Cholesterol Gallstones Research Articles

Inhibition of PCSK9 prevents and alleviates cholesterol gallstones through PPARα-mediated CYP7A1 activation

Background & aimsDysregulated cholesterol metabolism is the major factor responsible for cholesterol gallstones (CGS). Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol homeostasis and its inhibitors secure approval for treating various cholesterol metabolic disorders such as hypercholesterolemia and cardiovascular diseases, but its role in CGS remains unclear. Our study aims to clarify mechanisms by which PCSK9 promotes CGS formation and explore the application of the PCSK9 inhibitor, alirocumab, in preventing and treating CGS. Approach & resultsThe expressions of PCSK9 were notably increased in CGS patients' serum, bile, and liver tissues compared to those without gallstones. Moreover, among CGS patients, hepatic PCSK9 was positively correlated with hepatic cholesterol and negatively correlated with hepatic bile acids (BAs), suggesting PCSK9 was involved in disrupted hepatic cholesterol metabolism related to CGS. Mechanistically, in vitro experiments demonstrated that inhibition of PCSK9 enhanced nuclear expression of PPARα by diminishing its lysosomal degradation and subsequently activated CYP7A1 transcription. Finally, inhibition of PCSK9 prevented CGS formation and dissolved the existing stones in CGS mice by elevating the conversion of cholesterol into BAs through PPARα-mediated CYP7A1 activation. Additionally, serum PCSK9 level may function as a prognostic signature to evaluate the therapeutic efficacy of PCSK9 inhibitors. ConclusionsInhibition of PCSK9 exerts preventive and therapeutic effects on CGS by activating PPARα-mediated CYP7A1 expression and facilitating the conversion of cholesterol into BAs, which highlights the potential of PCSK9 inhibition as a promising candidate for preventing and treating CGS in clinical applications. Impact and implicationsPCSK9 plays a pivotal role in cholesterol metabolism and its inhibitors are approved for clinical use in cardiovascular diseases. Our study observes inhibition of PCSK9 prevents and dissolves CGS by activating PPARα-mediated CYP7A1 expression and facilitating the conversion of cholesterol into BAs. Mechanistically, PCSK9 inhibition enhanced the nuclear expression of PPARα by diminishing its lysosomal degradation and subsequently activated CYP7A1 transcription. Our study sheds light on the new function and mechanism of PCSK9 in CGS, providing a novel preventive and therapeutic target with potential clinical applications.

Laparoscopic cholecystectomy for symptomatic cholelithiasis in children and adolescents: analysis of 50 cases from a single institution.

To investigate the clinical characteristics of symptomatic cholecystolithiasis and laparoscopic cholecystectomy complications in pediatric patients. The medical records of 50 children and adolescents who underwent laparoscopic cholecystectomy were analyzed. We evaluated gender, age, body mass index, preoperative clinical aspects, perioperative complications, and gallstone composition. Among the patients, 33 (66%) were female, and 17 (34%) were male. The mean age was 11.4 ± 3.6. All patients were diagnosed with cholecystolithiasis by abdominal ultrasonography. Twelve patients (24%) had hematological disease: eight (16%) with sickle cell anemia and four (8%) with hereditary spherocytosis. Thirteen patients (26%) were obese. Twelve patients (24%) had complicated biliary disease. During the intraoperative period, three patients (6%) had excessive bleeding in the hepatic hilum, and one had an accidental injury to the common bile duct. Three (6%) postoperative complications (acute pancreatitis, common bile duct stenosis, and intestinal obstruction) were observed. Among 28 patients (56%), 25 (50%) had cholesterol gallstones, and three (6%) had bile pigment gallstones. The evolution of cholecystolithiasis in the pediatric population can present serious complications, emphasizing the need to avoid temporizing cholecystolithiasis in children and adolescents because laparoscopic cholecystectomy in this group is safe, with low complication rates.

Quantitative phase analysis and molecular structure of human gallstones using synchrotron radiation X-ray diffraction and FTIR spectroscopy

Human gallstones are the most common disorder in the biliary system, affecting up to 20 % of the adult population. The formation of gallstones is primarily due to the supersaturating of cholesterol in bile. In order to comprehend gallstone disease in detail, it is necessary to have accurate information about phase identification and molecular structure. Different types of gallstone samples were collected from the Middle East area after surgical operations including; cholesterol, pigment, and mixed gallstones. To estimate the basic information about the stone formation and the pathophysiology of cholelithiasis as well as to classify the collected human gallstones, attenuated total reflection Fourier transform Infrared spectrometry (ATR-FTIR) was used to analyze the different gallstone structures in the wavenumber range from 400 to 4000 cm−1. Calcium bilirubinate was specified by the bands at 1662 cm−1, 1626 cm−1, and 1572 cm−1, while cholesterol rings were designated by the bands at 1464, 1438, 1055, and 1022 cm−1. It can be assumed that all samples consist of mixed gallstones based on the doublets at 1375 cm−1 and 1365 cm−1. The levels of calcium bilirubin and various minerals varied among the analyzed samples, indicating the heterogeneity in their composition and suggesting potential implications for gallstone formation. Based on the quantitative phase analysis using synchrotron radiation X-ray diffraction (SR-XRD), two phases of anhydrous cholesterol as a major content and one phase of monohydrate cholesterols as trace content represent the main components of most of the gallstones. Additional phases of calcium carbonate in the form of calcite, vaterite, aragonite, and bilirubinate were also quantified. According to the outcomes of the FTIR and the SR-XRD measurements, there exists a statistical correlation between the different types of chemical constituents of the gallstones.

Discovery and exploration of novel somatostatin receptor subtype 5 (SSTR5) antagonists for the treatment of cholesterol gallstones

The shortage of cholesterol gallstones treatment intensifies the need to discover of effective small molecule drugs. Clinical follow-up and studies have found that activation of somatostatin receptor subtype 5 (SSTR5) reduce gallbladder contraction and thus increase the risk of cholesterol gallstones, implying that antagonizing SSTR5 may promote gallbladder emptying and reduce the formation of gallstones. Herein, we discovered novel SSTR5 antagonists and firstly investigated its effects on cholesterol gallstone. From loperamide, a reported seed structure with micromole activity, we identified optimal compound 23 as an SSTR5 antagonist exhibiting single-digit nanomolar potency, low hERG inhibition and oral availability. Further in vivo evaluation revealed that 23 significantly promoted gallbladder emptying. Moreover, in a mouse cholesterol gallstone model, 23 (3 mg/kg) effectively reduced the cholesterol gallstones formation, showing better efficacy than the clinical first-line drug UDCA (60 mg/kg), providing a new insight into the development of anti-gallstone drugs.

Insight into the mechanism of gallstone disease by proteomic and metaproteomic characterization of human bile.

Cholesterol gallstone disease is a prevalent condition that has a significant economic impact. However, the role of the bile microbiome in its development and the host's responses to it remain poorly understood. In this study, we conducted a comprehensive analysis of microbial and human bile proteins in 40 individuals with either gallstone disease or gallbladder polyps. We employed a combined proteomic and metaproteomic approach, as well as meta-taxonomic analysis, functional pathway enrichment, and Western blot analyses. Our metaproteomic analysis, utilizing the lowest common ancestor algorithm, identified 158 microbial taxa in the bile samples. We discovered microbial taxa that may contribute to gallstone formation, including β-glucuronidase-producing bacteria such as Streptococcus, Staphylococcus, and Clostridium, as well as those involved in biofilm formation like Helicobacter, Cyanobacteria, Pseudomonas, Escherichia coli, and Clostridium. Furthermore, we identified 2,749 human proteins and 87 microbial proteins with a protein false discovery rate (FDR) of 1% and at least 2 distinct peptides. Among these proteins, we found microbial proteins crucial to biofilm formation, such as QDR3, ompA, ndk, pstS, nanA, pfIB, and dnaK. Notably, QDR3 showed a gradual upregulation from chronic to acute cholesterol gallstone disease when compared to polyp samples. Additionally, we discovered other microbial proteins that enhance bacterial virulence and gallstone formation by counteracting host oxidative stress, including sodB, katG, rbr, htrA, and ahpC. We also identified microbial proteins like lepA, rtxA, pckA, tuf, and tpiA that are linked to bacterial virulence and potential gallstone formation, with lepA being upregulated in gallstone bile compared to polyp bile. Furthermore, our analysis of the host proteome in gallstone bile revealed enhanced inflammatory molecular profiles, including innate immune molecules against microbial infections. Gallstone bile exhibited overrepresented pathways related to blood coagulation, folate metabolism, and the IL-17 pathway. However, we observed suppressed metabolic activities, particularly catabolic metabolism and transport activities, in gallstone bile compared to polyp bile. Notably, acute cholelithiasis bile demonstrated significantly impaired metabolic activities compared to chronic cholelithiasis bile. Our study provides a comprehensive metaproteomic analysis of bile samples related to gallstone disease, offering new insights into the microbiome-host interaction and gallstone formation mechanism.

Cholesterol Gallstone among Patients with Cholelithiasis Admitted to the Department of Surgery of a Tertiary Care Center.

Gallstone is one of the most common pathological conditions found mostly in females. The incidence of cholesterol gallstones is developing higher nowadays which is increasing the burden of cost in society. This study aimed to find out the prevalence of cholesterol gallstones among patients with cholelithiasis admitted to the Department of Surgery of a tertiary care centre. A descriptive cross-sectional study was conducted among patients with cholelithiasis who presented in the Department of Surgery of tertiary care centre for cholecystectomy (laparoscopic or open) from 23 December 2022 to 22 September 2023 after obtaining ethical approval from the Institutional Review Committee. Patients who were diagnosed by use of abdominal ultrasound or CT scan were included. Those patients with gallbladder polyps, cholangitis, and gallbladder tumours were excluded from the study. A convenience sampling method was used. The point estimate was calculated at a 95% Confidence Interval. Among 190 patients, the prevalence of cholesterol gallstones was seen in 85 (44.74%) (37.67-51.81, 95% Confidence Interval). The female to male ratio was 6.72:1. The prevalence of cholesterol gallstones was found to be higher as compared to other studies done in similar settings. cholesterol; gallbladder; gallstone; prevalence.

Abstract 16444: Gut Microbiota Derived Metabolite, Ursodeoxycholic Acid, and the Risk of Cardiovascular Disease

Introduction: Gut microbial metabolites have been recognized as risk factors for cardiometabolic diseases. The associations between plasma levels of the gut microbiota derived metabolites with cardiovascular disease (CVD) have not been comprehensively examined. Our objective was to investigate the association between 6 secondary bile acids including ursodeoxycholic acid and CVD. Ursodeoxycholic acid is used to dissolve cholesterol gall stones to treat cholestatic liver diseases. Hypothesis: Gut microbiota derived metabolites are associated with CVD. Methods: We performed a secondary analysis of data from the cross-sectional Metabolic Syndrome in Men (METSIM) cohort. Our study included 915 men aged 45-73 years (age = 54.94 ± 5.04 years, BMI = 27.31 ± 3.44), randomly selected from the population register of Kuopio, Eastern Finland, from 2005 to 2010. The associations between 6 secondary bile acids (ursodeoxycholic acid, glycoursodeoxycholate, glycolithocholate sulfate, taurocholenate sulfate, taurochenodeoxycholate, and glycocholenate sulfate) and the risk of CVD were investigated. Kaplan-Meier analysis with Cox proportional-hazards regressions were conducted for the time-to-event analysis to determine hazard ratios (HR) and 95% confidence intervals (CI) for CVD. Adjustments were made for traditional cardiovascular risk factors (age, systolic blood pressure, low-density and high-density lipoprotein cholesterol levels, triglyceride levels, smoking status, high-sensitivity C-reactive protein, estimated glomerular filtration rate, and alcohol consumption). Results: Participants were stratified into groups according to quartile metabolites levels and CVD. During a median follow-up of 16 years, n = 62 (7%) of patients experienced CVD. Only ursodeoxycholic acid was significantly associated with CVD risk. Cox regression analyses showed that the adjusted HR for CVD was higher in patients in quartile 4 (HR: 2.8, 95% [CI]: 1.35-4.6; p =0.008) than in quartile 1. The Kaplan-Meier analysis indicated that patients in quartile 4 had a significantly lower event-free survival (p = 0.037) compare to quartile 1. Conclusions: Ursodeoxycholic acid, a secondary bile acid, was positively associated with an increased risk of CVD.

Elemental studies and mapping of cholesterol and pigment gallstones using scanning electron microscopy–energy dispersive spectroscopy

AbstractGallstone formation is one of the most severe human diseases, with regional differences in gallstone composition worldwide. The formation of gallstones inside the gallbladder is a complex process and is still under debate despite advances in instrumentation. This study was an in‐depth analysis of the chemical, structural, and elemental composition of cholesterol and pigment‐type gallstones using Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy–energy dispersive X‐ray spectroscopy (SEM–EDS). Thermal gravimetric and differential scanning calorimetry (TG‐DSC) analysis was also carried out on gallstones to predict their thermal behavior. FTIR spectroscopy was employed to distinguish the cholesterol and pigment gallstones. Using SEM, we performed the morphological studies of gallstone and EDS were carried out to analyze elemental distribution within the gallstones. Elemental imaging and mapping of the major and minor elements within the cholesterol and black pigment gallstones were carried out, revealing the stone's heterogeneous nature. The level of heavy and toxic elements was found to be higher in pigment stones than in cholesterol gallstones. The results obtained from TG‐DSC are well correlated and supported by the results from FTIR spectroscopy.

Gallstone Disease and Its Correlation With Thyroid Disorders: A Narrative Review.

Over the years, several studies have revealed an important link between thyroid disorders and gallstone disease. According to these studies, hypothyroidism and hyperthyroidism are associated with cholesterol gallstone disease. This association between thyroid hormone disorders and cholesterol gallstone disease is due to the importance of thyroid hormones on cholesterol synthesis, bile functioning and content, and gallbladder motility. Several genes and receptors have been found on the thyroid gland, liver, and gallbladder to verify this association. These genes affect thyroid hormone secretion, lipid metabolism, and bile secretion. Defects in these various gene expression and protein functions lead to bile duct diseases. Other causes that lead to cholesterol gallstone disease are supersaturation of the bile with cholesterol and impaired gallbladder motility, which leads to bile stasis. This article has discussed these factors in detail while highlighting the association between thyroid hormones and cholesterol gallstone disease.

Correlation of serum biochemical characteristics and ABCG8 genetic variant (rs 11887534) with gall stone compositions and risk of gallstone disease in a group of Egyptian patients.

The overexpression of the cholesterol transporter: ATP-binding cassette transporter (ABCG8) due to the effect of ABCG8 genetic variant (rs11887534) leads to the precipitation of cholesterol crystals and gallstone disease (GSD). To evaluate the chemical composition of gallstones and the role of ABCG8 (rs11887534) in the susceptibility to GSD. We enrolled 77 patients with GSD treated with standard laparoscopic or open cholecystectomy and 75 age and sex-matched healthy controls. Chemical analysis of the extracted gallstones was performed. Analysis of the rs11887534 was performed by real-time PCR TaqMan technique for both cases and controls. Pure cholesterol stones were the main type of stones in GSD patients. The CC genotype carriers of rs11887534 were more prone to gallstone formation than other genotypes. The CC genotype carriers were 100 folds at increased risk to develop pure cholesterol stones. The most prevalent type of gallbladder stones is pure cholesterol stone. ABCG8 (rs11887534) could be associated with increased risk for cholesterol gallstones formation, this risk was more pronounced in female patients.

In-vitro Dissolution Study of Gallstone with Medicinal Plant Extracts

Background: Gallstone disease poses a substantial economic burden on healthcare systems globally, necessitating safer alternatives to current treatments like dissolution therapy and cholecystectomy. Natural compounds from plants offer a potential solution, but research on their cholelitholytic activity is limited. In vitro dissolution studies are crucial for identifying effective plant-based therapies. Objective: This study aims to investigate the in vitro cholelitholytic activity of six plants and Ayurvedic medicines, selected based on ethnopharmacological knowledge and folk medicinal practices. Methods: Gallstone samples were categorized as combined cholesterol gallstones (CCGS) or black pigment gallstones based on external morphology and cross-sectional analysis. In vitro dissolution studies were conducted using extracts from Bergenia ciliata, Berberis asiatica, Cuscuta europaea, Kalanchoe pinnata, Teraxacum officinale, Macrotyloma uniflorum, and Ayurvedic medicines (Cystone®, Gokshuradi, and Calcury). The samples were immersed in the extracts and controls separately and incubated in a shaking water bath. The gallstone dissolution capacity was assessed by recording the dry weight of the samples at multiple time points. Results: T. officinale was highly effective in dissolving black pigment gallstones, while B. asiatica exhibited superior efficacy for CCGS. M. uniflorum and C. europaea also demonstrated significant dissolution activity against black pigment gallstones. However, K. pinnata was less effective for both gallstone types. B. ciliata and C. europaea exhibited equal effectiveness against both types. Ayurvedic medicine extracts were less effective compared to plant extracts. Conclusion: This in vitro study showed the plants can dissolve GS effectively. However, the effectiveness of the plant to dissolve GS depends on the type of the stone. The findings from this study serve as a basis for further in vivo research

Metabolic dysfunction-associated gallstone disease: expecting more from critical care manifestations.

About 20% of adults worldwide have gallstones which are solid conglomerates in the biliary tree made of cholesterol monohydrate crystals, mucin, calcium bilirubinate, and protein aggregates. About 20% of gallstone patients will definitively develop gallstone disease, a condition which consists of gallstone-related symptoms and/or complications requiring medical therapy, endoscopic procedures, and/or cholecystectomy. Gallstones represent one of the most prevalent digestive disorders in Western countries and patients with gallstone disease are one of the largest categories admitted to European hospitals. About 80% of gallstones in Western countries are made of cholesterol due to disturbed cholesterol homeostasis which involves the liver, the gallbladder and the intestine on a genetic background. The incidence of cholesterol gallstones is dramatically increasing in parallel with the global epidemic of insulin resistance, type 2 diabetes, expansion of visceral adiposity, obesity, and metabolic syndrome. In this context, gallstones can be largely considered a metabolic dysfunction-associated gallstone disease, a condition prone to specific and systemic preventive measures. In this review we discuss the key pathogenic and clinical aspects of gallstones, as the main clinical consequences of metabolic dysfunction-associated disease.

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.

Biliary Amino Acids and Telocytes in Gallstone Disease.

The role of amino acids in cholesterol gallstone formation is not known. Therefore, the aim of the study was to determine the amino acid profile in the bile of patients with and without cholecystolithiasis in relation to bile lithogenicity and telocyte numbers within the gallbladder wall. The study included 23 patients with cholecystolithiasis and 12 gallstone-free controls. The levels of free amino acids in the bile were measured, and telocytes were identified and quantified in the gallbladder muscle wall. The mean values of valine, isoleucine, threonine, methionine, phenylalanine, tyrosine, glutamic acid, serine alanine, proline and cystine were significantly higher in the study group than in the controls (p from 0.0456 to 0.000005), and the mean value of cystine was significantly lower in patients with gallstone disease than in the controls (p = 0.0033). The relationship between some of the amino acids, namely alanine, glutamic acid, proline, cholesterol saturation index (CSI) and the number of telocytes was significant (r = 0.5374, p = 0.0051; r = 0.5519, p = 0.0036; and r = 0.5231, p = 0.0071, respectively). The present study indicates a potential relationship between the altered amino acid composition of bile and the reduced number of telocytes in the gallbladder muscle wall in cholelithiasis.

Targeted bile acids metabolomics in cholesterol gallbladder polyps and gallstones: From analytical method development towards application to clinical samples

Bile acids (BAs) are synthesized by the liver from cholesterol through several complementary pathways and aberrant cholesterol metabolism plays pivotal roles in the pathogeneses of cholesterol gallbladder polyps (CGP) and cholesterol gallstones (CGS). To date, there is neither systematic study on BAs profile of CGP or CGS, nor the relationship between them. To explore the metabolomics profile of plasma BAs in healthy volunteers, CGP and CGS patients, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous determination of 42 free and conjugated BAs in human plasma. The developed method was sensitive and reproducible to be applied for the quantification of BAs in the investigation of plasma samples. The results show that, compared to healthy volunteers, CGP and CGS were both characterized by the significant decrease in plasma BAs pool size, furthermore CGP and CGS shared aberrant BAs metabolic characteristics. Chenodeoxycholic acid, glycochenodeoxycholic acid, λ-muricholic acid, deoxycholic acid, and 7-ketolithocholic acid were shared potential markers of these two cholesterol gallbladder diseases. Subsequent analysis showed that clinical characteristics including cysteine, ornithine and body mass index might be closely related to metabolisms of certain BA modules. This work provides metabolomic information for the study of gallbladder diseases and analytical methodologies for clinical target analysis and efficacy evaluation related to BAs in medical institutions.

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.

A study of H. pylori infection as risk factor for the gall stone disease

Background: Gallstones are one of the widely occurring digestive/hepatobiliary disorders. Chronic cholecystitis, cholelithiasis or symptomatic gallbladder is a prolonged mechanical or functional disorder of abnormal gallbladder emptying. Nearly eighty percent of the gallstones are cholesterol gallstones, and 20% are pigmented stones consisting of bilirubin and calcium, the two components present in the bile. The diagnosis is established by a variety of diagnostic tools like ultrasonography, CT scan, ERCP, liver function tests and pancreatic enzyme studies. The hypothesis of the presence of H. pylori in the biliary epithelium of the patients with hepatobiliary ailments has been sporadically investigated. Helicobacter pylori (H. pylori) is a spiral, microaerophilic, gram-negative bacterium. There are many suggestive evidences that the DNA components of H. pylori is found in the bile, gallbladder tissue and/or cholesterol gallstones. The association of gallstones with Helicobacter pylori has been investigated by many authors, but not clearly established. Methods: The present study is aimed to identify the Helicobacter pylori infection as a risk factor for gallstone disease. The histopathological samples of gall bladder mucosa were examined by staining with modified giemsa stain, haematoxylin, and eosin. Results: Our study showed the presence of H. pylori in only one case which is not statistically significant. Moreover, the present study was based on H and E stains only and they should have been substantiated with immunohistochemistry and PCR studies. Thus, further studies are required to establish a causal relationship between the Helicobactor pylori infection and gallstone formation. Conclusions: Helicobacter pylori as a risk factor for gallstones is yet to be proven.

Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. herb pair suppresses breast cancer liver metastasis by targeting ECM1-mediated cholesterol biosynthesis pathway

BackgroundLiver metastasis is a frequent event in breast cancer that causes low survival rate and poor prognosis. Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. (CR), a traditional Chinese herb pair, is used for the treatment of breast cancer liver metastasis or cholesterol gallstone disease in clinics. PurposeThis study attempted to investigate the potential therapeutic target and mechanism of CR herb pair on breast cancer liver metastasis. MethodsThe anti-metastatic and cholesterol-lowering activities of CR extract were evaluated in triple-negative breast cancer (TNBC) cell lines and an experimental liver metastasis model. The role of extracellular matrix protein 1 (ECM1) in the cholesterol biosynthesis pathway was determined by the knockdown and overexpression of ECM1 gene of TNBC cells. Changes in the gene and protein expression levels of ECM1 and the cholesterol biosynthesis pathway after CR treatment were detected in vitro and in vivo by real-time PCR and Western blot. ResultsThe invasive and metastatic potentials and hypercholesterol levels of TNBC cells were positively associated with ECM1 expression. ECM1 knockdown reduced tumor cholesterol levels via downregulating cholesterol biosynthesis genes, including ACAT2, HMGCS1, HMGCR, MVK, and MVD, whereas ECM1 overexpression elicited the opposite effects. CR herb pair exerts the potential therapeutic effects on TNBC liver metastasis, which is partially mediated by disrupting ECM1-activated cholesterol biosynthesis process in TNBC cells. ConclusionThis study reveals that ECM1 is a novel target for the activation of cholesterol biosynthesis to promote TNBC liver metastasis occurrence. CR herb pair, an ECM1 inhibitor, maybe be considered to serve as an adjuvant therapeutic drug for liver metastasis in clinical practice.

Metabolic endotoxemia caused by dysbiosis-induced elevated lipopolysaccharide (LPS) levels increases susceptibility to cholesterol gallstone formation in mice

Cholesterol gallstone formation is a complex process intervened by genetic and environmental factors. Clinical and animal studies have clearly demonstrated that alterations in the gut microbiota are associated with the formation of cholesterol gallstones, suggesting that gut dysbiosis may be a high risk factor for gallstone disease. However, the mechanisms underlying the roles of gut microbiota in the pathogenesis of gallstone formation are not fully understood. Western diet high in fat and cholesterol can increase the proportion of LPS-expressing bacteria in the gut, resulting in a 0.5- to 2-fold increase in plasma LPS levels and low-grade chronic systemic inflammation, also known as metabolic endotoxemia. Our aims are to investigate whether metabolic endotoxemia caused by dysbiosis-induced elevated LPS levels plays a critical role in increasing gallstone formation by disrupting biliary cholesterol metabolism in mice. Methods: The biliary and gallstone phenotypes were studied in male C57BL/6 mice fed a lithogenic diet and treated with LPS (from Escherichia coli 055:B5) at 7.5 μg/day (~300 μg/kg/day) or 0.9% NaCl (as control) for 8 wk. Plasma and bile LPS concentrations were determined by ELISA. Results: Our findings show for the first time that in metabolic endotoxemia (plasma LPS=77.6±8.3 ng/mL), LPS is present in hepatic bile (476±334 pg/mL) of mice treated with LPS at 7.5 μg/day. However, LPS cannot be detected in bile of control mice receiving no LPS. Model bile studies show that LPS reduces cholesterol solubility in bile by disturbing the physical state of cholesterol carriers, i.e., biliary vesicles. Metabolic endotoxemia also disrupts biliary cholesterol homeostasis by inhibiting expression of hepatic cholesterol 7α-hydroxylase and the classical pathway of bile acid biosynthesis via the LPS-stimulated Toll like receptor 4 (TLR4) signaling cascade in the liver. These abnormalities enhance hepatic cholesterol hypersecretion and promote the aggregation and fusion of unilamellar vesicles to form unstable multilamellar vesicles in bile. As a result, this accelerates heterogenous cholesterol crystallization, thereby leading to rapid formation of numerous classical plate-like cholesterol monohydrate crystals in supersaturated gallbladder bile. Metabolic endotoxemia via the LPS/TLR4 pathway also impairs gallbladder emptying and refilling, causing gallbladder stasis. These abnormalities promote the accumulation of excessive mucin gels and rapid growth and agglomeration of solid cholesterol monohydrate crystals into biliary sludge, a precursor of gallstones. Conclusions: Metabolic endotoxemia induced by gut dysbiosis is a risk environmental factor, and its lithogenic mechanism involves driving the initiation of supersaturated bile and heterogeneous cholesterol crystallization, two key steps in the early stages of cholesterol gallstone formation. NIH R01 DK126369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.