The Effect Of Licorice Extract On Lipid Metabolism And TNF Alpha In Megalobrama Amblycephala

In recent years,with the rapid development of high density intensive aquaculture,lipid as a source of energy,was used in feed production to spare protein gradually.However,the wide use of low nitrogen high energy feed,generally caused the fish body lipid metabolism disorders and abnormal physiological problems,which can seriously cause fatty liver,ultimately lead to the slowly growth,increased feed conversion rate,receded disease-resistant ability,high mortality and may also trigger body’s inflammation.Therefore,looking for preventing or alleviating the method of fat metabolism disorders for sustainable and healthy development of aquaculture is particularly important.Considering those reasons,this experiment was conducted to explore tumor necrosis factor(TNF-a)cloning and molecular biology analysis of Megalobrama amblycephala,moreover licorice extract of TNF-a mediated Megalobrama amblycephala regulating lipid metabolism and related genes.This study mainly includes three experiments:Experiment 1:Molecular characterization of tumor necrosis factor alpha and its potential connection with lipid metabolism in Megalobrama amblycephalaTumor necrosis factor alpha(TNF-α)is one kind of cytokines which related to inflammation and lipid metabolism.TNF-a cDNA was cloned from liver of blunt snout bream(Megalobrama amblycephala)through real-time PCR and rapid amplification of cDNA ends(RACE)methods.The full-length cDNA of TNF-a covered 1466 bp,with an open reading frame(ORF)of 723 bp,which encodes 240 amino acids.Predict the protein molecular weight(Mw)of 26457.90 kDa,isoelectric point(PI)of 6.37.It possessed the TNF family signature IIIPDDGIYFVYSQ.After the lipopolysaccharide(LPS)challenge test,a graded tissue-specific expression pattern of TNF-a was observed and high expression abundance in the kidney,brain and liver.After 8 weeks feeding trial,liver samples,two groups fed with 6%and 11%lipid levels,were collected.The results showed that feed with high-fat diet,the triglyceride of serum and lipid content of liver were elevated.Furthermore,TNF-a and peroxisome proliferator-activated receptors(PPARa,p)mRNA expression of fish fed 11%lipid diet were significantly up-regulated(P<0.05).Lipoprotein lipase(LPL)and PPARy mRNA expression of fish fed 11%lipid lever diet were significantly decreased than those of fish fed 6%(P<0.05).The differences between the various expression of related genes in high and low fat group,demonstrating that TNF-a played a key role in lipid metabolism,which may have influence on fat metabolism through reducing fat synthesis and strengthening the P-oxidation of fatty acid.These discrepancies warrant further research.Experiment 2:Effects of licorice extract on growth performance,body composition and fat deposition of blunt snout bream(Megalobrama amblycephala)In order to further explore the regulation effect of licorice extract on blunt snout bream lipid metabolism,developing applicable regulatory agent for fish.In this study licorice extract supplementation was added to the feeds to blunt snout bream(100.55±0.21)g to investigate the growth,body composition and fat deposition.Water extract-alcohol precipitation method was used to licorice root extract.240 fish were randomly divided into 3 groups,4 replications and fed diets containing different licorice extract levels(0 g/kg,0.3 g/kg,0.6 g/kg)feeding 8 weeks.The results demonstrated that the weight gain rate,specific growth rate,feed conversion rate with licorice extract supplementation of blunt snout bream had no significant differences among groups(P>0.05).Licorice extract can reduce hepatosomatic index(HSI),viscerosomatic index(VSI),rate of survival and the fat content of liver and muscle significantly(P<0.05),but for dressing percentage(DP),intraperitoneal fat ratio(IFR)had no significant influence(P>0.05).Between the experimental groups plasma triglyceride,total cholesterol,free fatty acids,high-density lipoprotein cholesterol content were significantly lower than control group(P<0.05),but the added groups was no significant difference(P>0.05),while the content of low density lipoprotein cholesterol increased with increasing dietary licorice extract levels(P<0.05).Results of the above showed that the licorice extract in blunt snout bream took lipid-lowering effect on blood plasma,liver,and muscle,this may be associated with lipid-lowering active ingredient in licorice extract,through regulating blood lipid,in turn,affects Megalobrama amblycephala in vivo fat metabolism,thus play a lipid-lowering effect.Experiment 3:Tumor necrosis factor alpha mediated licorice extract on lipid metabolism regulation in blunt snout bream(Megalobrama amblycephala)with high-fat dietEctopic lipid metabolism has been observed in fish fed with high-fat diet.This study aimed to investigate the molecular mechanisms underlying the influence of licorice extract supplementation on lipid metabolism in blunt snout bream fed high-fat diet.Blunt snout bream fingerling(45.57 ±0.98)g were randomly assigned to five groups four replicates,control diet(6%lipid lever),high-fat diet(11%lipid lever),high-fat + licorice extract(3.5 g/kg,7 g/kg,14 g/kg)for 8 weeks.The results showed that growth rate decreased in high-fat groups compared to 6%lipid group.After adding licorice extract,had no significant effect on weight gain rate,but a relief on abdominal fat deposition,carcass rate also increased with the increase of concentration of licorice extract showed a trend of rising.Gene expression in liver showed,the regulatory factors peroxisome proliferator activated receptors and he fat intake gene(FATP,LPL)showed a rising trend,fatty acid beta oxidation genes associated CPT1 increased significantly(P<0.05),apo lipoprotein B(apoB)and MTTP up-regulation significantly(P<0.05).Moreover the relative expression of TNF-a was down regulated after licorice extract supplied.In summary,licorice extract were added to the high-fat diets had no significant effect on the growth,but can reduce abdominal fat rate,increase the proportion of edible part of fish,transport and promote the fish body fat and fatty acids of beta oxidation decomposition,facilitate fish fat catabolism.In addition,the high lipid induced the expression of TNF alpha,but after add licorice extract relative expression of TNF alpha is restrained,it can be conducted that the licorice extract regulated lipid as well as anti-inflammatory indirectly.