Effects Of Fucoidan And Galactoligosaccharides On Dyslipidemia And Gut Microbiota In SD Rats

ObjectiveWe investigated the effects of fucoidan(FUC)extracted from Undaria pinnatifida and galactooligosaccharides(GOS)produced from lactose on preventing dyslipidemia,regulating gut microbiota in high-fat-diet-induced dyslipidemia rats.This study provides new ideas and experimental basis for clarifying the use of prebiotics in regulating dyslipidemia.Method1.Animals and dietsTwenty-four male inbred SD rats aged 6 weeks were randomly divided into the normal(NFD),high-fat diet(HFD),FUC and GOS groups,with n=6 per group.The rats in NFD group were injected with 0.5 ml corn oil in 4 days.The rats in HFD,FUC and GOS groups were injected with vitamin D3(700 000 IU/kg)dissolved in 0.5 ml corn oil in 4 days.The NFD group was fed a normal diet.The HFD,FUC and GOS groups were fed high-fat diets.GOS and FUC were respectively dissolved in normal saline(0.9% sodium chloride).The NFD and HFD groups were orally gavaged with 2 ml normal saline solution once daily for 8 weeks,the FUC group was orally gavaged with 2 ml fucoidan solution(100mg/kg)once daily for 8 weeks,and the GOS group was orally gavaged with 2 ml galactooligosaccharides solution(800 mg/kg)once daily for 8 weeks.During the feeding period,the daily drinking water and food intake were recorded,the fasting weight was measured weekly,and the fasting blood glucose was measured every 2 weeks.2.Explore the effects of FUC and GOS on serum lipids in SD ratsTC was measured through the reaction of glycerol cholesterol oxidase-peroxidase method.TG was measured using glycerol phosphate oxidase-peroxidase method.LDL-C was measured by the direct method.HDL-C was measured using ELISA method.3.Explore the effects of FUC and GOS on serum lipopolysaccharide(LPS)in SD ratsThe serum LPS was measured using ELISA method.4.Explore the effects of FUC and GOS on bile acid metabolism in SD ratsThe serum TBA was measured using ELISA method.Western blot and RT-PCR were used to measured the protein and m RNA levels of bile acid synthesis rate-limiting enzyme7-alpha hydroxylase(CYP7A1)in the "classical pathway" of liver tissue.5.Explore the effects of FUC and GOS on liver tissue and aortic arch in SD ratsAfter fixation in 10% formaldehyde,paraffin-embedded liver tissue and aortic arches were sectioned(5 ?m)and used for hematoxylin and eosin(HE)staining.Sections were photographed at 100× magnification using optical microscopy.6.Explore the effects of FUC and GOS on gut microbiota in SD ratsDegrading gradient gel electrophoresis(PCR-DGGE)and 16 S r RNA gene high throughput sequencing were used to measured the difference of Beta diversity,Alpha diversity and species differences among groups.7.Explore the effects of FUC and GOS on intestinal bile salt hydrolase(BSH)activity inSD ratsBSH activity was measured by enzymatic reaction.8.Explore the relationship between the bacteria and BSH enzyme activityThe relationship between the bacteria and BSH enzyme activity was examined using Spearman's rank correlation test.9.Explore the effects of FUC and GOS on Lactobacillus casei DM8121 growth and BSH enzyme activity in vitroGlucose(20 g/L),fucoidan(20 g/L)and GOS(20 g/L)were dissolved in special sugarfree MRS broth to obtain three distinct MRS broths(M-GLU,M-FUC and M-GOS).Lactobacillus casei DM8121 was inoculated into the M-FUC,M-GOS and M-GLU broths with an inoculum size of 1% and incubated anaerobically at 37? for 24 h.Colonyforming units(CFUs)were determined from the samples at different time points(6,12,18 and 24 h).Growth rates were calculated for the CFUs.The Lactobacillus casei DM8121 strain's BSH enzyme activity was analyzed in the broths as previously described.Result1.Fucoidan and galactoligosaccharides had no significant effect on blood glucose and body weight of SD ratsDuring the 8-week feeding,blood glucose level,body weight,food intake and water intake did not differ among high fat diet groups.2.Fucoidan and galactoligosaccharides significantly alleviated dyslipidemia in SD ratsThe HDL-C was significantly increased in the GOS and FUC groups compared with the HFD group(P<0.05).The TC and LDL-C were significantly increased in the HFD group compared with the NFD group(P<0.05).The TC and LDL-C were significantly increased in the GOS and FUC groups compared with the NFD group(P<0.05).However,the GOS and FUC groups showed significant decreases in TC and LDL-C compared with the HFD group(P<0.05).TG did not differ among groups.3.Fucoidan and galactoligosaccharides significantly decreased serum LPS levels in SD ratsThe LPS was significantly increased in the HFD group compared with the NFD group and significantly decreased in the GOS and FUC groups compared with the HFD group(P<0.05).4.Fucoidan and galactoligosaccharides regulated bile acid metabolism in SD ratsThe TBA was significantly increased in the HFD group compared with the NFD group(P<0.05)and significantly decreasedin the GOS and FUC groups compared with the HFD group(P<0.05).The HFD group yielded significantly decreased m RNA and protein expressions of cholesterol 7-alpha hydroxylase(CYP7A1)in the livercompared with the NFD group(P<0.05);however,GOS and FUC supplementation increased these expressions in the liver compared with those of the HFD group(P<0.05).5.Fucoidan and galactoligosaccharides alleviated the pathological process of hepatic tissue steatosis and aortic arch in SD ratsIn the HFD group,smooth muscle fascicle arrangement in the aortic arch was disordered and showed foam cell infiltration.However,FUC and GOS-supplemented rats showed improved aortic arches with less disorder in the smooth muscle cells and less foam cell infiltration than that in the HFD group.Hepatic tissue in HFD-fed rats showed more steatosis than that in the NFD group,but the GOS and FUC groups showed improved hepatic tissue with less steatosis than that in the HFD group.6.Fucoidan and galactoligosaccharides regulated gut microbiota in SD ratsCluster analysis of PCR-DGGE showed that the intestinal flora structure of SD rats in HFD group was significantly different from that of rats in NFD group,FUC group and GOS group.The intestinal flora structure of rats in NFD group,FUC group and GOS group was similar.High throughput sequencing of 16 S r RNA gene showed that the Simpson and ACE indices revealed no significant difference between the HFD and NFD groups,but the Shannon index revealed a significant decrease in the HFD group compared with the NFD group.The Chao1,Simpson,ACE and Shannon indices showed no significant differences between the FUC and HFD groups or between the GOS and HFD groups.The gut community composition structure at the genus level was determined from the PCA score plot.At the phylum level,Cyanobacteria was significantly decreased in the HFD group compared with the NFD group(P<0.05).Compared with the HFD group,Actinobacteria was significantly decreased in the FUC group,while Verrucomicrobia and Bacteroidetes were significantly increased,and Firmicutes was significantly decreased in the GOS group(P<0.05).At the genus level,compared with the NFD group,Ruminococcus,Adlercreutzia,Coprococcus,and Yaniella were significantly decreased and Aerococcus and Brevibacterium were significantly increased in the HFD group(P<0.05).Compared with the HFD group,in the FUC group,the relative abundance of Aerococcus was significantly decreased,and in the GOS group,Blautia and Akkermansia were significantly increased compared with the HFD group(P<0.05).7.Fucoidan and galactoligosaccharides significantly increased BSH activity in intestinal contents of SD ratsBSH activity in the HFD group's ileal contents was significantly decreased compared with that of the NFD group,while in the GOS and FUC groups,BSH activity was significantly increasedcompared with the HFD group(P<0.05).8.The correlation between gut microbiota and BSH activitySpearman analysis showed that Adlercreutzia and Oscillospira were positively correlated with BSH enzyme activity,while Aerococcus and Brevibacterium were negatively correlated with BSH enzyme activity.9.Fucoidan and galactoligosaccharides significantly increased BSH activity of Lactobacillus casei DM8121Lactobacillus casei DM8121 grew in M-GOS,M-FUC and M-GLU broths.CFU values did not differ among groups in vitro.However,the 24-hour growth rates of Lactobacillus casei DM8121 in M-GOS and M-FUC were significantly decreased compared with those in M-GLU.BSH activity in the M-GOS and M-FUC groups were significantly increasedcompared with that in the M-GLU group(P<0.05).Conclusion1.Fucoidan and galactoligosaccharides ameliorated high-fat diet induced dyslipidemia,hepatic tissue steatosis and aortic arch injury in SD rats.2.Fucoidan and galactoligosaccharides regulated the gut microbiota of high-fat diet induced dyslipidemia rats,increased normal flora and reduced conditional pathogens.3.Fucoidan and galactoligosaccharides reduced the serum total bile acid by increased cholesterol 7-alpha hydroxylase(CYP7A1)expression in the liver and BSH activity in the small intestinal contents.4.In vitro experiment,GOS and FUC increased Lactobacillus casei DM8121's BSH activity.