| Berberine possesses a series of biological activities,including anti-inflammatory,reducing blood glucose level,improving immune performance,and so on.It has been proved that berberine could reduce plasma cholesterol level and improve lipid deposition.It means that berberine plays important role in aspect of regulating lipid metabolism.However,there are few reports on berberine in the regulation of fish immunity,oxidative stress and nutrient metabolism.The current study demonstrated that berberine added in high-fat diet could mitigate oxidative stress,inhibit the apoptosis and enhance disease resistance of fish.However,it was found that the growth performance of fish fed with normal diet supplied berberine was significantly enhanced but immunity was not improved.In order to solve the problem,this study was aimed to examine the effects of dietary berberine optimal feeding mode on growth performance,immune performance,antioxidant activities,hepatoceyte apoptosis and lipid metabolism of blunt snout bream(Megalobrama amblycephala)fed with high-fat diet in combination with vivo and vitro experiments.The experiment included four parts:Experiment 1 The effect of berberine on oxidative stress and apoptosis in grass carpThe study was aimed to explore the effect of different concentrations of berberine on cell activity,oxidative stress and apoptosis of grass carp(Ctenopharyngodon idellus)hepatopancreas cell in vitro experiment.Cultured cells were treated with different concentrations of berberine(0 μmol/L,5 μmol/L,25 μmol/L,50 μmol/L,100 μmol/L)for 0 h,6 h,12 h,24 h.Then,the optimal concentration of berberine was showed by detecting cell viability in CCK-8 assay.Results showed that the cell activity was affected by interaction of time and berberine concentration(P<0.05).Berberine significantly(P<0.05)affected cell activity in a dose-dependent manner.According to the cell activity data,the remaining indicators were selected to data of 12 hours for anal)-zing related indicators,including glutamate oxalate transaminase(GOT)and glutamate pyruvate transaminase(GPT)activity,malondialdehyde(MDA)and ROS content,apoptosis,caspase-3,caspase-8 and caspase-9 mRNA expression.Compared with control group,GOT and GPT activity in group of 100 0.mol/L berberine concentration were significantly(P<0.05)increased.Meanwhile,the content of MDA and ROS were significantly increased with increasing concentration of berberine.The tendency of content of MDA and ROS were same as that of GOT and GPT activity(P<0.05).In addition,compared with control group,the total cell apoptosis rate in group of 25 μmol/L and 50 μmol/L berberine concentration were significantly(P<0.05)increased while cell apoptosis rate in 100 μmol/L berberine group vvas significantly(P<0.05)decreased.At last,the mRNA expression of caspase-3 in group of 50 μmol/L and 100 μmol/L berberine group were significantly enhanced(P<0.05).Experiment 2 Molecular characterization of Microtubule-associated protein 1-Iight chain 3 B(LC3B)and physiological characterization in Megalobrama amblycephalaThis study tested molecular characterization of Microtubule-associated protein 1-light chain 3 B and physiological characterization.The full-length cDNA of LC3B was 1871 bp with an open reading frame of 378 bp encoding 125 amino acids.The complete ORF encoded a protein of 125 amino acids with a calculated molecular mass(MM)of 14.74kDa and isoelectric point of 8.76.High homology at nucleotide and amino acid sequences to carp LC3B was revealed though sequence analysis.The protein sequence of Megalobrama amblycephala LC3B contained a similar architecture with mammals,including a ubiquitin domain of GABA-receptor-associated protein(GABARAP),C-terminal glycine residue,the tubulin binding sites,ATG7 binding sites and lipidation sites.The phylogenetic tree illustrated that blunt snout bream,grass carp(Ctenopharyngodon Idellus)and common carp{Cyprinus carpio)were in the same group forming a clade which was different from that of endotherms.LC3B was mainly(P<0.05)expressed in hepatopancreas but lower in several peripheral tissues,including gill,intestine,kidney and spleen.Experiment 3 Growth performance,oxidative stress and innate immune responses of blunt snout bream,Megalobrama amblycephala adapted to different berberine-dietary feeding modesHigh-fat diets not only might have negative effects on growth and cost,but usually induce excessive fat deposition and liver damage.This study explored that the effect of different feeding modes of berberine on growth performance,innate immune responses and autophagy related protein of fingerling blunt snout bream(Megalobrama amblycephala)fed with high-fat diet.Then,an 8-week feeding trial was conducted to select the optimal feeding mode of berberine.Fish(average initial weight 4.70 ± 0.02 g)were fed two fat levels(5%and 10%)diets in three berberine-feeding modes(supplementing 50 mg/kg berberine continuously,two-week or four-week intervals)with four replicates,respectively.Then,fish were challenged by Aeromonas hydrophila and mortality was recorded for the next 96h after feeding trial.The results showed that different feeding modes of berberine significantly influenced growth,innate immunity and antioxidant capability of fish.The weight gain(WG)and hepatosomatic index(HSI)of fish was significantly decreased,viscerosomatic index(VSI)was significantly increased with increasing the dietary lipid level(P<0.05).When dietary lipid level was enhanced from 5%to 10%,plasma TC and TG contents of fish were significantly(P<0.05)increased.At the same lipid level,the plasma TC and TG contents of fish in berberine at twro-week,four-week and eight-week interval group were significantly(P<0.05)decreased.When dietary lipid level was enhanced,the lysozyme(LYZ)activities,complement component 3(C3)and complement component 4(C4)concentrations,superoxide dismutase(SOD)and catalase(CAT)activities of fish were significantly(P<0.05)decreased and hepatopancreas malondialdehyde(MDA)and lipid peroxide(LPO)contents was significantly increased(P<0.05),Fish fed diet with 50 mg/kg berberine at two-week interval mode not only exhibited low hepatopancreas MDA and LPO contents,but significantly(P<0.05)improved LYZ,C3,C4,SOD and CAT activities.When dietary lipid level was enhanced,the number of apoptosis and LC3B expression levels were significantly(P<0.05)increased.The cell apoptosis ratio of fish in group of different feeding modes of berberine were significantly(P<0.05)decreased,while LC3B expression levels was significantly(P<0.05)increased in group of berberine at eight-week interval.Experiment 4 The influence of optimal feeding mode of berberine on growth and intestinal immune response in blunt snout bream fed with high energy dietsBased on the second experiment,this study used a 2 ×3 factorial design to study the influence of optimal feeding mode of berberine on growth performance and intestinal immune response in blunt snout bream fed with high energy diets.Fish(average initial weight 44.83±0.06 g)were fed six experiments with three kinds of diet(basic diet,high-fat diet,high carbohydrate diet)and two berberine supplementation(0 and 50 mg/kg)for 8 weeks.Besides,feeding mode of berberine was adopted to two-week interval.The result showed that the growth performance of fish was affected by dietary berberine supplementation.Compared with diet no supplemented with berberine,fish fed diet supplemented with 50 mg/kg berberine obtained great weight gain and specific growth rate,and lower feed conservation ratio(P<0,05).Fish protein efficiency ratio and nitrogen retention ratio were significantly enhanced(P<0.05)with increasing dietary lipid level and decreasing dietary protein content.Plasma immune indictors of fish were influenced(P<0.05)by dietary berberine supplementation.Plasma IgG and IgM content of fish fed diet supplemented with berberine were significantly(P<0.05)improved.The intestinal permeability,inflammation and intestinal immune paraments were significantly(P<0.05)affected by interaction of diet and dietary berberine supplementation.The D-lactate(D-LA),diamine oxidase(DAO),Tumor necrosis factor alpha(TNF-α)and Interleukins-6(IL-6)expression were significantly(P<0.05)enhanced when fish fed high-fat and high carbohydrate diet.Berberine could suppress inflammation and improve intestinal permeability through decreasing D-LA,DAO,TNF-α and IL-6 expression.The major histocompatibility complex class(MHC),mucin-2(Muc-2),mucin-5b(Muc-5b)expression of fish fed high-fat diet or high-carbohydrate diet were significantly(P<0.05)decreased but improved in group supplemented with berberine(P<0.05).In general,the optimal concentration of berberine in fish liver cells should be less than 50 μmol/L.When the concentration is more than 50 μmol/L,it will have toxicity to noramal growth cells and affect cell firnction.Those feeding experiments suggested that dietary lipid and carbohydrate could save protein and enhance feed efficiency.However,high energy diet could weaken the intestinal permeability,cause inflaumation response,and damage intestinal immune system.Based on fish healthy improvement and feeding cost saving,blunt snout bream fed diet with 50 mg/kg berberine at two-week interval was optimal feeding mode.It not only saves feeding cost,but promotes growth,regain intestinal permeability and immune barrier,inhibits inflammation response and improves immune performance. |
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