Elucidating the beneficial effects of gut microbiota-derived metabolites in treating hepatocellular carcinoma: an integrated network pharmacology, molecular docking, and single-cell sequencing analysis

Abstract

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-related mortality worldwide. While accumulating evidence has highlighted the beneficial effects of gut microbiota (GM)-derived metabolites in HCC treatment, the underlying mechanisms remain unclear. We retrieved GM-derived metabolites from gutMGene v2.0, acquired their targets using the Similarity Ensemble Approach (SEA) and Swiss Target Prediction (STP), and identified HCC-related targets from public databases. Overlapping targets were then obtained via Venn diagram analysis. We constructed protein–protein interaction (PPI) networks, analyzed signaling pathways, and established a comprehensive microbiota-metabolite-target-disease (M-M-T-D) network. We also examined the correlation between core gene expression and overall survival in HCC patients using UALCAN and GEPIA2. Single-cell transcriptomic data from TISCH2 were analyzed to explore the cellular distribution of core genes. To identify candidate compounds, we evaluated the ADMET properties of metabolites and determined the compound with the highest binding affinity via molecular docking. Ultimately, 8 core targets (AKT1, IL6, PPARG, IL1B, JUN, NFKB1, CASP3, and PTGS2) were identified. These targets interacted with seven core metabolites (10-oxo-12(Z)-octadecenoic acid, genipin, 3-indolepropionic acid, succinate, acetate, propionate, and butyrate) through the M-M-T-D network to exert inhibitory effects on HCC. Single-cell RNA sequencing (scRNA-seq) analysis revealed that core gene expression was correlated with the tumor microenvironment (TME) of HCC. Molecular docking confirmed that 3-indolepropionic acid exhibited the strongest binding affinity to the core targets. Collectively, GM-derived metabolites exert beneficial effects against HCC by regulating multiple signaling pathways and targets, supporting their potential as therapeutic candidates.

Graphical abstract

Keywords

Gut microbiota / Metabolites / Network pharmacology / Hepatocellular carcinoma / 3-indolepropionic acid

References

[1] Agus A, Planchais J, Sokol H. Gut microbiota regulation of tryptophan metabolism in health and disease. Cell Host Microbe, 2018, 23(6716-724

[2] Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: pathophysiological basis for therapy. J Hepatol, 2020, 72(3): 558-577

[3] Baffy G, Brunt EM, Caldwell SH. Hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace. J Hepatol, 2012, 56(6): 1384-1391

[4] Banerjee P, Kemmler E, Dunkel M, Preissner R. ProTox 3.0: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res, 2024, 52: W513-W520

[5] Basolo, A., Hohenadel, M., Ang, Q. Y., Piaggi, P., Heinitz, S., Walter, M., Walter, P., Parrington, S., Trinidad, D. D., von Schwartzenberg, R. J., Turnbaugh, P. J. & Krakoff, J. (2020)

[6] Bindels LB, Porporato P, Dewulf EM, Verrax J, Neyrinck AM, Martin JC, Scott KP, Buc Calderon P, Feron O, Muccioli GG, Sonveaux P, Cani PD, Delzenne NM. Gut microbiota-derived propionate reduces cancer cell proliferation in the liver. Br J Cancer, 2012, 107(8): 1337-1344

[7] Caradonna E, Abate F, Schiano E, Paparella F, Ferrara F, Vanoli E, Difruscolo R, Goffredo VM, Amato B, Setacci C, Setacci F, Novellino E. Trimethylamine-N-Oxide (TMAO) as a rising-star metabolite: implications for human health. Metabolites, 2025, 15(4): 220

[8] Chandrashekar DS, Karthikeyan SK, Korla PK, Patel H, Shovon AR, Athar M, Netto GJ, Qin ZS, Kumar S, Manne U, Creighton CJ, Varambally S. UALCAN: an update to the integrated cancer data analysis platform. Neoplasia, 2022, 25: 18-27

[9] Che Y, Chen G, Guo Q, Duan Y, Feng H, Xia Q. Gut microbial metabolite butyrate improves anticancer therapy by regulating intracellular calcium homeostasis. Hepatology, 2023, 78(1): 88-102

[10] Chen W, Wen L, Bao Y, Tang Z, Zhao J, Zhang X, Wei T, Zhang J, Ma T, Zhang Q, Zhi X, Li J, Zhang C, Ni L, Li M, Liang T. Gut flora disequilibrium promotes the initiation of liver cancer by modulating tryptophan metabolism and up-regulating SREBP2. Proc Natl Acad Sci U S A, 2022, 119(52 e2203894119

[11] Chidambaranathan-Reghupaty S, Fisher PB, Sarkar D. Hepatocellular carcinoma (HCC): epidemiology, etiology and molecular classification. Adv Cancer Res, 2021, 149: 1-61

[12] Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep, 2017, 7: 42717

[13] Eferl R, Ricci R, Kenner L, Zenz R, David JP, Rath M, Wagner EF. Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell, 2003, 112(2): 181-192

[14] Gao H, Sun M, Li A, Gu Q, Kang D, Feng Z, Li X, Wang X, Chen L, Yang H, Cong Y, Liu Z. Microbiota-derived IPA alleviates intestinal mucosal inflammation through upregulating Th1/Th17 cell apoptosis in inflammatory bowel disease. Gut Microbes, 2025, 17(1): 2467235

[15] Gaul S, Leszczynska A, Alegre F, Kaufmann B, Johnson CD, Adams LA, Wree A, Damm G, Seehofer D, Calvente CJ, Povero D, Kisseleva T, Eguchi A, McGeough MD, Hoffman HM, Pelegrin P, Laufs U, Feldstein AE. Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. J Hepatol, 2021, 74(1): 156-167

[16] Gong W, Sun P, Li X, Wang X, Zhang X, Cui H, Yang J. Investigating the molecular mechanisms of resveratrol in treating cardiometabolic multimorbidity: a network pharmacology and bioinformatics approach with molecular docking validation. Nutrients, 2024, 16(15): 2488

[17] Hong M, Lee S, Clayton J, Yake W, Li J. Genipin suppression of growth and metastasis in hepatocellular carcinoma through blocking activation of STAT-3. J Exp Clin Cancer Res, 2020, 39(1): 146

[18] Hopkins AL. Network pharmacology. Nat Biotechnol, 2007, 25(10): 1110-1111

[19] Hsu CL, Schnabl B. The gut-liver axis and gut microbiota in health and liver disease. Nat Rev Microbiol, 2023, 21(11): 719-733

[20] Hu P, Song W, Shan Y, Du M, Huang M, Song C, Zhang L. Lactobacillus paracasei subsp. paracasei M5L induces cell cycle arrest and calreticulin translocation via the generation of reactive oxygen species in HT-29 cell apoptosis. Food Funct, 2015, 6(7): 2257-2265

[21] Hu C, Xu B, Wang X, Wan WH, Lu J, Kong D, Jin Y, You W, Sun H, Mu X, Feng D, Chen Y. Gut microbiota-derived short-chain fatty acids regulate group 3 innate lymphoid cells in HCC. Hepatology (Baltimore, MD), 2023, 77(1): 48-64

[22] Jia D, Wang Q, Qi Y, Jiang Y, He J, Lin Y, Sun Y, Xu J, Chen W, Fan L, Yan R, Zhang W, Ren G, Xu C, Ge Q, Wang L, Liu W, Xu F, Wu P, Wang Y, Chen S, Wang L. Microbial metabolite enhances immunotherapy efficacy by modulating T cell stemness in pan-cancer. Cell, 2024, 187(7): 1651-1665 e1621

[23] Kang Y, Cai Y, Yang Y. The gut microbiome and hepatocellular carcinoma: implications for early diagnostic biomarkers and novel therapies. Liver Cancer (Basel), 2022, 11(2): 113-125

[24] Kurlishchuk Y, Cindric Vranesic A, Jessen M, Kipping A, Ritter C, Kim K, Cramer P, von Eyss B. A non-canonical repressor function of JUN restrains YAP activity and liver cancer growth. EMBO J, 2024, 43(20): 4578-4603

[25] Li C, Tang Z, Zhang W, Ye Z, Liu F. GEPIA2021: integrating multiple deconvolution-based analysis into GEPIA. Nucleic Acids Res, 2021, 49(W1): W242-W246

[26] Li N, Yamamoto G, Fuji H, Kisseleva T. Interleukin-17 in liver disease pathogenesis. Semin Liver Dis, 2021, 41(4): 507-515

[27] Li S, Duan Y, Luo S, Zhou F, Wu Q, Lu Z. Short-chain fatty acids and cancer. Trends Cancer, 2025, 11(2): 154-168

[28] Liu B, Zhou Z, Jin Y, Lu J, Feng D, Peng R, Sun H, Mu X, Li C, Chen Y. Hepatic stellate cell activation and senescence induced by intrahepatic microbiota disturbances drive progression of liver cirrhosis toward hepatocellular carcinoma. J Immunother Cancer, 2022

[29] Liu Y, Yang X, Gan J, Chen S, Xiao ZX, Cao Y. CB-Dock2: improved protein-ligand blind docking by integrating cavity detection, docking and homologous template fitting. Nucleic Acids Res, 2022, 50(W1): W159-W164

[30] Lv Y, Jiang M, Ouyang Y, Zheng X, Zhang L, Yao J, Hu L, Zhao J, Li Z, Wang S. Sodium butyrate-loaded microspheres with enhanced bioavailability for targeted treatment of intestinal barrier injury. Adv Healthc Mater, 2025, 14(9 e2402773

[31] Ma HY, Yamamoto G, Xu J, Liu X, Karin D, Kim JY, Alexandrov LB, Koyama Y, Nishio T, Benner C, Heinz S, Rosenthal SB, Liang S, Sun M, Karin G, Zhao P, Brodt P, McKillop IH, Quehenberger O, Dennis E, Saltiel A, Tsukamoto H, Gao B, Karin M, Brenner DA, Kisseleva T. IL-17 signaling in steatotic hepatocytes and macrophages promotes hepatocellular carcinoma in alcohol-related liver disease. J Hepatol, 2020, 72(5): 946-959

[32] Ma H, Yang L, Liang Y, Liu F, Hu J, Zhang R, Li Y, Yuan L, Feng F. B. thetaiotaomicron-derived acetic acid modulate immune microenvironment and tumor growth in hepatocellular carcinoma. Gut Microbes, 2024, 16(1): 2297846

[33] Mann ER, Lam YK, Uhlig HH. Short-chain fatty acids: linking diet, the microbiome and immunity. Nat Rev Immunol, 2024, 24(8): 577-595

[34] McDonald TA, Holland NT, Skibola C, Duramad P, Smith MT. Hypothesis: phenol and hydroquinone derived mainly from diet and gastrointestinal flora activity are causal factors in leukemia. Leukemia, 2001, 15(1): 10-20

[35] Nie Q, Luo X, Wang K, Ding Y, Jia S, Zhao Q, Li M, Zhang J, Zhuo Y, Lin J, Guo C, Zhang Z, Liu H, Zeng G, You J, Sun L, Lu H, Ma M, Jia Y, Zheng MH, Pang Y, Qiao J, Jiang C. Gut symbionts alleviate MASH through a secondary bile acid biosynthetic pathway. Cell, 2024, 187(11): 2717-2734 e2733

[36] Nogales C, Mamdouh ZM, List M, Kiel C, Casas AI, Schmidt H. Network pharmacology: curing causal mechanisms instead of treating symptoms. Trends Pharmacol Sci, 2022, 43(2): 136-150

[37] Pang Z, Lu Y, Zhou G, Hui F, Xu L, Viau C, Spigelman AF, MacDonald PE, Wishart DS, Li S, Xia J. MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res, 2024, 52(W1): W398-W406

[38] Patitucci C, Couchy G, Bagattin A, Caneque T, de Reynies A, Scoazec JY, Rodriguez R, Pontoglio M, Zucman-Rossi J, Pende M, Panasyuk G. Hepatocyte nuclear factor 1alpha suppresses steatosis-associated liver cancer by inhibiting PPARgamma transcription. J Clin Invest, 2017, 127(5): 1873-1888

[39] Pinzi L, Rastelli G. Molecular docking: shifting paradigms in drug discovery. Int J Mol Sci, 2019

[40] Ponziani FR, Bhoori S, Castelli C, Putignani L, Rivoltini L, Del Chierico F, Sanguinetti M, Morelli D, Paroni Sterbini F, Petito V, Reddel S, Calvani R, Camisaschi C, Picca A, Tuccitto A, Gasbarrini A, Pompili M, Mazzaferro V. Hepatocellular carcinoma is associated with gut microbiota profile and inflammation in nonalcoholic fatty liver disease. Hepatology, 2019, 69(1): 107-120

[41] Sangro B, et al.. EASL clinical practice guidelines on the management of hepatocellular carcinoma. J Hepatol, 2025, 82(2): 315-374

[42] Schwabe RF, Greten TF. Gut microbiome in HCC - Mechanisms, diagnosis and therapy. J Hepatol, 2020, 72(2): 230-238

[43] Scott SA, Fu J, Chang PV. Microbial tryptophan metabolites regulate gut barrier function via the aryl hydrocarbon receptor. Proc Natl Acad Sci U S A, 2020, 117(32): 19376-19387

[44] Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res, 2003, 13(11): 2498-2504

[45] Tilg H, Adolph TE, Trauner M. Gut-liver axis: pathophysiological concepts and clinical implications. Cell Metab, 2022, 34(11): 1700-1718

[46] To JC, Chiu AP, Tschida BR, Lo LH, Chiu CH, Li XX, Kuka TP, Linden MA, Amin K, Chan WC, Bell JB, Moriarity BS, Largaespada DA, Keng VW. ZBTB20 regulates WNT/CTNNB1 signalling pathway by suppressing PPARG during hepatocellular carcinoma tumourigenesis. JHEP Rep, 2021, 3(2): 100223

[47] Tripathi A, Debelius J, Brenner DA, Karin M, Loomba R, Schnabl B, Knight R. The gut-liver axis and the intersection with the microbiome. Nat Rev Gastroenterol Hepatol, 2018, 15(7): 397-411

[48] Wang R, Tang R, Li B, Ma X, Schnabl B, Tilg H. Gut microbiome, liver immunology, and liver diseases. Cell Mol Immunol, 2021, 18(1): 4-17

[49] Wang X, Zhang B, Jiang R. Microbiome interplays in the gut-liver axis: implications for liver cancer pathogenesis and therapeutic insights. Front Cell Infect Microbiol, 2025, 15: 1467197

[50] Wang Y, Peng X, Qian B, Wang L, Wang J. The integration of metabolites from Forsythia suspensa and gut microbiota ameliorates drug-induced liver injury: network pharmacology and molecular docking studies. Artif Cells Nanomed Biotechnol, 2025, 53(1): 105-121

[51] Wang Y, Wang X, Zhang X, Pan M, Sun M, Chen Z, Song Y. Utilizing network pharmacology and other tools to examine active components and mechanism of action of Magnolia officinalis rheum rhabarbarum decoction in treating Streptococcus pyogenes skin infections. Bioresour Bioprocess, 2025, 12(1): 92

[52] Xia J, Ding H, Liu S, An R, Shi X, Chen M, Ren H. C-type lectin receptors-triggered antifungal immunity may synergize with and optimize the effects of immunotherapy in hepatocellular carcinoma. J Inflamm Res, 2023, 16: 19-33

[53] Yao J, Ning B, Ding J. The gut microbiota: an emerging modulator of drug resistance in hepatocellular carcinoma. Gut Microbes, 2025, 17(1): 2473504

[54] Yao W, Huo J, Liu K, Tao P. Unveiling the therapeutic potential of gut microbiota metabolites for the treatment of renal fibrosis based on network pharmacology study. Bioresour Bioprocess, 2026, 13(1): 94

[55] Yeo YH, Abdelmalek M, Khan S, Moylan CA, Rodriquez L, Villanueva A, Yang JD. Current and emerging strategies for the prevention of hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol, 2025, 22(3): 173-190

[56] Yu J, Zhu P, Shi L, Gao N, Li Y, Shu C, Xu Y, Yu Y, He J, Guo D, Zhang X, Wang X, Shao S, Dong W, Wang Y, Zhang W, Zhang W, Chen WH, Chen X, Liu Z, Yang X, Zhang B. Bifidobacterium longum promotes postoperative liver function recovery in patients with hepatocellular carcinoma. Cell Host Microbe, 2024, 32(1): 131-144 e136

[57] Yu Q, Zhu L, Ding X, Lou Y. Integration of network pharmacology and experimental validation to explore the pharmacological mechanism of andrographolide against asthma. Bioresour Bioprocess, 2025, 12(1): 30

[58] Zhan X, Wu R, Kong XH, You Y, He K, Sun XY, Huang Y, Chen WX, Duan L. Elevated neutrophil extracellular traps by HBV-mediated S100A9-TLR4/RAGE-ROS cascade facilitate the growth and metastasis of hepatocellular carcinoma. Cancer Commun (Lond), 2023, 43(2): 225-245

[59] Zhang X, Coker OO, Chu ES, Fu K, Lau HCH, Wang YX, Chan AWH, Wei H, Yang X, Sung JJY, Yu J. Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites. Gut, 2021, 70(4): 761-774

[60] Zhao L, Zhang H, Li N, Chen J, Xu H, Wang Y, Liang Q. Network pharmacology, a promising approach to reveal the pharmacology mechanism of Chinese medicine formula. J Ethnopharmacol, 2023, 309: 116306

Funding

Guangdong Basic and Applied Basic Research (2024A1515030013)

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