Gut microbiome and metabolome characteristics of patients with cholesterol gallstones suggest the preventive potential of prebiotics

Abstract

Cholesterol gallstones (CGS) still lack effective noninvasive treatment. The etiology of experimentally proven cholesterol stones remains underexplored. This cross-sectional study aims to comprehensively evaluate potential biomarkers in patients with gallstones and assess the effects of microbiome-targeted interventions in mice. Microbiome taxonomic profiling was conducted on 191 samples via V3−V4 16S rRNA sequencing. Next, 60 samples (30 age- and sex-matched CGS patients and 30 controls) were selected for metagenomic sequencing and fecal metabolite profiling via liquid chromatography-mass spectrometry. Microbiome and metabolite characterizations were performed to identify potential biomarkers for CGS. Eight-week-old male C57BL/6J mice were given a lithogenic diet for 8 weeks to promote gallstone development. The causal relationship was examined through monocolonization in antibiotics-treated mice. The effects of short-chain fatty acids such as sodium butyrate, sodium acetate (NaA), sodium propionate, and fructooligosaccharides (FOS) on lithogenic diet-induced gallstones were investigated in mice. Gut microbiota and metabolites exhibited distinct characteristics, and selected biomarkers demonstrated good diagnostic performance in distinguishing CGS patients from healthy controls. Multi-omics data indicated associations between CGS and pathways involving butanoate and propanoate metabolism, fatty acid biosynthesis and degradation pathways, taurine and hypotaurine metabolism, and glyoxylate and dicarboxylate metabolism. The incidence of gallstones was significantly higher in the Clostridium glycyrrhizinilyticum group compared to the control group in mice. The grade of experimental gallstones in control mice was significantly higher than in mice treated with NaA and FOS. FOS could completely inhibit the formation of gallstones in mice. This study characterized gut microbiome and metabolome alterations in CGS. C. glycyrrhizinilyticum contributed to gallstone formation in mice. Supplementing with FOS could serve as a potential approach for managing CGS by altering the composition and functionality of gut microbiota.

Keywords

cholesterol gallstone / gut microbiota / metabolomics / microbiome-targeted therapies / transplantation

References

[1] Portincasa, Piero, Antonio Moschetta, and Giuseppe Palasciano. 2006. “Cholesterol Gallstone Disease.” The Lancet 368: 230-239. https://doi.org/10.1016/S0140-6736(06)69044-2

[2] Rebholz, Charlotte, Marcin Krawczyk, and Frank Lammert. 2018. “Genetics of Gallstone Disease.” European Journal of Clinical Investigation 48: e12935. https://doi.org/10.1111/eci.12935

[3] Di Ciaula, Agostino, David Q. H. Wang, and Piero Portincasa. 2018. “An Update on the Pathogenesis of Cholesterol Gallstone Disease.” Current Opinion in Gastroenterology 34: 71-80. https://doi.org/10.1097/MOG.0000000000000423

[4] Wang, Qihan, Long Jiao, Chuanqi He, Haidong Sun, Qu Cai, Tianquan Han, and Hai Hu. 2017. “Alteration of Gut Microbiota in Association With Cholesterol Gallstone Formation in Mice.” BMC Gastroenterology 17: 74. https://doi.org/10.1186/s12876-017-0629-2

[5] Cai, Jie, Lulu Sun, and Frank J. Gonzalez. 2022. “Gut Microbiota-Derived Bile Acids in Intestinal Immunity, Inflammation, and Tumorigenesis.” Cell Host & Microbe 30: 289-300. https://doi.org/10.1016/j.chom.2022.02.004

[6] Staley, Christopher, Alexa R. Weingarden, Alexander Khoruts, and Michael J. Sadowsky. 2017. “Interaction of Gut Microbiota With Bile Acid Metabolism and Its Influence on Disease States.” Applied Microbiology and Biotechnology 101: 47-64. https://doi.org/10.1007/s00253-016-8006-6

[7] Agus, Allison, Karine Clément, and Harry Sokol. 2021. “Gut Microbiota-Derived Metabolites as Central Regulators in Metabolic Disorders.” Gut 70: 1174-1182. https://doi.org/10.1136/gutjnl-2020-323071

[8] Kenny, Douglas J., Damian R. Plichta, Dmitry Shungin, Nitzan Koppel, A. Brantley Hall, Beverly Fu, Ramachandran S. Vasan, et al. 2020. “Cholesterol Metabolism by Uncultured Human Gut Bacteria Influences Host Cholesterol Level.” Cell Host & Microbe 28: 245-257.e246. https://doi.org/10.1016/j.chom.2020.05.013

[9] Hu, Hai, Wentao Shao, Qian Liu, Ning Liu, Qihan Wang, Jin Xu, Xin Zhang, et al. 2022. “Gut Microbiota Promotes Cholesterol Gallstone Formation by Modulating Bile Acid Composition and Biliary Cholesterol Secretion.” Nature Communications 13: 252. https://doi.org/10.1038/s41467-021-27758-8

[10] Ye, Xin, Dan Huang, Zhixia Dong, Xiaoxin Wang, Min Ning, Jie Xia, Shuang Shen, et al. 2022. “FXR Signaling-Mediated Bile Acid Metabolism Is Critical for Alleviation of Cholesterol Gallstones by Lactobacillus Strains.” Microbiology Spectrum 10: e00518-e00522. https://doi.org/10.1128/spectrum.00518-22

[11] The British-Italian Gallstone Study Group, M. L. Petroni, R. P. Jazrawi, P. Pazzi, A. Lanzini, M. Zuin, M. G. Pigozzi, et al. 2001. “Ursodeoxycholic Acid Alone or With Chenodeoxycholic Acid for Dissolution of Cholesterol Gallstones: A Randomized Multicentre Trial.” Alimentary Pharmacology & Therapeutics 15: 123-128. https://doi.org/10.1046/j.1365-2036.2001.00853.x

[12] Hall, Lewis, James Halle-Smith, Richard Evans, Giles Toogood, Tom Wiggins, Sheraz R. Markar, Spyros Kapoulas, et al. 2023. “Ursodeoxycholic Acid in the Management of Symptomatic Gallstone Disease: Systematic Review and Clinician Survey.” BJS Open 7: zrac152. https://doi.org/10.1093/bjsopen/zrac152

[13] Rice, Christopher P., Krishnamurthy B. Vaishnavi, Celia Chao, Daniel Jupiter, August B. Schaeffer, Whitney R. Jenson, Lance W. Griffin, and William J. Mileski. 2019. “Operative Complications and Economic Outcomes of Cholecystectomy for Acute Cholecystitis.” World Journal of Gastroenterology 25: 6916-6927. https://doi.org/10.3748/wjg.v25.i48.6916

[14] Feng, Xiaoxi (Chelsea), Edward Phillips, and Daniel Shouhed. 2021. “Management of Postcholecystectomy Complications.” Surgical Clinics of North America 101: 889-910. https://doi.org/10.1016/j.suc.2021.06.012

[15] Wang, Gang, Yang Yu, Yu-Zhu Wang, Jun-Jie Wang, Rui Guan, Yan Sun, Feng Shi, Jing Gao, and Xing-Li Fu. 2019. “Role of SCFAs in Gut Microbiome and Glycolysis for Colorectal Cancer Therapy.” Journal of Cellular Physiology 234: 17023-17049. https://doi.org/10.1002/jcp.28436

[16] Liu, Yao-Jiang, Bo Tang, Feng-Chao Wang, Li Tang, Yuan-Yuan Lei, Ya Luo, Sheng-Jie Huang, et al. 2020. “Parthenolide Ameliorates Colon Inflammation through Regulating Treg/Th17 Balance in a Gut Microbiota-Dependent Manner.” Theranostics 10: 5225-5241. https://doi.org/10.7150/thno.43716

[17] Pujo, Julien, Camille Petitfils, Pauline Le Faouder, Venessa Eeckhaut, Gaelle Payros, Sarah Maurel, Teresa Perez-Berezo, et al. 2021. “Bacteria-Derived Long Chain Fatty Acid Exhibits Anti-Inflammatory Properties in Colitis.” Gut 70: 1088-1097. https://doi.org/10.1136/gutjnl-2020-321173

[18] Liao, Minjing, Yuanfang Zhang, Yilan Qiu, Zhengchun Wu, Zhihong Zhong, Xiaoqi Zeng, Yiliang Zeng, et al. 2021. “Fructooligosaccharide Supplementation Alleviated the Pathological Immune Response and Prevented the Impairment of Intestinal Barrier in DSS-Induced Acute Colitis Mice.” Food & Function 12: 9844-9854. https://doi.org/10.1039/D1FO01147B

[19] Huang, Xiaoqing, Qiongyun Chen, Yanyun Fan, Renzhi Yang, Guoyu Gong, Changsheng Yan, and Song Yang, et al. 2023. “Fructooligosaccharides Attenuate Non-Alcoholic Fatty Liver Disease by Remodeling Gut Microbiota and Association With Lipid Metabolism.” Biomedicine & Pharmacotherapy 159: 114300. https://doi.org/10.1016/j.biopha.2023.114300

[20] Hughes, Riley L., David A. Alvarado, Kelly S. Swanson, and Hannah D. Holscher. 2022. “The Prebiotic Potential of Inulin-Type Fructans: A Systematic Review.” Advances in Nutrition 13: 492-529. https://doi.org/10.1093/advances/nmab119

[21] Zhai, Lixiang, Chunhua Huang, Ziwan Ning, Yijing Zhang, Min Zhuang, Wei Yang, Xiaolei Wang, et al 2023. “Ruminococcus Gnavus Plays a Pathogenic Role in Diarrhea-Predominant Irritable Bowel Syndrome by Increasing Serotonin Biosynthesis.” Cell Host & Microbe 31: 33-44.e5. https://doi.org/10.1016/j.chom.2022.11.006

[22] Lopez-Siles, Mireia, Sylvia H. Duncan, L. Jesús Garcia-Gil, and Margarita Martinez-Medina. 2017. “Faecalibacterium Prausnitzii: From Microbiology to Diagnostics and Prognostics.” The ISME Journal 11: 841-852. https://doi.org/10.1038/ismej.2016.176

[23] Wang, Hua, Junfeng Gong, Jingyi Chen, Wei Zhang, Yanjun Sun, and Dengqun Sun. 2024. “Intestinal Microbiota and Biliary System Diseases.” Frontiers in Cellular and Infection Microbiology 14: 1362933. https://doi.org/10.3389/fcimb.2024.1362933

[24] Hu, Junqing, Jichao Tang, Xinpeng Zhang, Kaijin Yang, Ayan Zhong, Qin Yang, Yanjun Liu, Yi Li, and Tongtong Zhang. 2023. “Landscape in the Gallbladder Mycobiome and Bacteriome of Patients Undergoing Cholelithiasis With Chronic Cholecystitis.” Frontiers in Microbiology 14: 1131694. https://doi.org/10.3389/fmicb.2023.1131694

[25] Wang, Qiang, Chenjun Hao, Wenchao Yao, Defu Zhu, Haifeng Lu, Long Li, Biao Ma, et al. 2020. “Intestinal Flora Imbalance Affects Bile Acid Metabolism and Is Associated With Gallstone Formation.” BMC Gastroenterology 20: 59. https://doi.org/10.1186/s12876-020-01195-1

[26] Han, Xu, Juan Wang, Yingnan Wu, Hao Gu, Ning Zhao, Xing Liao, and Miao Jiang. 2024. “Predictive Value of Bile Acids as Metabolite Biomarkers for Gallstone Disease: A Systematic Review and Meta-Analysis.” PLoS One 19: e0305170. https://doi.org/10.1371/journal.pone.0305170

[27] Stewart, Lygia, J. McLeod Grifiss, Gary A. Jarvis, and Lawrence W. Way. 2006. “Biliary Bacterial Factors Determine the Path of Gallstone Formation.” The American Journal of Surgery 192: 598-603. https://doi.org/10.1016/j.amjsurg.2006.08.001

[28] Nakano, Toru, Jiro Yanagisawa, and Fumio Nakayama. 1988. “Phospholipase Activity in Human Bile.” Hepatology 8: 1560-1564. https://doi.org/10.1002/hep.1840080615

[29] Proffitt, Ceri, Gholamreza Bidkhori, Sunjae Lee, Abdellah Tebani, Adil Mardinoglu, Mathias Uhlen, David L. Moyes, and Saeed Shoaie. 2022. “Genome-Scale Metabolic Modelling of the Human Gut Microbiome Reveals Changes in the Glyoxylate and Dicarboxylate Metabolism in Metabolic Disorders.” iScience 25: 104513. https://doi.org/10.1016/j.isci.2022.104513

[30] Beaumont, Michelle, Julia K. Goodrich, Matthew A. Jackson, Idil Yet, Emily R. Davenport, Sara Vieira-Silva, Justine Debelius, et al. 2016. “Heritable Components of the Human Fecal Microbiome Are Associated With Visceral Fat.” Genome Biology 17: 189. https://doi.org/10.1186/s13059-016-1052-7

[31] Lammert, Frank, Kurinchi Gurusamy, Cynthia W. Ko, Juan-Francisco Miquel, Nahum Méndez-Sánchez, Piero Portincasa, Karel J. van Erpecum, Cees J. van Laarhoven, and David Q. H. Wang. 2016. “Gallstones.” Nature Reviews Disease Primers 2: 16024. https://doi.org/10.1038/nrdp.2016.24

[32] Maniscalco, Mauro, Debora Paris, Dominique J. Melck, Maria D'Amato, Anna Zedda, Matteo Sofia, Cristiana Stellato, and Andrea Motta. 2017. “Coexistence of Obesity and Asthma Determines a Distinct Respiratory Metabolic Phenotype.” Journal of Allergy and Clinical Immunology 139: 1536-1547.e5. https://doi.org/10.1016/j.jaci.2016.08.038

[33] Crost, Emmanuelle H., Erika Coletto, Andrew Bell, and Nathalie Juge. 2023. “Ruminococcus Gnavus: Friend or Foe for Human Health.” FEMS Microbiology Reviews 47: fuad014. https://doi.org/10.1093/femsre/fuad014

[34] Sakuma, Keita, Maki Kitahara, Ryoko Kibe, Mitsuo Sakamoto, and Yoshimi Benno. 2006. “Clostridium glycyrrhizinilyticum Sp. Nov., a Glycyrrhizin-hydrolysing Bacterium Isolated From Human Faeces.” Microbiology and Immunology 50: 481-485. https://doi.org/10.1111/j.1348-0421.2006.tb03818.x

[35] Wang, Yiqun, Shengxia Lv, Tianbai Shen, Minchao Ye, Dehe Wang, Pan Zhou, Ying Li, and Wei Zhang. 2021. “Qinghua Fang Inhibits High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Modulating Gut Microbiota.” Annals of Palliative Medicine 10: 3219-3234. https://doi.org/10.21037/apm-21-448

[36] Ye, Xin, Shuang Shen, Zhengjie Xu, Qian Zhuang, Jingxian Xu, Jingjing Wang, Zhixia Dong, and Xinjian Wan. 2021. “Sodium Butyrate Alleviates Cholesterol Gallstones by Regulating Bile Acid Metabolism.” European Journal of Pharmacology 908: 174341. https://doi.org/10.1016/j.ejphar.2021.174341

[37] Grigor'eva, Irina N., and Tatyana I. Romanova. 2020. “Gallstone Disease and Microbiome.” Microorganisms 8: 835. https://doi.org/10.3390/microorganisms8060835

[38] Rau, Monika, Ateequr Rehman, Marcus Dittrich, Albert K. Groen, Heike M. Hermanns, Florian Seyfried, Niklas Beyersdorf, et al. 2018. “Fecal SCFAs and SCFA-producing Bacteria in Gut Microbiome of Human NAFLD as a Putative Link to Systemic T-Cell Activation and Advanced Disease.” United European Gastroenterology Journal 6: 1496-1507. https://doi.org/10.1177/2050640618804444

[39] Cao, Xinlu, Oksana Zolnikova, Roman Maslennikov, Maria Reshetova, Elena Poluektova, Arina Bogacheva, Maria Zharkova, and Vladimir Ivashkin. 2023. “Differences in Fecal Short-Chain Fatty Acids Between Alcoholic Fatty Liver-Induced Cirrhosis and Non-Alcoholic (Metabolic-Associated) Fatty Liver-Induced Cirrhosis.” Metabolites 13: 859. https://doi.org/10.3390/metabo13070859

[40] Ni, Yueqiong, Lingling Qian, Sara Leal Siliceo, Xiaoxue Long, Emmanouil Nychas, Yan Liu, Marsena Jasiel Ismaiah, et al. 2023. “Resistant Starch Decreases Intrahepatic Triglycerides in Patients With NAFLD via Gut Microbiome Alterations.” Cell Metabolism 35: 1530-1547.e8. e1538. https://doi.org/10.1016/j.cmet.2023.08.002

[41] Hu, Haijuan, Shanshan Zhang, Fengxia Liu, Peipei Zhang, Zafarullah Muhammad, and Siyi Pan. 2019. “Role of the Gut Microbiota and Their Metabolites in Modulating the Cholesterol-Lowering Effects of Citrus Pectin Oligosaccharides in C57BL/6 Mice.” Journal of Agricultural and Food Chemistry 67: 11922-11930. https://doi.org/10.1021/acs.jafc.9b03731

[42] Kaur, Narinder, and Anil K. Gupta. 2002. “Applications of Inulin and Oligofructose in Health and Nutrition.” Journal of Biosciences 27: 703-714. https://doi.org/10.1007/BF02708379

[43] Lin, Huai, Qing Wang, Lei Liu, Zeyou Chen, Ranjit Das, Yanhui Zhao, Daqing Mao, and Yi Luo. 2020. “Colonization of Mice With Amoxicillin-Associated Klebsiella Variicola Drives Inflammation via Th1 Induction and Treg Inhibition.” Frontiers in Microbiology 11: 1256. https://doi.org/10.3389/fmicb.2020.01256

[44] Singh, Vishal, Beng San Yeoh, Benoit Chassaing, Xia Xiao, Piu Saha, Rodrigo Aguilera Olvera, John D. Lapek,, et al. 2018. “Dysregulated Microbial Fermentation of Soluble Fiber Induces Cholestatic Liver Cancer.” Cell 175: 679-694.e22. e622. https://doi.org/10.1016/j.cell.2018.09.004

[45] Akiyoshi, T., K. Uchida, H. Takase, Y. Nomura, and N. Takeuchi. 1990. “Cholesterol Gallstones in Alloxan-Diabetic Mice.” Journal of Lipid Research 27: 915-924. https://doi.org/10.1016/S0022-2275(20)38774-5

RIGHTS & PERMISSIONS

2025 The Author(s). iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.