| Berberine can reduce blood sugar content of fish and improve fish health by regulating intestinal microflora.Largemouth bass(Micropterus salmoides)had very low carbohydrate load and scavenging ability.In this study,largemouth bass was selected as the object.By adding berberine,antibiotics and agonists,high-throughput sequencing,q RT-PCR,liquid chromatography-mass spectrometry and other techniques were adopted,and FXR inhibitors were used to clarify the hypoglycemic target effect of FXR.The direct effect of BBR on intestinal flora was verified by in vitro culture of intestinal microorganisms,in order to provide theoretical basis for the application of BBR to regulate the blood glucose of largemouth bass.1.Effects of dietary berberine on growth performance,physiology and biochemistry of Largemouth bassIn order to investigate the effects of dietary berberine and bile acid on largemouth bass,largemouth bass(133.7±1.43g)was divided into Control group,berberine group(BBR group,dietary 1g/kg BBR)and bile acid group(BAs group,dietary 300 mg/kg BAs).They were fed largemouth bass for 50 days.The results showed that dietary supplementation of berberine and bile acid could significantly improve the growth performance of largemouth bass.Compared with the control group(1.34±0.32%/d),the specific growth rate of berberine group(1.54±0.21%/d)and bile acid group(1.62±0.16%/d)was significantly increased,and the hepatosomatic ratio was significantly decreased(P < 0.05).Blood glucose content was significantly decreased from 59.11±8.95mmol/L in control group to 25.72±6.6mmol/L in berberine group and 32.75±11.48 mmol/L in bile acid group(P < 0.05).The proportion of intestinal microorganisms Firmicutes in berberine group and bile acid group increased,Proteobacteria decreased and Pseudomonas decreased.The vacuolation of liver tissue was significantly improved,the cellular fat was reduced,the intestinal villi was longer,the intestinal wall was thicker,the number of goblet cells was increased,and the number of eosinophilic granulosa cells was less.After addition of berberine,the metabolomics analysis of intestinal contents showed that bile acids such as β-MCA and DCA were significantly decreased(P <0.05),while the contents of THCA,THDCA and CA were significantly increased(P < 0.05).The addition of berberine and bile acid can improve the growth performance,reduce inflammatory response,improve the sugar metabolism of largemouth bass,and change the relative abundance and diversity of intestinal flora.2.The intestinal flora mediated berberine to regulate the blood glucose of largemouth bassIn order to investigate whether the intestinal flora was the pathway by berberine to regulate the glucose metabolism of largemouth bass,the largemouth bass(133.7±1.43g)was divided into Control group,berberine group(BBR group,supplemented with 1g/kg BBR)and mixed antibiotics group(ATB group,supplemented with 0.9g/kg mixed antibiotics).In the BBR+ATB group,the breeding cycle was 50 days.The results showed that compared with the control group,the use of BBR improved the growth performance of largemouth bass and decreased the hepatosomatic ratio and viscerosomatic ratio.Serum TC(8.75±0.56mmol/L in control group to6.13±0.21mmol/L in BBR group)and GLU(40.16±1.94mmol/L in control group to27.27±2.11mmol/L in BBR group)were significantly decreased(P < 0.05).Serum TBA(9.20±0.34μmol/L in control group to 13.49±0.59μmol/L in BBR group)was significantly increased(P < 0.05);The activities of HK,PK,G6 P and GOT in liver were significantly increased(P < 0.05);The content of Firmicutes in intestinal tract was significantly decreased while Bacteroidota was significantly increased.In vitro culture results of intestinal flora showed that the application of BBR significantly increased the number of culturable bacteria.Strain identification results showed that the characteristic bacterium of BBR group was Enterobacter cloacae,and API analysis showed that the bacterium had the function of metabolizing sugar.In the BBR group,hepatocytes became smaller,vacuolation decreased,nuclei were centered,and lipid distribution decreased.By comparing the results of addition of berberine and addition of antibiotics,it was found that berberine regulates sugar metabolism of largemouth bass by acting on intestinal flora.3.Berberine regulates the blood glucose of largemouth bass through FXR molecular mechanismIn order to explore the mechanism of berberine regulating the glucose metabolism of largemouth bass,largemouth bass(133.7±1.43g)was divided into Control group and berberine group(BBR group).Diet supplemented with 1g/kg BBR),FXR agonist group(GW4064)(diet supplemented with GW4064 0.1g/kg),FXR inhibitor group(MCA)(diet supplemented with MCA 250mg/kg)and berberine + MCA group,the breeding cycle was 50 days.The results showed that compared with the control group,the use of BBR improved the growth performance of largemouth bass and decreased the hepatosomatic ratio and viscerosomatic ratio.Serum TC(8.75±0.56mmol/L in control group to 6.13±0.21mmol/L in BBR group)and GLU(40.16±1.94mmol/L in control group to 27.27±2.11mmol/L in BBR group)were significantly decreased(P < 0.05).Serum TBA(9.20±0.34μmol/L in control group to 13.49±0.59μmol/L in BBR group)was significantly increased(P < 0.05);The activities of HK,PK and G6 P in liver were significantly increased(P < 0.05).Serum TBA content,serum LDL,HK,PK and G6 P were significantly increased in GW4064 group.Compared with the control group,the content of Firmicutes in the gut of the BBR group was significantly decreased and Bacteroidota was significantly increased.The hepatocytes became smaller,the vacuolation decreased,the nucleus was centered,and the lipid distribution decreased.Expressions of FXR(1.03±0.09 to BBR group 1.49±0.09)in liver,FXR(1.03±0.07 to BBR group 4.50±0.73)and FGFR4(1.04±0.18 to BBR group 2.72±0.36)in intestinal tract were increased.By comparing the results of addition of berberine with those of FXR agonist GW4064 and FXR inhibitor MCA,it was found that the target of berberine regulated sugar metabolism of largemouth bass through FXR. |
