Molecular Mechanism Of Lotus Seed Resistant Starch Mediating The Bile Acids Metabolism By Regulating Gut Microbiota

Lotus seed is an important special cash crop in China,which belongs to high starch food.Lotus seed starch is easy to regenerate to form type 3 resistant starch during processing,namely lotus seed resistant starch(LRS).As a new type of dietary fiber,resistant starch can mediate the metabolism of bile acids by regulating gut microbiota.Different bile acids play important regulatory roles in various physiological processes by activating or inhibiting the expression of farnesoid X receptor(FXR).Bile acids are synthesized in the liver using cholesterol as raw material,and establishing a hyperlipidemic animal model to explore the metabolism of bile acids in the body is currently one of the common research methods.Based on this,SD rats were used as subjects to establish hyperlipidemic rat model.Through a joint analysis of multiple omics,the effects of LRS on blood lipid levels,bile acid pool composition,gut microbiota,liver related gene/protein expression,as well as intestinal FXR and sodium dependent bile acid transporter(ASBT)expression in hyperlipidemic rats were systematically studied.The nutritional geometric axis of "LRS-gut microbiota-bile acids" was constructed.On this basis,the pseudo sterile rat model was used to further verify the regulation of LRS on bile acids metabolism and FXR signaling pathway.The molecular mechanism of LRS regulated bile acid metabolism by gut microbiota had been revealed.The research results will provide a theoretical basis for the study of precise gut nutrition with functional carbohydrates.The main research findings are as follows:(1)Effect of lotus seed resistant starch on lowering blood lipid and serum bile acids in hyperlipidemic ratsBy establishing a hyperlipidemic rat model,the effects of LRS on physiological indicators and serum bile acid profiles in hyperlipidemic rats were studied.The results showed that compared to normal rats(NC group),hyperlipidemic rats(MC group)had an increase in body weight and liver index,dense round fat vacuoles inside liver cells,and damage to intestinal tissue.Meanwhile the contents of TG,TCHO and LDL-C in MC group increased.After intervention with simvastatin(PC group)or LRS(MC＿LRS group),the weight gain of rats was slowed down,and liver fat accumulation was decreased,and blood lipid level was ameliorated,indicating that LRS had a lipid-lowering effect.In addition,LRS could reduce the contents of serum taurine-primary bile acids(TCA,TCDCA,T-α-MCA,T-β-MCA),free primary bile acids(CA,α-MCA,βMCA)and secondary bile acids(GLCA,TLCA,TDCA,THCA,3β-CA)in hyperlipidemic rats.Furthermore,taurine-primary or secondary bile acids were negatively correlated with HDL-C,and positively correlated with TCHO and LDL-C.Meanwhile β-MCA,3β-CA were negatively correlated with HDL-C,but positively correlated with TG,TCHO,and LDL-C.This results indicated that LRS reduced the contents of serum combined bile acids(TCA,TCDCA,T-α-MCA,T-β-MCA,GLCA,TLCA,TDCA,THCA)and free bile acids(CA,α-MCA,β-MCA,HDCA,3β-CA),achieving the goal of restoring blood lipids in hyperlipidemic rats.(2)Effects of lotus seed resistant starch on intestinal flora and bile acids metabolism in hyperlipidemic ratsFurthermore,the 16 S rRNA technology and bile acids targeted metabolomics were used to study the effects of LRS on the composition of small intestinal flora,bile acids profiles in hyperlipidemic rats.The relationship between small intestinal microbiota,bile acids,and blood lipid indicators was constructed.The results showed that LRS increased the abundance and diversity of small intestinal microbiota in hyperlipidemic rats.LRS promoted the proliferation of Psychrobacter and Turicibacte,and inhibited Allobaculum,Blautia,unclassified＿f＿＿Lachnospiraceae growth.In addition,compared with NC group,the total bile acids,primary and secondary bile acids contents of small intestine in MC group increased.After LRS intervention,the contents of small intestinal conjugated primary bile acids(GCA,GCDCA,T-α-MCA,T-β-MCA),taurine-secondary bile acids(TUDCA,THDCA,T-ω-MCA),glycine-secondary bile acids(GUDCA,GDCA)in MC＿LRS group were decreased.Further,the contents of GCDCA and GDCA were positively correlated with the relative abundance of Blautia,unclassified＿f＿＿Lachnospiraceae,and blood lipid indicators(TCHO,LDL-C).This indicated that LRS regulated the relative abundance of Blautia,unclassified＿f＿＿Lachnospiraceae in the small intestine,promoted the excretion of glycine-bile acids(GCDCA,GDCA),and inhibited the reabsorption of bile acids,which ameliorated blood lipids in hyperlipidemic rats.(3)Effects of lotus seed resistant starch on colonic flora and bile acids metabolism in hyperlipidemic ratsOn the basis of small intestinal flora and bile acids,the effects of LRS on the composition of colonic flora and bile acids in hyperlipidemic rats were studied.The relationship between colonic microbiota,bile acids,and blood lipid indicators was constructed.The results indicated that LRS could improve the disorder of colonic microbiota,promote the proliferation of Lactobacillus,Bifidobacterium,Corynebacterium,and inhibit the growth of potentially harmful bacteria such as Allobaculum in hyperlipidemic rats.In addition,compared with NC group,the contents of total colonic bile acids,primary and secondary bile acids in MC group increased.After LRS intervention,the contents of free primary bile acids(α-MCA,β-MCA)and taurine-bile acids(TCDCA,T-α-MCA,T-β-MCA,THDCA,TDCA,TUDCA)were decreased.Meanwhile free secondary bile acids content was increased,among which,the contents of UDCA,3β-UDCA were increased,and 12-keto-LCA,3β-CA were decreased in colon of rats in MC＿LRS group.Furthermore,3β-CA was negatively correlated with HDL-C,while it was positively correlated with THCO,LDL-C,and Allobaculum.Taurine combined bile acids(THDCA,TUDCA,TCDCA)showed a positive correlation with TCHO.Corynebacterium was positively correlated with HDL-C,while negatively correlated with THCO and LDL-C.This result indicated that LRS ameliorated blood lipid levels by regulating the relative abundance of Allobaculum,Corynebacterium,promoting the excretion of taurine combined bile acids(THDCA,TUDCA,TCDCA)and 3β-CA,inhibiting the reabsorption of bile acids in the colon of hyperlipidemic rats.(4)Effects of lotus seed resistant starch on hepatic bile acids metabolism and genes in hyperlipidemic ratsThrough bile acids targeted metabolomics and transcriptome technology,the liver bile acids profiles and its related genes were measured.The correlation between differential genes,bile acids,gut microbiota,and blood lipid indicators was constructed.The results showed that compared with the NC group,the total bile acids,primary and secondary bile acids contents in the liver of the MC group decreased,and the synthesis rate of bile acids slowed down.After LRS intervention,the contents of glycine-primary bile acids(GCA,GCDCA)and free primary bile acids(CDCA)in the liver of rats in MC＿LRS group increased,and the speed of cholesterol decomposition into bile acids accelerated.According to the results of transcriptome,compared with MC group,genes such as Apoa2,Msmo1,Ebp,Fabp1,Abcg8,Cyp8b1,and Tm7sf2 in the liver of rats in the MC＿LRS group were upregulated,while Scd,Ch25 h,Cyp7b1,Soat1,and Cyp51 were downregulated.Furthermore,Genes Abcg8,Dhcr24,Tm7sf2,and Msmo1 were positively correlated with HDL-C,and negatively correlated with THCO and LDL-C.Among them,genes Msmo1,Dhcr24,Tm7sf2 were also negatively correlated with TG.Gene Cyp7b1 was negatively correlated with HDL-C and positively correlated with THCO,LDL-C,and TG.In addition,TG was positively correlated with Blautia and Allobaculum,while THCO,LDL-C,TG were negatively correlated with Corynebacterium.3β-UDCA was negatively correlated with the gene Cyp8b1 while unclassified＿f＿＿Lachnospiraceae showed a positive correlation.CDCA was positively correlated with Abcg8 and Tm7sf2,while GCDCA was highly correlated with differential genes such as Abcg8,Ebp,Cyp51,Fabp1.This result indicated that LRS reduced the relative abundance of Blautia and unclassified＿f＿＿Lachnospiraceae in the small intestine,and regulated the relative abundance of Corynebacterium and Allobaculum in the colon,ao as to change the types and contents of bile acids pool in the body.After bile acid hepatointestinal circulation,the expression of liver genes Ebp,Abcg8,Dhcr24,Tm7sf2,Msmo1,Cyp8b1,Cyp7b1,and Cyp51 gradually changed.The decomposition of liver cholesterol into glycine-primary bile acids(GCDCA)and free primary bile acids(CDCA)accelerated,and the blood lipid levels in hyperlipidemic rats were ameliorated.(5)Molecular mechanism of lotus seed resistant starch regulating bile acids metabolism in hyperlipidemic ratsThe expression of bile acids metabolism related enzymes(BSH,HSDH)and FXR,ASBT in the intestinal tissues of rats in each group were measured by ELISA kit and Western blot.Meanwhile the expression of liver bile acids metabolism related proteins were measured by proteomics technology.The molecular mechanism of LRS regulating bile acids metabolism in hyperlipidemic rats had been revealed by combining gut microbiota,bile acid distribution,and differential gene expression.The results showed that compared to the NC group,the activity of small intestinal microbiota enzymes(BSH,HSDH)and the expression levels of FXR and ASBT in the MC group rats increased.After LRS intervention,the activity of BSH and HSDH in the small intestine of rats in MC＿LRS group,as well as the expression levels of FXR and ASBT,were lower than those of MC group rats.In addition,the differential proteins in the liver of MC＿LRS and MC group rats included Msmo1,Dhcr24,Tm7sf2,Dhcr7,and Hsd17b7,which were expressed in both transcriptome and proteome.LRS regulated the relative abundance of Blautia and unclassified＿f＿＿Lachnospiraceae in the small intestine,reduced the activity of microbial enzymes(BSH,HSDH)and the content of conjugated bile acids in hyperlipidemic rats.At the same time,small intestinal secondary bile acids(GDCA,GUDCA,THCA,TUDCA,T-ω-MCA)content was decreased.The expression of FXR was inhibited,and the breakdown of liver cholesterol into bile acids was accelerated,promoting the transport of conjugated bile acids to the colon.LRS regulated the relative abundance of Corynebacterium,Allobaculum and then secreted BSH,HSDH,reduced the content of conjugated bile acids(TCDCA,THDCA,TDCA,TUDCA)in the colon.LRS inhibited the reabsorption of bile acids into the liver and promoted their excretion from the body.The continuous circulation of "liver-gut bile acids" changed the types and contents of bile acids in the body’s bile acids pool,and the expression of genes/proteins(such as Msmo1,Dhcr24,Tm7sf2)in the liver gradually changes,jointly regulating the metabolism of bile acids in the body and restoring blood lipids in hyperlipidemic rats.(6)Effect mechanism of lotus seed resistant starch regulating bile acids metabolism in pseudo sterile hyperlipidemic ratsA pseudo sterile rat model was established to verify the effect of LRS on bile acids metabolism in rats.The results showed that LRS had different effects on bile acids metabolism between pseudo sterile normal rats(NC＿A group)and pseudo sterile hyperlipidemic rats(MC＿A group).LRS had no significant effect on reducing blood lipids in pseudo sterile hyperlipidemic rats,but could reduce the volume of bile acids pool.Intragastric administration of LRS to pseudo sterile normal rats(NC＿A＿LRS group)or pseudo sterile hyperlipidemic rats(MC＿A＿LRS group),the expression levels of FXR and ASBT proteins in the small intestine of NC＿A＿LRS group rats were higher than those in NC＿A group.There was no significant change in the expression of FXR protein in the small intestine of MC＿A＿LRS group and MC＿A group,while the expression of ASBT protein in MC＿A＿LRS group was lower than that of MC＿A group.The proteome results indicated that there were differences in protein expression in the liver of NC＿A＿LRS group and MC＿A＿LRS group,mainly manifested in Msmo1,Dhcr24,Cyp51,and Dhcr7.LRS promoted the reabsorption of bile acids in the small intestine,and reduced the rate of liver cholesterol synthesis of bile acids in pseudo sterile normal rats,making the body’s bile acids pool in dynamic equilibrium.LRS inhibited the reabsorption of bile acids in the small intestine,promoted the excretion of bile acids,and reduced the volume of bile acids pool in the body in pseudo sterile hyperlipidemic rats.The continuous circulation of "liver-gut bile acids" gradually changed the expression of genes/proteins(Msmo1,Dhcr24,Cyp51,Dhcr7)in the liver of pseudo sterile rats,and jointly regulated the metabolism of bile acids in the body.In summary,LRS had a lipid-lowering effect on hyperlipidemic rats,while its lipid-lowering effect on pseudo sterile hyperlipidemic rats was not significant.Moreover,LRS also had different effects on bile acids metabolism in hyperlipidemic rats or pseudo sterile hyperlipidemic rats,which indicated that gut microbiota played an important role in the process of LRS regulating bile acids metabolism and reducing blood lipid levels in hyperlipidemic rats.LRS regulated the small intestinal microbiota and reduced the content of combined secondary bile acids in hyperlipidemic rats.Small intestine bile acids reabsorption and FXR expression were inhibited,and the decomposition of liver cholesterol into bile acids was accelerated.At the same time,LRS regulated the colonic microbiota,reduced the content of conjugated bile acids,and inhibited their reabsorption,excreted them from the body.The continuous circulation of "liver-gut bile acids" altered the expression of genes/proteins in the liver,jointly regulating the metabolism of bile acids in the body and restoring blood lipid levels in hyperlipidemic rats.In pseudo sterile rat model,LRS could inhibit the intestinal bile acids reabsorption of pseudo sterile hyperlipidemic rats,promoted the excretion of bile acids,and reduced the volume of the body’s bile acids pool,while it did not significantly promote the decomposition of bile acids by liver cholesterol.