver fat accumulation.In this experiment,the E.coli prokaryotic expression system of AM protein was first constructed,and the target protein was obtained through protein expression and purification process.Then the zebrafish was used as a model to set treatment groups,which were LFD,HFD,HFD+25 μg/g,HFD+50 μg/g,HFD+100 μg/g,and HFD+200 μg/g to determine the optimal amount of addition.Each treatment group was in parallel with 4 treatments,18 fishes in parallel,quantitative feeding at 9:00 and 16:00 daily.The breeding period was 14 days and the water temperature was maintained at 28°C±3℃.During the cultivation period,whether or not was there any death.After the cultivation was completed,the fish in each tank were individually weighed.The growth results showed that the fish survival rate was 100%,and the end weight and weight gain rate of HFD were the highest,significantly higher than LFD(P<0.05).At the same time,the ability of body weight loss was improved with the increase of AM protein added.The weight gain rate was the lowest when the amount of 100 μg/g AM protein was added(P<0.01),and then the indices of the 200 μg/g supplemented group increased slightly.The feed conversion rate was the most significant in the 100 μg/g AM protein supplement group,with a value of 1.60,significantly higher than that of HFD(P<0.01).The further detection of TAG content in liver tissue was the highest in the HFD group,which was significantly higher than that in LFD(P<0.05).The level of added protein in the AM protein-treated group also decreased first and then increased.The content of TAG in the 100 μg/g AM group was the lowest,which was 17.91 μg/mg,P<0.01.The whole fish HE slice results further confirmed that the 100 μg/g AM treatment group showed a significant reduction in lipid droplets relative to HFD.These results all confirmed the lipid-lowering efficacy of the AM protein,and the optimal level of addition was 100 μg/g.In order to further analyze the lipid-lowering efficacy of the AM protein,we chose to further study the mechanism of action at 100 μg/g addition level.Fluorescent quantitative PCR of TNF-α and TGF-β inflammatory factors was first performed on zebrafish liver and intestine.The results showed that compared with HFD,the AM protein group and HFD group showed a consistent expression level,it confirmed that the weight-reducing effect of AM protein is not mediated by inflammation.The Toll-like receptor(TLR)family was screened and it was found that in the intestine,relative to HFD,the mRNA expression levels of TLR1 and TLR4 ba mRNA in the AM protein group were increased by 1.72-fold(P<0.01)and 1.54-fold(P<0.05).It is speculated that TLR1 or TLR4 ba is the pathway of AM protein.Finally,lipid metabolism-related genes were also tested.The results showed that the AM protein mainly reduces hepatic triglyceride content by up-regulating β-oxidation-related genes PPARα,ACOX3 and CPT1.In conclusion,the results of this study suggest that AM protein has a role in alleviating fatty liver in fish and is mediated through the receptor TLR1 and TLR4 ba pathways to further regulate lipid metabolism in the liver.As above,lipid metabolism is controlled by β-oxidation to achieve the purpose of reducing fat accumulation under conditions of high fat induction. |
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