Exploring The Alterations Of Bile Acids And Their Mechanisms Under Three Different Pathophysiological Conditions In Rats And Mice

Obj ectiveBile acids(BAs),the important endogenous substances,have extensive biological activities and play an important role in various physiological and pathological processes in vivo.BAs as the pivotal signaling molecules,combined with the nuclear receptors,farnesoid X receptor(Fxr),pregnane X receptor(Pxr),constitutive androstane receptor(Car)and G protein-coupled bile acid receptor(Tgr5),regulate the gene expressions of the target enzymes,transporters and the others relevant functional proteins at transcriptional level,which altogether form the signaling pathways networks to control and maintain the substances metabolic balance and the physiological homeostasis.It has been reported that BAs play a key role in regulating the homeostasis of BAs,glucose and lipids in vivo,and BAs metabolic disorder can induce the physiologic derangement even diseases including intrahepatic cholestasis,diabetes especially the type 2 diabetes mellitus(T2DM),obesity,nonalcoholic fatty liver disease,atherosclerosis,etc..In addition,the alterations of the concentrations and compositions of BAs in different pathophysiological conditions exhibit significantly distinctive characters,which can be employed to diagnose and classify the relevant diseases at an early stage.It is,however,that,the associations between BAs and the relevant diseases still have not been identified clearly,and more comprehensive and systematic studies are needed.As a result,in the present study,rats and mice were employed respectively as our experimental animals.We primarily elucidated the alterations of BAs and their mechanisms in the context of cholecystectomy which is the disease resulting in the physiologic derangement,and under the pathological conditions including estrogen-induced intrahepatic cholestasis and T2DM.Thereafter,we explored the relevance between BAs metabolic disorder and the occurrence and the development of the diseases mentioned above.The findings from this study are expected to provide valuable cues for identifying novel and effective therapeutic targets for amelioration or treatment of the relevant diseases.MethodsThere are three parts of the experiments in the current study:(1)The alterations and the circadian rhythms of BAs and their underlying mechanisms in mice with cholecystectomyThe cholecystectomy and sham operations were executed in Kunming male mice,respectively.After the surgery,the mice recovered for 2 weeks,and then the specimens including serum,liver,ileum,gallbladder,small intestine containing contents and feces were collected.The ultraviolet spectrophotometry was used to determine the total BAs(TBAs)concentrations in all of the specimens and the BAs pool size.The pathology of liver and ileum was investigated applying the histopathological method.The concentrations and compositions of individual BAs in serum,liver tissue,ileum tissue,ileum contents and feces were determined by HPLC/M/MS.The Real Time-quantification PCR(RT-qPCR)and Western Blotting were respectively used to determine the mRNA and protein expressions of the relevant nuclear receptors,enzymes and transporters.Furtermore,the circadian rhythms of BAs and the mRNA expressions of the functional genes were evaluated.(2)Exploring the pathogenic,compensatory and decompensatory mechanisms of estrogen-induced intrahepatic cholestasis via BAs and the "intestine-liver axis" and the "liver-kidney axis" in ratsIn this work,we employed Wistar male rats and HepaRG cells as our experimental objects.Firstly,the pathogenic,compensatory and decompensatory stages of intrahepatic cholestasis model were respectively induced by the subcutaneously administration with 17a-ethynylestradiol(EE)in rats for 7,14 and 28 days.Secondary,after EE administration,the specimens of serum,liver,ileum,kidney,bile,feces and urine were collected and analyzed.The analyses included the levels of transaminases and bilirubin in serum,the TBAs concentrations in all of the specimens,BAs pool size,the liver pathology,the individual BAs concentrations and compositions in serum,bile,liver tissue and ileum tissue,and the mRNA expressions of the relevant nuclear receptors,enzymes and transporters in liver,ileum and kidney.Furthermore,the molecular docking and HepaRG cells studies were employed for verification.(3)Exploring the mechanisms about BAs alterations and preliminarily investigating the effects of the altered BAs on glucose metabolic disorder in rats with T2DMT2DM model was induced in Wistar male rats by high-fat diet combined with the low dose of streptozotocin(STZ).Briefly,rats were fed with a high-fat diet for 7 weeks to induce obesity,and then a low dose(30 mg/kg)of streptozotocin was intraperitoneally injected into rats,followed by another one-week high-fat diet to induce T2DM.Following the intervention scheme,the specimens of serum,liver,ileum,colon,pancreas,small intestine with the contents and feces were collected and evaluated.The evaluation includes the levels of transaminases,glucose,cholesterol and triglyceride in serum;the TBAs concentrations in serum,liver tissue,small intestine with the contents and feces,BAs pool size,the liver pathology,the individual BAs concentrations and compositions in serum and tissues specimens of liver,ileum,colon and pancreas,and the mRNA expressions of the relevant nuclear receptors,enzymes and transporters in liver,ileum,colon and pancreas.ResultsThe following results were found after the circumstance of cholecystectomy in mice.The BAs pool size in vivo reduced significantly,which could be ascribed to the down-regulated protein expression of ileum apical sodium-dependent bile acid transporter(Asbt),and the down-regulated Asbt resulted in the BAs ileal malabsorption and an abundance of BAs losing via feces.The disappearance of constriction circadian rhythms of gallbladder attenuated the circadian rhythms of BAs ileum reabsorption and resulted in the circadian rhythmicity of BAs pool flatting.The circadian rhythmicity of the Cyp7al gene expression flattened,which triggered the disappearance of the hepatic biosynthesis rhythmicity of the BAs.The circadian rhythms of the mRNA expressions of Fxr and its downstream gene fibroblast growth factor(Fgf15)in ileum altered significantly which eventually determined the rhythmicity of hepatic Cyp7al.In addition,the mRNA and protein expressions of hepatic Cyp3a11 down-regulated remarkably,and the circadian rhythms of the mRNA expressions of Cyp3a11,Oatp2 and Mrp2 in liver attenuated remarkably.Under the condition of EE-induced intrahepatic cholestasis in rats,the following results can be found.The results from the animal experiment,molecular docking study and HepaRG cells experiment all indicated that EE inhibited Fxr to down regulate the gene expression of Bsep in the liver,which resulted in biliary excretion of BAs decreasing profoundly to induced the intrahepatic cholestasis.At the compensatory stage,the distinct alterations of BAs concentrations and compositions in ileum and liver tissue contributed to the adaptive regulations of Fxr-mediated"intestine-liver axis",which increased the hepatic biosynthesis of CDCAs via up-regulating mRNA expressions of Cyp7al and Cyp27a1,and down-regulating the mRNA expression of Cyp8b1 in liver,and the increased hepatic CDCAs level blocked the antagonistic effect of EE on Fxr-Bsep pathway,and then promoted BAs biliary excretion to alleviate the EE-induced cholestasis.The adaptive alterations of"liver-kidney axis",including the activation of hepatic Pxr-sulfotransferase family 2a member 1(S ult2a 1)/multidrug resistance protein 3(Mrp3)pathway,the up-regulation of gene expressions of renal efflux transporter Mrp4 and the down-regulation of the renal reabsorption transporters including Asbt and organic solute transporter β(Ostβ),were the potential mechanism of the greatly elevated BAs urinary excretion.At the decompensatory stage,the liver lesion and the necrosis of hepatocytes aggravated apparently,and the mRNA expressions of most of the functional genes in liver down-regulated significantly.Meanwhile,in liver tissue,the concentrations of CDCAs that has higher hepatotoxicity and the composition ofβ-MCAs that is the Fxr antagonist increased,which might result in the EE-induced cholestasis developing from the compensatory stage to the decompensatory stage.Based on the results from molecular docking study,the binding mode and the docked value between EE-Fxr and estradiol-Fxr were similar.In rats with T2DM,the concentrations and compositions of the individual BAs in serum exhibited significantly different characteristics compared to the control,and the most apparent BAs characteristics in the serum of T2DM rats was the significantly increased ratio of 12a-hydroxylate(OH)BAs to non-12a-OH BAs.Moreover,the mRNA expression ratio of hepatic Cyp8b1 increased profoundly,which could be the key factor contributing to the significantly increased ratio of 12a-OH BAs,and the secondary BAs level in T2DM rats decreased remarkably.In colon,pancreas and liver tissue of rats with T2DM,the levels of BAs that are the agonist of Fxr and Tgr5 decreased remarkably.Furthermore,the mRNA expressions of some functional proteins that belong to the Fxr and Tgr5-mediated signaling pathways down-regulated significantly,which might have negative effects on the glucose metabolic disorder in rats with T2DM and promote the development of T2DMConclusionUnder the circumstance of cholecystectomy,estrogen-induced intrahepatic cholestasis and T2DM,the concentrations and compositions of BAs in vivo exhibited significant metabolic disorder,which could be ascribed to the alterations of the signaling pathways that are consist of the relevant nuclear reporters,enzymes and transporters.The alterations of the BAs and they-mediated signaling pathways in the context of these three pathophysiological conditions were further associated with the occurrence and the development of the cholecystectomy relevant diseases,estrogen-induced intrahepatic cholestasis and T2DM.The findings from the current study provided new evidence for the illumination of the relationships between BAs metabolic disorder and the relevant diseases.