Lithogenic Diet Research Articles

Hepatocyte aquaporin 8-mediated water transport facilitates bile dilution and prevents gallstone formation in mice

Although water channel aquaporin-8 (AQP8) has been implicated in hepatic bile formation and liver diseases associated with abnormal bile flow in human and animal studies, direct evidence of its involvement in bile secretion is still lacking. This study aimed to determine the role of AQP8 in bile secretion and gallstone formation. We generated various transgenic knock-in and knockout mouse models and assessed liver AQP8 expression by immunostaining and immunoblotting, hepatic bile secretion by cannulation of the common bile duct, cholesterol gallstone formation by feeding a high-fat lithogenic diet, and identified regulatory small molecules by screening the organic fractions of cholagogic Chinese herbs and biochemical characterization. We identified a novel expression pattern of AQP8 protein in the canalicular membrane of approximately 50% of the liver lobules. AQP8-deficient mice exhibited impaired hepatic bile formation, characterized by the secretion of concentrated bile with a lower flow rate and higher levels of bile lipids than that of wild-type littermates. AQP8-/- mice showed accelerated gallstone formation, which was rescued by AAV-mediated hepatic expression of AQP8 or AQP1. Moreover, we identified a small molecule, scutellarin, that upregulates hepatocyte AQP8 expression in vitro and in vivo. In AQP8+/+ mice, scutellarin significantly increased bile flow, decreased bile lipid concentrations, and prevented gallstone formation compared to AQP8-/- mice. Molecular studies revealed that scutellarin promoted the ubiquitination and degradation of HIF-1α, a transcriptional negative regulator of AQP8, by disrupting its interactions with HSP90. AQP8 plays a crucial role in facilitating water transport and bile dilution during hepatic bile formation, thereby mitigating gallstone formation in mice. Small-molecule intervention validated hepatocyte AQP8 as a promising drug target for gallstone therapy. The incidence of gallstone disease is high, and current drug treatments for gallstones are very limited, necessitating the identification of novel drug targets for developing new drugs with universal applicability. To our knowledge, this is the first study to provide direct evidence that hepatic water channel AQP8 plays a key role in bile dilution and gallstone formation. Modulation of hepatic water transport may provide a universal therapeutic strategy for all types of gallstone diseases.

Inhibiting miRNA-146a suppresses mouse gallstone formation by regulating LXR/megalin/cubilin-media cholesterol absorption

BackgroundmiRNA has been implicated in regulating cholesterol homeostasis, a critical factor in gallstone formation. Here, we focused on elucidating the role of miR-146a in this pathological process. MethodsC57BL/6 mice were fed with lithogenic diet (LD) and injected with miR-146 antagomir (anta-146) via the tail vein for various weeks. The gallbladders and liver tissues were collected for cholesterol crystal imaging, gallstone mass quantification, and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and metabolic parameters in serum and bile were assessed by ELISA. A 3′ UTR reporter gene assay was used to verify the direct target genes for miR-146. The relative expression of metabolism genes was analyzed by quantitative real-time PCR and immunoblotting. ResultsmiR-146a-5p expression was reduced in mice and clinical samples with gallstones. Anta-146 treatment effectively prevented LD-induced gallstone formation in mice without hepatic and cholecystic damage. The mice treated with anta-146 exhibited beneficial alterations in bile cholesterol and bile acids and lipid levels in the blood. A key biliary cholesterol transporter-Megalin was identified as a direct target of miR-146. Anta-146 administration upregulated megalin expression, thereby ameliorating impaired gallbladder cholesterol absorption associated with the LXR-megalin/cubilin pathway. ConclusionThe data demonstrates that miR-146 modulates gallbladder cholesterol absorption by targeting megalin, and prevents the pathogenesis of cholesterol gallstones.

Hif-1α expression targets the TMA/Fmo3/TMAO axis to participate in gallbladder cholesterol stone formation in individuals living in plateau regions

The incidence of gallbladder cholesterol stones (GCS) increases rapidly among people living in high-altitude hypoxic environments compared to those in normoxic areas. Upregulation of hepatic hypoxia inducible factor 1α (Hif-1α) plays a key role in the formation of GCS. High plasma trimethylamine-N-oxide (TMAO) levels are positively correlated with the occurrence of GCS. We hypothesized that HIF-1α may upregulate TMAO levels by promoting the transcription of flavin-containing monooxygenase 3 (Fmo3), which eventually leads to GCS formation. Our study shows that in women, high plasma total cholesterol and apolipoprotein B were positively correlated with cholecystolithiasis and hypoxia. Hif-1α binds to the Fmo3 promoter and promotes Fmo3 expression. Hypoxia and lithogenic diet induce the expression of Hif-1α, Fmo3, TMAO and cholesterol tube transporters in the livers of mice, disturb the proportion of bile and plasma components, and induce the formation of GCS. In cell experiments, silencing Hif-1α downregulates the expression of Fmo3, TMAO and cholesterol tube transporters. In a mouse model of hypoxic cholecystolithiasis, silencing Hif-1α downregulates the expression of related genes, restores the proportion of bile and plasma lipid components, and reduces the formation of GCS. Our study shows that Hif-1α binds to the promoter region of Fmo3 and promotes Fmo3 transcription. Thus, it mediates the transcriptional activation of the TMA/Fmo3/TMAO pathway, upregulates the expression of ATP-binding cassettes (Abc) g5 and g8, and participates in the regulation of the occurrence of GCS in the plateau region.

Ganoderma lucidum polysaccharide ameliorates cholesterol gallstone formation by modulating cholesterol and bile acid metabolism in an FXR-dependent manner

BackgroundCholesterol gallstone (CG) disease is a worldwide common disease characterized by cholesterol supersaturation in gallbladder bile. Ganoderma lucidum polysaccharide (GLP) has been shown to possess various beneficial effects against metabolic disorders. However, the role and underlying mechanism of GLP in CG formation are still unknown. This study aimed to determine the role of GLP in ameliorating lithogenic diet (LD)-induced CG formation.MethodsMice were fed either a normal chow diet, a LD, or LD supplemented with GLP. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression of genes involved in cholesterol and bile acid (BA) metabolism. The BA concentrations in the ileum were quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). The microbiota in cecal contents were characterized using 16S ribosomal RNA (16S rRNA) gene sequencing.ResultsGLP effectively alleviated CG formation induced by LD. Specifically, GLP reduced the total cholesterol (TC) levels, increased the total BA levels, and decreased the cholesterol saturation index (CSI) in gallbladder bile. The protective effect of GLP was attributed to the inhibition of farnesoid X receptor (FXR) signaling, increased hepatic BA synthesis and decreased hepatic cholesterol synthesis and secretion. GLP also altered the BA composition in the ileum, reducing FXR-agonistic BAs and increasing FXR-antagonistic BAs, which may contribute to the inhibition of intestinal FXR signaling. Additionally, GLP improved dysbiosis of the intestinal flora and reduced the serum levels of hydrogen sulfide (H2S), a bacterial metabolite that can induce hepatic FXR, thereby inhibiting hepatic FXR signaling. Moreover, the protective effect of GLP against CG formation could be reversed by both the global and gut-restricted FXR agonists.ConclusionsTaken together, GLP ameliorates CG formation by regulating cholesterol and BA metabolism in an FXR-dependent manner. Our study demonstrates that GLP may be a potential strategy for the prevention against CG disease.

Involvement of the vagus nerve and hepatic gene expression in serum adiponectin concentrations in mice.

Several humoral factors, such as adiponectin and urate, have been suggested to affect metabolic syndromes. Previously, we reported a reduction in blood adiponectin concentrations after a high-fructose diet partially via the vagus nerve in rats. Although a lithogenic diet (LD), i.e., supplementation of a normal control diet (CT) with 0.6% cholesterol and 0.2% sodium cholate, reduced blood adiponectin concentrations, the involvement of the vagus nerve in this mechanism remains unclear. To estimate the involvement of the vagus nerve in the regulation of blood adiponectin concentrations using an LD, male imprinting control region mice that had been vagotomized (HVx) or only laparotomized (Sham) were administered a CT or an LD for 10weeks. Serum adiponectin concentrations in the Sham-LD, HVx-CT, and HVx-LD groups were reduced by half compared with the Sham-CT group. The hepatic mRNA levels of fibroblast growth factor 21 (Fgf21), which reportedly stimulates adiponectin secretion from white adipose tissue, were lower in the LD groups compared with the CT groups. HepG2 hepatoma cells showed that various bile acids reduced the mRNA expression of FGF21. Moreover, the LD increased serum urate concentrations and reduced hepatic expressions of the acyl-CoA oxidase 1 (Acox1) mRNA and glucokinase, suggesting insufficient regeneration of ATP from AMP. In conclusion, serum adiponectin concentration may be regulated via the vagus nerve in normal mice, whereas a reduction of hepatic Fgf21 mRNA by bile acids may also lower serum adiponectin levels. Moreover, the LD may promote hepatic AMP accumulation and subsequently increase the serum urate concentration in mice.

SAT009 Investigating The Role Of Hepatic Small Heterodimer Partner In Regulating Gallstone Formation

Abstract Disclosure: N.M. Dang: None. S. Anakk: None. Nuclear receptor, Small Heterodimer partner (Shp) regulates bile acid homeostasis by inhibiting transcription of Cholesterol 7 alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme involved in converting cholesterol to bile acids, within the liver. Bile acid regulation prevents the formation of gallstones as cholesterol is excreted into bile along with bile acids and phospholipids. How hepatic Shp affects the development of gallstones and the mechanisms underlying these effects remain unclear. Here we used a liver-specific knockout mouse model and a lithogenic diet model to uncover potential liver-to-gallbladder crosstalk that contributes to gallstone development. Previously, we had shown that the knockout of Shp alters bile acid composition, resulting in higher percentages of secondary bile acid, deoxycholic acid. This increased the hydrophobic index of hepatic bile acids and also implicated a trend for a subsequent increase in the hydrophobicity of biliary bile. Higher hydrophobicity is sufficient to increase susceptibility to developing gallstones by promoting cholesterol nucleation. Of note, the expression of transporter genes Abcg5 and Aqp1 were altered in the gallbladder of hepatic Shp knockout mice, indicating a potential change in their bile composition. The enterokine, Fgf15 is known to stimulate gallbladder filling through its interaction with its receptor, Fgfr4. Recently, the Fgf15-Fgfr4 axis was shown to prevent degradation of the SHP protein such that impeding this axis led to higher Cyp7a1 transcript, increased bile acid levels, and altered gallbladder filling. Taken together, these findings suggest that hepatic Shp acts as a central node that regulates bile acid transport, homeostasis, and composition within the gall bladder and thus can prevent gallstone development. Elucidating Shp-mediated liver-to-gallbladder crosstalk is important for understanding the contribution of liver dysfunction towards gallbladder diseases and gallstones. Presentation: Saturday, June 17, 2023

Effect of Clostridium butyricum on the formation of primary choledocholithiasis based on intestinal microbiome and metabolome analysis.

To investigate the function and probable mechanism of Clostridium butyricum in the development of choledocholithiasis. The lithogenic diet group and the lithogenic diet+C. butyricum group were used to develop the choledocholithiasis model. During the experiment, C. butyricum suspension was administered to the rats in the lithogenic diet+C. butyricum group. The findings demonstrated that the C. butyricum intervention decreased the Firmicutes/Bacteroidetes ratio in the colon of experimental animals given a lithogenic diet. The relative levels of Desulfovibrio (0.93%) and Streptococcus (0.38%) fell, whereas Lactobacillus (22.36%), Prevotella (14.09%), and bacteria that produce short-chain fatty acids increased. Finally, 68 distinct metabolic products were found based on nontargeted metabonomics, and 42 metabolic pathways associated to the various metabolites were enriched. We found that C. butyricum decreased the development of choledocholithiasis. It keeps the equilibrium of the rat's gut microbiome intact and lowers the danger of bacterial infections of the gastrointestinal and biliary systems. It is hypothesized that by controlling lipid metabolism, it may also have an impact on the development of cholelithiasis.

Hyodeoxycholic acid attenuates cholesterol gallstone formation via modulation of bile acid metabolism and gut microbiota

Background & aimsHyodeoxycholic acid (HDCA), a hydrophilic bile acid (BA), may prevent and suppress the formation of cholesterol gallstones (CGs). However, the mechanism by which HDCA prevents CGs formation remains unclear. This study aimed to investigate the underlying mechanism of HDCA in preventing CG formation. MethodsC57BL/6J mice were fed either a lithogenic diet (LD), a chow diet, or LD combined with HDCA. The concentration of BAs in the liver and ileum were determined using liquid chromatography-mass spectrometry (LC-MS/MS). Genes involved in cholesterol and BAs metabolism were detected using polymerase chain reaction (PCR). The gut microbiota in the faeces was determined using 16S rRNA. ResultsHDCA supplementation effectively prevented LD-induced CG formation. HDCA increased the gene expression of BA synthesis enzymes, including Cyp7a1, Cyp7b1, and Cyp8b1, and decreased the expression of the cholesterol transporter Abcg5/g8 gene in the liver. HDCA inhibited LD-induced Nuclear farnesoid X receptor (Fxr) activation and reduced the gene expression of Fgf15 and Shp in the ileum. These data indicate that HDCA could prevent CGs formation partly by promoting BA synthesis in the liver and reduced the cholesterol efflux. In addition, HDCA administration reversed the LD-induced decrease in the abundance of norank_f_Muribaculaceae, which was inversely proportional to cholesterol levels. ConclusionsHDCA attenuated CG formation by modulating BA synthesis and gut microbiota. This study provides new insights into the mechanism by which HDCA prevents CG formation. Lay summaryIn this study, we found that HDCA supplementation suppressed LD-induced CGs in mice by inhibiting Fxr in the ileum, enhancing BA synthesis, and increasing the abundance of norank_f_Muribaculaceae in the gut microbiota. HDCA can also downregulate the level of total cholesterol in the serum, liver, and bile.

Dietary diosgenin transcriptionally down-regulated intestinal NPC1L1 expression to prevent cholesterol gallstone formation in mice

BackgroundCholesterol gallstone disease is a common disease. Reducing cholesterol burden is important to prevent/treat gallstone. In this study, we investigated the application of diosgenin (DG) to prevent the formation of gallstone in mice.MethodsAdult male C57BL/6J mice were fed with the lithogenic diet (LD) only or LD supplemented with DG or ezetimibe for 8 weeks. Incidences of gallstone formation were documented. Intestine and liver tissues were collected to measure the lipid contents and expression of genes in cholesterol metabolism. Caco2 cells were treated with DG to monitor the regulation on cholesterol absorption and the transcriptional regulation of Npc1l1 gene. Changes of gut microbiota by DG was analyzed. Intraperitoneal injection of LPS on mice was performed to verify its effects on STAT3 activation and Npc1l1 expression in the small intestine.ResultsLD led to 100% formation of gallstones in mice. In comparison, dietary DG or ezetimibe supplementary completely prevents gallstones formation. DG inhibited intestinal cholesterol absorption in mice as well as in Caco2 cells by down-regulation of Npc1l1 expression. DG could directly inhibit phosphorylation of STAT3 and its transcriptional regulation of Npc1l1 expression. Furthermore, DG could modulate gut microbiota profiles and LPS mediated STAT3 activation and Npc1l1 expression.ConclusionOur results demonstrated that dietary DG could inhibit intestinal cholesterol absorption through decreasing NPC1L1 expression to prevent cholesterol gallstone formation.

Macrophage inhibitory cytokine-1 aggravates diet-induced gallstone formation via increased ABCG5/ABCG8 expression.

Macrophage inhibitory cytokine 1 (MIC-1), which is overproduced in various human cancers and associated with cachexia, acts on the hypothalamus to suppress appetite and reduce body weight. We investigated the mechanisms through which MIC-1 affects bile acid metabolism and gallstone formation, which are poorly understood. Over 6 weeks, male C57BL/6 mice fed either standard chow or a lithogenic diet were intraperitoneally injected with phosphate-buffered saline (PBS) or MIC-1 (200 μg/kg/week). Among lithogenic diet-fed mice, MIC-1 treatment resulted in increased gallstone formation compared with PBS treatment. Compared with PBS treatment, MIC-1 treatment decreased hepatic cholesterol and bile acid levels and reduced expression of HMG-CoA reductase (HMGCR), the master cholesterol metabolism regulator sterol regulatory element-binding protein 2, cholesterol 7α-hydroxylase (CYP7A1), mitochondrial sterol 27-hydroxylase, and oxysterol 7α-hydroxylase. Compared with PBS treatment, MIC-1 treatment had no effect on small heterodimer partner, farnesoid X receptor, or pregnane X receptor expression, and extracellular signal-related kinase and c-Jun N-terminal kinase phosphorylation decreased, suggesting that these factors do not contribute to the MIC-1-induced reduction in CYP7A1 expression. Compared with PBS treatment, MIC-1 treatment increased AMP-activated protein kinase (AMPK) phosphorylation. Treatment with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reduced CYP7A1 and HMGCR expression, whereas the AMPK inhibitor Compound C reversed MIC-1-induced reductions in CYP7A1 and HMGCR expression. Furthermore, in MIC-1-treated mice, total biliary cholesterol levels increased together with increased ATP-binding cassette subfamily G (ABCG)5 and ABCG8 expression. Compared with PBS treatment, MIC-1 treatment did not affect expression of liver X receptors α and β, liver receptor homolog 1, hepatocyte nuclear factor 4α, or NR1I3 (also known as constitutive androstane receptor), which are upstream of ABCG5/8; however, MIC-1 treatment increased ABCG5/8 expression and promoter activities. Our study indicates that MIC-1 influences gallstone formation by increasing AMPK phosphorylation, reducing CYP7A1 and HMGCR expression, and increasing ABCG5 and ABCG8 expression.

Glutaredoxin 1 regulates cholesterol metabolism and gallstone formation by influencing protein S-glutathionylation

ObjectiveCholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are increasingly being observed to drive various physiological and pathological processes, especially in metabolic diseases such as diabetes, obesity and fatty liver. However, Glrx1 has been minimally explored in cholesterol metabolism and gallstone disease. MethodsWe first investigated whether Glrx1 plays a role in gallstone formation in lithogenic diet-fed mice using immunoblotting and quantitative real-time PCR. Then a whole-body Glrx1-deficient (Glrx1−/−) mice and hepatic-specific Glrx1-overexpressing (AAV8-TBG-Glrx1) mice were generated, in which we analyzed the effects of Glrx1 on lipid metabolism upon LGD feeding. Quantitative proteomic analysis and immunoprecipitation (IP) of glutathionylated proteins were performed. ResultsWe found that protein S-glutathionylation was markedly decreased and the deglutathionylating enzyme Glrx1 was greatly increased in the liver of lithogenic diet-fed mice. Glrx1−/− mice were protected from gallstone disease induced by a lithogenic diet because their biliary cholesterol and cholesterol saturation index (CSI) were reduced. Conversely, AAV8-TBG-Glrx1 mice showed greater gallstone progression with increased cholesterol secretion and CSI. Further studies showed that Glrx1-overexpressing greatly altered bile acid levels and/or composition to increase intestinal cholesterol absorption by upregulating Cyp8b1. In addition, liquid chromatography-mass spectrometry and IP analysis revealed that Glrx1 also affected the function of asialoglycoprotein receptor 1 (ASGR1) by mediating its deglutathionylation, thereby altering the expression of LXRα and controlling cholesterol secretion. ConclusionOur findings present novel roles of Glrx1 and Glrx1-regulated protein S-glutathionylation in gallstone formation through the targeting of cholesterol metabolism. Our data advises Glrx1 significantly increased gallstone formation by simultaneously increase bile-acid-dependent cholesterol absorption and ASGR1- LXRα-dependent cholesterol efflux. Our work suggests the potential effects of inhibiting Glrx1 activity to treat cholelithiasis.

Osteopontin (SPP1) expression on gallstone formation in rabbits fed with a lithogenic diet.

This research was designed to assess the influence of the administration of a lithogenic diet, hydrogen peroxide, and vitamin AD3E on rabbits' gallstone formation and to envisage the expression of osteopontin (OPN) in their hepatic tissues. Twenty-four healthy local mature rabbits of both genders were divided into four equal groups. At the end of the feeding period, samples of blood were taken from all rabbits after they had fasted overnight to estimate the serum lipid profile. And some of the hepatic tissue has been preserved at -28°C for molecular analysis and gene expression. The gallstones were formed 100% in the GIII and 50% in the GIV. The mRNA OPN expression showed a significant increase in the GIII when compared with other groups. In Groups III and II, the serum levels of total cholesterol, Triglyceride, L-C, low-density lipoprotein-choles, and VLDL-C were significantly increased when compared with GI, while in GIII, the serum level of high-density lipoprotein-cholesterol was significantly decreased when compared with GI. It was concluded that the expression of the mRNA OPN was increased in the hepatic tissue of gallstone-formed rabbits.

Preclinical evaluation of Hibiscus cannabinus Linn. in the treatment of urolithiasis and cholelithiasis

ObjectiveTo investigate the therapeutic effect of Hibiscus cannabinus Linn. (H. cannabinus) leaves on cholelithiasis and urolithiasis. MethodsThe study evaluated the effect of aqueous leaf extract of H. cannabinus on thiouracil and cholesterol cholic acid diet induced cholelithiasis in BALB/c mice and ethylene glycol induced urolithiasis in Wistar rats. Three doses of aqueous extract (40, 80, and 160 mg/kg) were selected to evaluate the effectiveness in cholelithiasis in mice; another three doses of aqueous extract (400, 800, and 1600 mg/kg) were administered for evaluating the effect on urolithiasis in rats. Biochemical parameters such as biliary cholesterol, biliary phospholipid, and bile acid were determined in cholelithiasis model. Similarly, 24-hour urine output, urinary parameters such as creatinine, uric acid, protein, urea, presence of calcium oxalate crystals, red blood cells (RBCs), and pyuria were determined in urolithiasis model. ResultsStatistically significant differences were noted in the biliary and urinary parameters after administrating three test doses of H. cannabinus aqueous extract (P < 0.05). ConclusionH. cannabinus was found to be effective against high fat lithogenic diet urolithiasis and cholelithiasis.

Effect of hepatic NPC1L1 on cholesterol gallstone disease and its mechanism

Cholesterol gallstone disease (CGD) is associated with bile cholesterol supersaturation. The Niemann-Pick C1-like 1 (NPC1L1), the inhibitory target of ezetimibe (EZE), is a critical sterol transporter of cholesterol absorption. Intestinal NPC1L1 facilitates the absorption of cholesterol, whereas hepatic NPC1L1 promotes cholesterol uptake by hepatocytes and reduces bile cholesterol supersaturation. The potential of hepatic NPC1L1 to prevent CGD has yet to be established due to its absence in the mice model. In this study, we generated mice expressing hepatic NPC1L1 using adeno-associated virus (AAV) gene delivery. The biliary cholesterol saturations and gallstone formations were explored under chow diet and lithogenic diet (LD) with or without EZE treatment. The long-term (8-week) LD-fed AAV-mNPC1L1 mice exhibited no significant differences in biliary cholesterol saturation and gallstone formation compared to WT mice. EZE effectively prevented CGD in both WT and AAV-mNPC1L1 mice. Mechanistically, prolonged LD feeding induced the degradation of hepatic NPC1L1, whereas short-term (2-week) LD feeding preserved the expression of hepatic NPC1L1. In conclusion, our findings suggest that hepatic NPC1L1 is unable to prevent CGD, whereas EZE functions as an efficient bile cholesterol desaturator during CGD development.

Effect of nicotine on cholesterol gallstone formation in C57BL/6J mice fed on a lithogenic diet.

Gallstones are diseases of the biliary system caused by cholesterol supersaturation and/or deficiency in bile salts in bile. Early studies have shown that symptomatic gallstones are primarily a disease of non-smokers, raising the possibility that nicotine can prevent gallstone formation. The present study investigated the effect of nicotine on the formation of cholesterol gallstone in C57BL/6J mice. C57BL/6J mice (eight-weeks-old) were fed a normal or lithogenic diet (basic feed 82.45%, fat 15.8%, cholesterol 1.25% and sodium cholate 0.5%) and divided into five groups: normal diet (ND); ND + high dose nicotine (H); lithogenic diet (LD); LD + low dose nicotine (L) and LD + nicotine (H). They were treated with or without nicotine injection for 10 weeks. Nicotine treatment did not change the rate of cholesterol gallstone formation. There was no difference in TNFα, IL-1β and IL-6 among the five groups. The LD group showed the highest cholesterol levels and there was significant suppression of the total cholesterol, low-density lipoprotein-cholesterol and total bile acid levels in the serum of the nicotine-treated mice. Quantitative PCR showed nicotine altered few bile acid metabolism-related genes expression in liver tissue and significantly altered cholesterol-metabolism genes in gallbladder tissue. Hematoxylin and eosin staining and western blotting showed that protein levels of farnesoid X receptor (FXR) and megalin in the gallbladder increased in the lithogenic-diet mice, which was significantly suppressed in the nicotine-treated mice. In vitro studies using gallbladder epithelial cells showed that chenodeoxycholic acids increased megalin expression, which could be attenuated by nicotine. Nicotine could regulate bile acid metabolism via the FXR-megalin/cubilin pathways, which potentially contribute to cholesterol nucleation and subsequent gallstone formation.

Adeno-associated virus-based caveolin-1 delivery via different routes for the prevention of cholesterol gallstone formation

BackgroundHepatic caveolin-1 (CAV1) is reduced in cholesterol gallstone disease (CGD). Mice with CAV1 deficiency were prone to develop CGD. However, it remains unknown whether restored hepatic CAV1 expression prevents the development of CGD.MethodsC57BL/6 mice were injected with adeno-associated virus 2/8 (AAV2/8) vectors carrying the CAV1 gene (AAV2/8CAV1) via intravenous (i.v.) or intraperitoneal (i.p.) route and then subjected to a lithogenic diet (LD) for 8 weeks. Uninjected mice were used as controls. The functional consequences of rescuing CAV1 expression by either i.v. or i.p. AAV2/8CAV1 treatment for CGD prevention and its subsequent molecular mechanisms were examined.ResultsCAV1 expression was reduced in the liver and gallbladder of LD-fed CGD mice. We discovered that AAV2/8CAV1 i.p. delivery results in higher transduction efficiency in the gallbladder than tail vein administration. Although either i.v. or i.p. injection of AAV2/8CAV1 improved liver lipid metabolic abnormalities in CGD mice but did not affect LD feeding-induced bile cholesterol supersaturation. In comparison with i.v. administration route, i.p. administration of AAV2/8CAV1 obviously increased CAV1 protein levels in the gallbladder of LD-fed mice, and i.p. delivery of AAV2/8CAV1 partially improved gallbladder cholecystokinin receptor (CCKAR) responsiveness and impeded bile cholesterol nucleation via the activation of adenosine monophosphate-activated protein kinase (AMPK) signaling, which induced a reduction in gallbladder mucin-1 (MUC1) and MUC5ac expression and gallbladder cholesterol accumulation.ConclusionCGD prevention by i.p. AAV2/8CAV1 injection in LD-fed mice was associated with the improvement of gallbladder stasis, which again supported the notion that supersaturated bile is required but not sufficient for the formation of cholesterol gallstones. Additionally, AAV treatment via the local i.p. injection offers particular advantages over the systemic i.v. route for much more effective gallbladder gene delivery, which will be an excellent tool for conducting preclinical functional studies on the maintenance of normal gallbladder function to prevent CGD.

FGF15 promotes hepatic NPC1L1 degradation in lithogenic diet-fed mice

BackgroundCholesterol gallstone disease (CGD) is accompanied by biliary cholesterol supersaturation. Hepatic Niemann-Pick C1-like 1 (NPC1L1), which is present in humans but not in wild-type (WT) mice, promotes hepatocyte cholesterol uptake and decreases biliary cholesterol supersaturation. In contrast, intestinal NPC1L1 promotes intestinal cholesterol absorption, increasing biliary cholesterol supersaturation. Ezetimibe (EZE) can inhibit both hepatic and intestinal NPC1L1. However, whether hepatic NPC1L1 can affect CGD progress remains unknown.MethodsMice expressing hepatic NPC1L1 (NPC1L1hepatic-OE mice) were generated using Adeno-associated viruses (AAV) gene delivery. The protein level and function of hepatic NPC1L1 were examined under chow diet, high fat-cholesterol diet (HFCD), and lithogenic diet (LD) feeding. Gallstone formation rates were examined with or without EZE treatment. Fibroblast growth factor 15 (FGF15) treatment and inhibition of fibroblast growth factor receptor 4 (FGFR4) were applied to verify the mechanism of hepatic NPC1L1 degradation.ResultsThe HFCD-fed NPC1L1hepatic-OE mice retained the biliary cholesterol desaturation function of hepatic NPC1L1, whereas EZE treatment decreased biliary cholesterol saturation and did not cause CGD. The ubiquitination and degradation of hepatic NPC1L1 were discovered in LD-fed NPC1L1hepatic-OE mice. Treatment of FGF15 during HFCD feeding and inhibition of FGFR4 during LD feeding could affect the protein level and function of hepatic NPC1L1.ConclusionsLD induces the ubiquitination and degradation of hepatic NPC1L1 via the FGF15-FGFR4 pathway. EZE may act as an effective preventative agent for CGD.

Gypenosides Prevent and Dissolve Cholesterol Gallstones by Modulating the Homeostasis of Cholesterol and Bile Acids.

Gypenosides (GPs), obtained from Gynostemma pentaphyllum (Thunb.) Makino, have been traditionally prescribed to treat metabolic disorders in Asians. This study assessed whether GPs could prevent lithogenic diet (LD)-induced cholesterol gallstone (CG) formation and enhance CG dissolution in mice. Gallstone-susceptible C57BL/6J mice were fed an LD or normal chow, with or without GPs. Bile acids (BAs) in gallbladder bile were analyzed by liquid chromatography-tandem mass spectrometry. Differentially expressed hepatic genes were identified by RNA sequencing, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. GPs were found to prevent LD-induced CG formation and to dissolve pre-existing LD-induced CGs. GPs reduced total cholesterol levels and increased BA levels in bile, as well as reducing the BA Hydrophobicity Index, ratio of 12α-hydroxylated (12α-OH) to non-12α-OH BAs, and Cholesterol Saturation Index in gallbladder bile. GO and KEGG pathway enrichment analyses indicated that GPs-induced genes were involved in BA biosynthesis and cholesterol metabolism. GPs increased the hepatic expression of genes encoding the cytochrome P450 (Cyp) enzymes Cyp7a1, Cyp7b1, and Cyp8b1, while decreasing the hepatic expression of genes encoding the adenosine triphosphate-binding cassette (Abc) transporters Abcg5 and Abcg8. GPs may be a promising strategy for preventing and dissolving CGs.

Circadian Rhythm Disruption Influenced Hepatic Lipid Metabolism, Gut Microbiota and Promoted Cholesterol Gallstone Formation in Mice.

BackgroundHepatic lipid metabolism regulates biliary composition and influences the formation of cholesterol gallstones. The genes Hmgcr and Cyp7a1, which encode key liver enzymes, are regulated by circadian rhythm-related transcription factors. We aimed to investigate the effect of circadian rhythm disruption on hepatic cholesterol and bile acid metabolism and the incidence of cholesterol stone formation.MethodsAdult male C57BL/6J mice were fed either a lithogenic diet (LD) only during the sleep phase (time-restricted lithogenic diet feeding, TRF) or an LD ad libitum (non-time-restricted lithogenic diet feeding, nTRF) for 4 weeks. Food consumption, body mass gain, and the incidence of gallstones were assessed. Circulating metabolic parameters, lipid accumulation in the liver, the circadian expression of hepatic clock and metabolic genes, and the gut microbiota were analyzed.ResultsTRF caused a dysregulation of the circadian rhythm in the mice, characterized by significant differences in the circadian expression patterns of clock-related genes. In TRF mice, the circadian rhythms in the expression of genes involved in bile acid and cholesterol metabolism were disrupted, as was the circadian rhythm of the gut microbiota. These changes were associated with high biliary cholesterol content, which promoted gallstone formation in the TRF mice.ConclusionDisordered circadian rhythm is associated with abnormal hepatic bile acid and cholesterol metabolism in mice, which promotes gallstone formation.

Aqueous Extract of Lysimachia christinae Hance Prevents Cholesterol Gallstone in Mice by Affecting the Intestinal Microflora.

With changes in human dietary patterns, the proportion of high-fat and high-cholesterol foods in the daily diet has increased. As a result, the incidence rate of cholelithiasis is increasing rapidly. Many studies have reported on the crucial role that the intestinal microflora plays in the progression of gallstones. Although the whole herb of Lysimachia christinae, a traditional Chinese medicine, has long been extensively used as a remedy for cholelithiasis in China, its effects on the intestinal microflora remain unknown. Hence, in this study, we investigated the ability of the aqueous extract of L. christinae (LAE) to prevent cholesterol gallstones (CGSs) in model animals by affecting the intestinal microflora. The effects of LAE on body weight, serum lipid profile, visceral organ indexes, and histomorphology were studied in male C57BL/6J mice, which were induced by a lithogenic diet. After the 8-week study, CGSs formation was greatly reduced after LAE treatment. LAE also reduced body weight gain and hyperlipidemia and restored the histomorphological changes. Moreover, the intestinal microflora exhibited significant variation. In the model group fed the lithogenic diet, the abundances of the genera unclassified Porphyromonadaceae, Lactobacillus and Alloprevotella decreased, but in contrast, Akkermansia dramatically increased compared with the control check group, which was fed a normal diet; the administration of LAE reversed these changes. These results imply that L. christinae can be considered an efficient therapy for eliminating CGSs induced by a high-fat and high-cholesterol diet, which may be achieved by influencing the intestinal microflora.