Rhizoma Coptidis Alkaloids Exert Their Anti-hyperlipidemic Effects Through Modulation Of Bile Acids Signaling And Gut Microbiota In Hyperlipidemia C57BL/6J Mice

Objective:Rhizoma Coptidis(RC,Coptis chinensis Franch),the dry rhizome of Ranunculaceae or Coptis chinensis,has been considered as a promising antihyperlipidemic candidate for clinic therapy.Modern pharmacology researches have revealed that the lipid–lowering activities of RC alkaloids were mainly achieved by increasing the expression of insulin receptor;ameliorating insulin resistance;stabilizing and increasing LDLR mRNA;reducing the inflammation reaction and by improving the antioxidant activity.Among numerous hepatic lipid metabolism signaling pathways,TGR5 and FXR signaling which belong to bile acids signaling have drawn more attention.The latest studies have revealed that natural or synthetic bile acid receptor agonists may show great clinical application prospects in the treatment of hyperlipidemia,diabetes,atherosclerosis and other metabolic abnormalities.At present,only oleanolic acid has been reported as a natural bile acid receptor agonist,therefore finding new bile acid receptor agonist will not only help to treat metabolic diseases,such as obesity,diabetes,dyslipidemia,but also beneficial for the treatment of inflammation and cancer.Pharmacokinetic studies by our group have demonstrated that the bioavailability of RC alkaloids is below 5 %,most of alkaloids are passed out of the rat body in the faeces.The absorbed alkaloids were mainly distributed in liver,intestine and lungs.This prompted us to hypothesize that the excellent hypolipidemic effects of RC alkaloids may achieved by two different mechanisms.One is the modulation of gut microbiota and further attenuates the metabolic syndrome.The other is the maintenance of dynamic equilibrium of lipids in obese subjects by interfering lipids metabolic pathways.Little attention has been paid to investigate whether RC alkaloids reduce lipid levels by affecting bile acids signaling.Thus,in the current study,illumina sequencing was used to investigate the differences in gut microbiota among normal,high fat high cholesterol diet(HFHC)induced dyslipidaemia and RC alkaloids treated C57BL/6 mice.Molecular technologies were applied to investigate glucose and lipid metabolism related genes and pathways,such as the SREBPs and bile acid signaling.Methods and results:(1)The effects of RC alkaloids on serum lipid levels in C57BL/6J mice were evaluated.HFHC diet led to a significant increase in TC,TG,LDL-C,TBA and LPS of C57BL/6j mice compared with the normal control group.RC alkaloids administration significantly decreased the levels of TC,TBA and LPS content in mice serum.Serum TC was significantly reduced 41.5 %,48.3 %,45.3 % and 47.5 % with berberine,coptisine,palmatine and TRCA treatment compared to HFHC group,respectively(P<0.05).The phenomenon was associated with a 11.2 % and 12.9 % reduction in cholesterol concentrations of the LDLC fraction in coptisine and TRCA treatment group(P<0.05).Serum TG was significantly reduced 32.1 %,37.2 % and 40.1 % by coptisine,palmatine and TRCA therapy(P<0.05).There was an upward trend in the serum HDLC level in RC alkaloids treated animals but no significant differences were observed compared with the HFHC group.Moreover,the LPS content was also elevated about 2 fold in obese mice,after treatment by berberine,coptisine,palmatine and TRCA,the LPS level deceased 32.4 %,42.3 %,31.0 % and 45.1 %,respectively.(2)Histological analysis of liver and adipose tissue using Oil Red O and HE staining staining.Oil Red O staining of the liver tissue sections showed RC alkaloids reduced HFHC-induced lipid accumulation in the liver.Quantitative analysis of the liver lipid contents revealed that the intake of palmatine,coptisine,berberine and TRAC suppressed the liver triglyceride content by 28.1 %,24.9 %,21.2 %,and 26.7 %,respectively.HE staining of of epididymal adipose tissue have showed that the administration of palmatine,coptisine,berberine and TRAC decreased adipocytes size by 37.2 %,17.4 %,15.6 % and 22.9 %,respectively.(3)The effects of RC alkaloids on expression of lipid metabolism related genes and bile acid signaling pathway involved proteins.QPCR analysis have revealed that the mRNA expression of FAS,TXNIP,GOT2,APOA4,FXR,EEF1B2,HMGCR,CYP7A1,SREBP2,LDLR,TGR5,ASBT and CYP8B1 can be reversed by RC alkaloids administration.Western blot results showed that compared with HFHC group,coptisine,berberine,palmatine and TRCA administration stimulated the protein expression of FXR,increased the expression of SREBP2,LDLR,CYP7A1 and ASBT.Besides,the expression of HMGCR and TXNIP was differentially suppressed by RC alkaloids treatment.(4)RC alkaloids reduced the inflammatory cytokines in C57BL/6J mouse ileum.Obesity is associated with a chronic inflammation response.Our Western blot data indicated that compared with HFHC mice,RC alkaloids significantly suppressed the expression of JNK and TLR4.A higher expression level of TLR4 and COX2 can also be found in hyperlipidemic mice,but RC alkaloids treatment reduced these two inflammatory cytokines in mouse ileum.(5)The effects of RC alkaloids on the enterohepatic circulation of bile acids in C57BL/6J mice.HFHC diet administration resulted to a significant increase in serum and liver TBA(Total bile acids)level,while,RC alkaloids treatment effectively decreased the TBA level in mice serum and increased the TBA level in feces.Mechanism study demonstrated that RC alkaloids increased the protein expression of CYP7A1 and BSEP.Since CYP7A1 catalyzes the first and rate-limiting step in the classical bile acid synthetic pathway in the liver,besides,BSEP is the primary transporter of bile acids from the hepatocyte to the biliary system.These two proteins accelerated the synthesis and transportation of bile acids.In ileum,the increased ASBT expression indicating an increased amount of bile acids were reabsorbed and transported back to the liver.Therefore,mice in therapy groups have a higher circulating level of bile acids.Additionally,RC alkaloids reduced the expression of integral membrane proteins Occludin and Claudin 1,lead to the increased intestinal permeability and enhanced the loss of bile acids through the bowel,therefore the lipid content in mice was decreased.(6)The effects of RC alkaloids on gut microflora of C57BL/6J mice were evaluated.16 S rRNA gene sequences assay was used to evaluate the composition of gut microbiome in mice of different groups.Totally,144218 usable pyrosequencing reads and 106 microbe species were identified from 32 stool samples.HFHC diet leads to a reduced bacterial species in obese C57BL/6j mice.Administration of RC alkaloids increased gut bacterial species to different extents,indicating that RC alkaloids treatment is capable of preventing the reduction of bacterial species and maintaining a stable and health gut microbiota.At the species level,RC alkaloids feeding significantly suppressed the presence of Escherichia coli(EC),Desulfovibrio C21_c20,Parabacteroides distasonis in obese mice,whereas,the abundance of Sporobacter termitidis,Alcaligenes faecalis,Akkermansia muciniphila(AF)is increased.UniFrac weighted hierarchical clustering of the bacterial community among different faeces samples revealed that RC alkaloids are capable of restoring the balance to a disrupted gut microflora caused by HFHC feeding.(7)The role of gut microflora on the expression of lipids metabolism related proteins in HepG2 cells was investigated.A cell and gut microbe co-culture model was developed to study the effect of gut microbe on the expression of lipids metabolism related proteins in HepG2 cells.Our results firstly demonstrated that Alcaligenes faecalis and Escherichia coli exhibited the “same” but “different” regulation activities.Both Alcaligenes faecalis and Escherichia coli are capable of regulating the lipids metabolism related proteins in HepG2 cells.Nevertheless,the expression levels of these targets elicited by the two microbes are quite different.Escherichia coli dose-dependently increased the CYP7A1 and ASBT expression.However,Alcaligenes faecalis showed a reverse tendency.Both EC and AF treatment increase the FXR level in HepG2 cells,nevertheless,the FXR up-regulation activity of the two strains decreased with the increase of concentration.Besides,EC dose-dependently inhibited the expression of LDLR and HMGCR.Three different concentrations of AF significantly reduced HMGCR levels.As for SREBP2,only EC improved the expression of this protein,treatment with AF exerted little effect under the same conditions.These results demonstrated that gut microbiota influence the glucose and lipid homeostasis by regulate the expression of lipid metabolism related proteins.Conclusion:Taken together,the obtained results firstly revealed that RC alkaloids can stimulate the expression of bile acid receptor FXR and TGR5.The hypolipidemic effects of RC alkaloids may achieved by regulation the bile acid receptor signaling in liver,promote the enterohepatic circulation of bile acids,ameliorate the inflammation status and by modulation of gut microbiota.