| This study was aimed to examine the effects of dietary berberine on growth performance,immune performance,antioxidantic activities,hepatocyte apoptosis and lipid metabolism of blunt snout bream(Megalobrama amblycephala)fed with high-fat diet.Combining in vitro cell toxicology test,to further explore the biological safety and regulation performance of berberine as functional feed additives.The experiment included three parts:Chapter 1 Effects of dietary berberine on growth performance,immune performance and antioxidantic activities of blunt snout bream response to high-fat dietAn 8-week feeding trial was conducted to investigate the effects of dietary berberine on growth performance,immune performance and antioxidantic activities of blunt snout bream fed with high-fat diet.320 fish(80.00 ± 0.90 g)were divided randomly into four trial groups(each with four replicates)and fed with 4 diets(normal diet,normal diet with 50 mg/kg berberine,high-fat diet,high-fat diet with 50 mg/kg berberine,and normal diet with 5%lipid while high-fat diet with 10%lipid),respectively.At the end of the feeding trial,ammonia stress test was carried out for 5 days.The results indicated that all of the blunt snout bream revealed a wholesome status and the survival rate were maintained at 100%during the feeding trial.Compared with normal diet group,Weight gain(WG)and specific growth rate(SGR)of fish fed with berberine supplemental diet were significantly increased,whereas feed coefficient rate(FCR)decreased significantly(P<0.05).No significant(P>0.05)difference was observed in fish fed high-fat diet.The immune parameters including plasm acid phosphatase(ACP)activities,lysozyme(LYZ)activities and complementary protein 3 and 4(C3,C4)contents were suppressed(P<0.05)in fish fed with high-fat diets but improved(P<0.05)in berberine diets compared with control(normal diet).Hepatopancreas oxidative status,the malondialdehyde(MDA),protein carbonyl(PC)and lipid peroxide(LPO)were increased significantly(P<0.05)when fish were fed with high-fat diets.Berberine could slow the progression of the oxidative stress induced by high-fat through increasing superoxide dismutase(SOD)activities and total sulfydryl(T-SH)levels of fish(P<0.05).And the hepatocyte apoptosis in the high-fat group could also be alleviated by berberine(P<0.05).In addition,it was elusive that significant improvement of fish growth performance was discovered but improvement effect of immunity was not showed when fish were fed with normal diet supplied berberine.After the ammonia stress test,the accumulative mortality was extremely(P<0.05)low in fish fed high-fat diet with berberine compared to other groups.Overall results showed that berberine could effectively improve the growth performance of Megalobrama Amblycephala,relieve the immune suppression caused by high-fat feeding,inhibit the peroxidation induced by high fat intake,and improved excessive apoptosis under unsuitable environment.Chapter 2 GPR43 gene cloning,tissue distribution of blunt snout bream and effect of berberine to its mRNA expressionThis study was conducted to explore the effects of berberine on the mRNA expression of GPR43 and lipid metabolism perfprmance of blunt snout bream fed with high-fat diet.The conceptual design was uniform to the first experiment.G protein coupled receptors 43(GPR43)of blunt snout bream was cloned and sequenced using a RACE assays.The effect of berberine on tissue GPR43 mRNA expressions was also investigated.The obtained full-length nucleotide sequence covered 2026 bp with an open reading frame of 981 bp,translating 326 amino acids.Moreover,GPR43 mRNAs were detected in different tissues of blunt snout bream.In blunt snout bream,the expression of GPR43 in intestine,hepatopancreas,gill and white muscle was highest and in the intestine of Blunt snout bream,GPR43 mRNA expression in the high-fat diet group significantly decreased(P<0.05)compared with control,while elevated effectively(P<0.05)with the addition of berberine.At the same time,a similar trend was also found in the hepatopancreas and muscle tissue,but the difference was not significant(P>0.05).Besides,we found that the contents of free fatty acids(FFA),triglycerides(TG),cholesterol(GHO)and the mRNA expression of Cytokinins protein kinase(p38)and Peroxidase value-added factor γ(PPARy)all exhibited an elevated trend(P<0.05)in the high-fat diet(HD)group compared with control group(ND)and received a sharp reduction(P<0.05)with the supplement of berberine,as well as the composition of Polyunsaturated fatty acids(PUFA)in the hepatopancreas and muscle tissues,while a completely inverse variation was presented in the Saturated fatty acids(SFA),Monounsaturated fatty acids(MUFA)compositions of both tissues and the mRNA expression of Carnitine palm acyltransferase Ⅰ(CPT Ⅰ),Peroxidase value-added factor a&β(PPARα&β)and AMP dependent protein kinase(AMPK).Nevertheless,the changes between berberine supplemental normal diet(NDB)group and control(ND)group was out of regular patterns,so we still had numerous work to do to explore the role of berberine in normal circumstances.Overall results showed berberine could obviously alleviate the hepatopancreas fat deposition induced by high-fat feeding,to improve the performance of lipid metabolism,and these effects might be related to GPR43 receptor.Chapter 3 The effect of berberine to decrease freshwater fish hepatocyte fatty deposits induced by free fatty acidIn order to further verify the function of berberine for fat deposition control and its biological safety.This study through in vitro cell abduction studies to explore the effect of berberine on grass carp hepatopancreas cell activity and cell apoptosis.And via hepatopancreas cell damage induced by Palmitic acid(PA)to induce fatty deposits,so as to explore the regulation function of berberine to improve the excessive fat deposition.Results showed that berberine effected with 6h could affect hepatopancreas cell activity,and when it played a regulatory role,the cell activity gradually reduced as the concentration of berberine growthed,5μM of berberine could do the most obvious downward trend(P<0.05).At the same time,the total cell apoptosis rate also increased gradually with the increased concentration of berberine,when the concentration increased to 5μM got the highest apoptosis rate(P<0.05),and then as the concentration of berberine continue to increase,the total apoptotic rate decreased while cell mortality rate increased significantly(P<0.05).Then different concentrations(0.5 mM and 1 mM,2 mM,4 mM)of PA was added to the cells,oil red staining found 0.5 mM of PA could significantly induce hepatocyte fatty deposits(P<0.05).The concentration of PA can be determined through cell activity because high concentration(greater than 0.5 mM)of PA could remarkably(P<0.05)inhibit the activity of hepatopancreas cells.Therefore 0.5 mM PA could be choose as the optimal concentration to cause hepatocyte fatty deposits.And berberine supplement could significantly reduce the fat deposition caused by PA intervention(P<0.05).Overall results showed that more than 5μM of berberine added in the cell could receive obvious toxic effect(P<0.05).In the fat deposition model induced by palmitate,5μM berberine can significantly reduce(P<0.05)the triglycerides deposition induced by fatty acid intake,keep the health status of tissue cells.To sum up,the supplement of berberine could increase the weight gain of blunt snout bream significantly,remit the peroxide stress caused by high-fat dietary,enhance their immune performance in unnatural environment,and relieve excessive apoptosis induced by too much fat intake.Besides,the role of berberine could effectively restrain the ectopic fat deposition induced by continuous high-fat feeding,so as to repair the damaged lipid homeostasis and keep the fish health. |
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