Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol-Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

Abstract

Gastric mucosal integrity is essential for maintaining systemic homeostasis, serving as the primary defense against external insults. Ethanol ingestion is a major clinical cause of gastric mucosal injury, yet effective prevention or treatment remains limited. This study investigates the protective role of nitrate against ethanol-induced gastric ulcers and its underlying mechanisms. In vivo, nitrate significantly ameliorated ethanol-induced gastric bleeding, edema, inflammation, and mucus layer thinning in rats, while strengthening the vascular endothelial barrier. Transcriptomic analyses and trefoil factor 2 (Tff2)-knockdown rats experiment identified Tff2 as the key gene responsible for mediating nitrate's protective effects against ethanol. In vitro, TFF2 was found to be a crucial target for nitrates, which enhance the migratory reparative capacities of human gastric epithelial cells. Further assays revealed that RBPJ regulates the TFF2 promoter, and NICD–RBPJ complex formation is critical for TFF2 transcriptional repression. We demonstrate for the first time that TFF2 is a central effector in nitrate-mediated gastric mucosal defense and repair and implicate the Notch signaling pathway in TFF2 regulation. These findings suggest nitrate exerts a protective effect on the gastric mucosa through multiple ways. TFF2 modulation as a potential preventive strategy for ethanol-induced gastric ulcers.

Keywords

epithelial restitution / ethanol / gastric mucosal barrier / migration / Notch / trefoil factor 2

References

[1] X. X. Wang, B. Dong, B. Hong, et al., “Long-term Prognosis in Patients Continuing Taking Antithrombotics After Peptic Ulcer Bleeding,” World Journal of Gastroenterology 23, no. 4 (2017): 723–729.

[2] C. Wang, Y. Yuan, H. Pan, et al., “Protective Effect of Ocotillol, the Derivate of Ocotillol-Type Saponins in Panax Genus, Against Acetic Acid-Induced Gastric Ulcer in Rats Based on Untargeted Metabolomics,” International Journal of Molecular Sciences 21, no. 7 (2020): 2577.

[3] T. Kawai, K. Oda, N. Funao, et al., “Vonoprazan Prevents Low-dose Aspirin-associated Ulcer Recurrence: Randomised Phase 3 Study,” Gut 67, no. 6 (2018): 1033–1041.

[4] R. Q. Yang, H. Mao, L. Y. Huang, P. Z. Su, and M. Lu, “Effects of Hydrotalcite Combined With Esomeprazole on Gastric Ulcer Healing Quality: A Clinical Observation Study,” World Journal of Gastroenterology 23, no. 7 (2017): 1268–1277.

[5] A. Lanas and F. K. L. Chan, “Peptic Ulcer Disease,” Lancet 390, no. 10094 (2017): 613–624.

[6] K. Bai, B. Hong, R. Tan, J. He, and Z. Hong, “Selenium Nanoparticles-Embedded Chitosan Microspheres and Their Effects Upon Alcohol-Induced Gastric Mucosal Injury in Rats: Rapid Preparation, Oral Delivery, and Gastroprotective Potential of Selenium Nanoparticles,” International Journal of Nanomedicine 15 (2020): 1187–1203.

[7] P. Dejban, F. Eslami, N. Rahimi, N. Takzare, M. Jahansouz, and A. R. Dehpour, “Involvement of Nitric Oxide Pathway in the Anti-inflammatory Effect of modafinil on Indomethacin-, Stress-, and Ethanol -induced Gastric Mucosal Injury in Rat,” European Journal of Pharmacology 887 (2020): 173579.

[8] K. Pohl, P. Moodley, and A. D. Dhanda, “Alcohol's Impact on the Gut and Liver,” Nutrients 13, no. 9 (2021): 3170.

[9] T. Smith, E. G. DeMaster, J. K. Furne, J. Springfield, and M. D. Levitt, “First-pass Gastric Mucosal Metabolism of Ethanol Is Negligible in the Rat,” Journal of Clinical Investigation 89, no. 6 (1992): 1801–1806.

[10] B. Tian, Q. Zhao, H. Xing, et al., “Gastroprotective Effects of Ganoderma Lucidum Polysaccharides With Different Molecular Weights on Ethanol-Induced Acute Gastric Injury in Rats,” Nutrients 14, no. 7 (2022): 1476.

[11] B. Crotty, “Ethanol and Upper Gastrointestinal Bleeding,” American Journal of Gastroenterology 90, no. 7 (1995): 1038–1039.

[12] R. Yang, J. Li, X. Xu, K. Xu, and J. Shi, “Preventive and Therapeutic Effects of Lactobacillus Rhamnosus SHA113 and Its Culture Supernatant on Alcoholic Gastric Ulcers,” Food & Function 12, no. 16 (2021): 7250–7259.

[13] K. Satoh, J. Yoshino, T. Akamatsu, et al., “Evidence-based Clinical Practice Guidelines for Peptic Ulcer Disease 2015,” Journal of Gastroenterology 51, no. 3 (2016): 177–194.

[14] J. Henriksnäs, M. Phillipson, M. Storm, L. Engstrand, M. Soleimani, and L. Holm, “Impaired Mucus-bicarbonate Barrier in Helicobacter pylori-infected Mice,” American Journal of Physiology Gastrointestinal and Liver Physiology 291, no. 3 (2006): G396–G403.

[15] S. Oncel and M. D. Basson, “Gut Homeostasis, Injury, and Healing: New Therapeutic Targets,” World Journal of Gastroenterology 28, no. 17 (2022): 1725–1750.

[16] J. Matúz, “Role of Mucus in Mucosal Protection Through Ethanol and Pepsin Damage Models,” Acta Physiologica Hungarica 80, no. 1-4 (1992): 189–194.

[17] B. Liu, X. Feng, J. Zhang, Y. Wei, and X. Zhao, “Preventive Effect of Anji White Tea Flavonoids on Alcohol-Induced Gastric Injury Through Their Antioxidant Effects in Kunming Mice,” Biomolecules 9, no. 4 (2019): 137.

[18] Y. Zhan, W. Su, and X. Liu, “Gut Microbiota Dysbiosis: The Hidden Roles in human Aging and Age-related Diseases,” Oral Science and Homeostatic Medicine 1, no. 1 (2025).

[19] A. Franke, S. Teyssen, and M. V. Singer, “Alcohol-related Diseases of the Esophagus and Stomach,” Digestive Diseases 23, no. 3-4 (2005): 204–213.

[20] S. Szabo, J. S. Trier, A. Brown, and J. Schnoor, “Early Vascular Injury and Increased Vascular Permeability in Gastric Mucosal Injury Caused by Ethanol in the Rat,” Gastroenterology 88, no. 1 Pt 2 (1985): 228–236.

[21] I. T. Beck, G. P. Morris, and M. G. Buell, “Ethanol-induced Vascular Permeability Changes in the Jejunal Mucosa of the Dog,” Gastroenterology 90, no. 5 Pt 1 (1986): 1137–1145.

[22] H. P. Parkman, W. L. Hasler, and R. S. Fisher, “American Gastroenterological Association Technical Review on the Diagnosis and Treatment of Gastroparesis,” Gastroenterology 127, no. 5 (2004): 1592–1622.

[23] F. Hoelzel and E. Da Costa, “Experimental Production of Pylorospasm and Gastric Retention in Rats,” Journal of Experimental Medicine 57, no. 4 (1933): 597–615.

[24] D. Taupin and D. K. Podolsky, “Trefoil Factors: Initiators of Mucosal Healing,” Nature Reviews Molecular Cell Biology 4, no. 9 (2003): 721–732.

[25] E. R. Lacy, “Epithelial Restitution in the Gastrointestinal Tract,” Journal of Clinical Gastroenterology 10 Suppl 1 (1988): S72–S77.

[26] W. Hoffmann, “Trefoil Factors TFF (trefoil factor family) Peptide-triggered Signals Promoting Mucosal Restitution,” Cellular and Molecular Life Sciences 62, no. 24 (2005): 2932–2938.

[27] W. Silen and S. Ito, “Mechanisms for Rapid Re-epithelialization of the Gastric Mucosal Surface,” Annual Review of Physiology 47 (1985): 217–229.

[28] L. Ma, L. Hu, X. Feng, and S. Wang, “Nitrate and Nitrite in Health and Disease,” Aging and Disease 9, no. 5 (2018): 938–945.

[29] J. Zhou, H. Liu, L. Hu, H. Kagami, and S. Wang, “Nitrate and Body Homeostasis,” Medicine Plus 1, no. 1 (2024): 100003.

[30] S. Wang and L. Qin, “Homeostatic Medicine: A Strategy for Exploring Health and Disease,” Current Medicinal Chemistry 1, no. 1 (2022): 16.

[31] J. O. Lundberg, M. Carlström, and E. Weitzberg, “Metabolic Effects of Dietary Nitrate in Health and Disease,” Cell Metabolism 28, no. 1 (2018): 9–22.

[32] X. Chen, G. Hu, L. Chang, et al., “Nitrate Ameliorates Myelin Loss and Cognitive Impairment in Alzheimer's disease Through Upregulation of Neuronal Sialin and Subsequent Inhibition of TPPP Phosphorylation,” Science Bulletin (Beijing) 70, no. 8 (2025): 1224–1229.

[33] X. Li, L. Hu, X. Wang, et al., “Salivary Nitrate Prevents Osteoporosis via Regulating Bone Marrow Mesenchymal Stem Cells Proliferation and Differentiation,” Journal of Orthopaedic Translation 45 (2024): 188–196.

[34] J. Gu, Z. Zhou, S. Xu, et al., “Topical Application of Nitrate Ameliorates Skin Fibrosis by Regulating ST2(+)CD4(+) T Cells in Systemic Sclerosis Mouse Model,” Journal of Investigative Dermatology 145, no. 2 (2025): 346–358.e5.

[35] S. Li, Y. Wang, Z. Zhang, et al., “Sodium Nitrate Protects Against Metabolic Syndrome by Sialin-mediated Macrophage Rebalance,” Signal Transduction and Targeted Therapy 10, no. 1 (2025): 323.

[36] S. Li, H. Jin, and G. Sun, “Dietary Inorganic Nitrate Protects Hepatic Ischemia-Reperfusion Injury through NRF2-Mediated Antioxidative Stress,” Frontiers in Pharmacology 12 (2021): 634115.

[37] W. Pan, G. Hu, S. Li, et al., “Nanonitrator: Novel Enhancer of Inorganic Nitrate's Protective Effects, Predicated on Swarm Learning Approach,” Science Bulletin (Beijing) 68, no. 8 (2023): 838–850.

[38] X. Wen, Y. Liu, C. Zhang, et al., “Nitrate Protects Against Methotrexate-induced Liver Injury by Activating Wnt/β-catenin Signaling in Mice,” Toxicology Research (Camb) 14, no. 4 (2025): tfaf107.

[39] Y. Xu, Y. Sa, C. Zhang, et al., “A Preventative Role of Nitrate for Hypoxia-induced Intestinal Injury,” Free Radical Biology and Medicine 213 (2024): 457–469.

[40] L. Hu, L. Jin, and D. Xia, “Nitrate Ameliorates Dextran Sodium Sulfate-induced Colitis by Regulating the Homeostasis of the Intestinal Microbiota,” Free Radical Biology and Medicine 152 (2020): 609–621.

[41] J. Petersson, C. Jädert, M. Phillipson, S. Borniquel, J. O. Lundberg, and L. Holm, “Physiological Recycling of Endogenous Nitrate by Oral Bacteria Regulates Gastric Mucus Thickness,” Free Radical Biology and Medicine 89 (2015): 241–247.

[42] J. Petersson, M. Phillipson, E. A. Jansson, A. Patzak, J. O. Lundberg, and L. Holm, “Dietary Nitrate Increases Gastric Mucosal Blood Flow and Mucosal Defense,” American Journal of Physiology Gastrointestinal and Liver Physiology 292, no. 3 (2007): G718–G724.

[43] E. A. Jansson, J. Petersson, C. Reinders, et al., “Protection From Nonsteroidal Anti-inflammatory Drug (NSAID)-induced Gastric Ulcers by Dietary Nitrate,” Free Radical Biology and Medicine 42, no. 4 (2007): 510–518.

[44] L. Jin, L. Qin, D. Xia, et al., “Active Secretion and Protective Effect of Salivary Nitrate Against Stress in human Volunteers and Rats,” Free Radical Biology and Medicine 57 (2013): 61–67.

[45] M. Miyoshi, E. Kasahara, A. M. Park, et al., “Dietary Nitrate Inhibits Stress-induced Gastric Mucosal Injury in the Rat,” Free Radical Research 37, no. 1 (2003): 85–90.

[46] S. Chari, S. Teyssen, and M. V. Singer, “Alcohol and Gastric Acid Secretion in Humans,” Gut 34, no. 6 (1993): 843–847.

[47] H. M. Hassan, N. M. Alatawi, A. Bagalagel, et al., “Genistein Ameliorated Experimentally Induced Gastric Ulcer in Rats via Inhibiting Gastric Tissues Fibrosis by Modulating Wnt/β-catenin/TGF-β/PKB Pathway,” Redox Report: Communications in Free Radical Research 28, no. 1 (2023): 2218679.

[48] L. Li, R. Rispoli, R. Patient, A. Ciau-Uitz, and C. Porcher, “Etv6 activates Vegfa Expression Through Positive and Negative Transcriptional Regulatory Networks in Xenopus Embryos,” Nature Communications 10, no. 1 (2019): 1083.

[49] R. S. Aziz, A. Siddiqua, M. Shahzad, A. Shabbir, and N. Naseem, “Oxyresveratrol Ameliorates Ethanol-induced Gastric Ulcer via Downregulation of IL-6, TNF-α, NF-ĸB, and COX-2 Levels, and Upregulation of TFF-2 Levels,” Biomedicine & Pharmacotherapy 110 (2019): 554–560.

[50] Y. Z. Lian, I. H. Lin, Y. C. Yang, and J. C. Chao, “Gastroprotective Effect of Lycium Barbarum Polysaccharides and C-phycocyanin in Rats With Ethanol-induced Gastric Ulcer,” International Journal of Biological Macromolecules 165, no. Pt A (2020): 1519–1528.

[51] L. Feng, L. A, and T. Bao, “An Integrated Network Analysis, RNA-seq and in Vivo Validation Approaches to Explore the Protective Mechanism of Mongolian Medicine Formulae Ruda-6 Against Indomethacin-induced Gastric Ulcer in Rats,” Frontiers in Pharmacology 14 (2023): 1181133.

[52] A. K. Sauer, J. Bockmann, K. Steinestel, T. M. Boeckers, and A. M. Grabrucker, “Altered Intestinal Morphology and Microbiota Composition in the Autism Spectrum Disorders Associated SHANK3 Mouse Model,” International Journal of Molecular Sciences 20, no. 9 (2019): 2134.

[53] E. Aihara, N. M. Medina-Candelaria, H. Hanyu, et al., “Cell Injury Triggers Actin Polymerization to Initiate Epithelial Restitution,” Journal of Cell Science 131, no. 16 (2018): jcs216317.

[54] Y. Jin and A. T. Blikslager, “The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases,” International Journal of Molecular Sciences 21, no. 10 (2020): 3550.

[55] K. E. Cunningham and J. R. Turner, “Myosin Light Chain Kinase: Pulling the Strings of Epithelial Tight Junction Function,” Annals of the New York Academy of Sciences 1258, no. 1 (2012): 34–42.

[56] I. Tan and T. Leung, “Myosin Light Chain Kinases: Division of Work in Cell Migration,” Cell Adhesion & Migration 3, no. 3 (2009): 256–258.

[57] B. D. Chen, C. H. He, X. C. Chen, et al., “Targeting Transgene to the Heart and Liver With AAV9 by Different Promoters,” Clinical and Experimental Pharmacology & Physiology 42, no. 10 (2015): 1108–1117.

[58] C. Hou, L. Liu, J. Ren, M. Huang, and E. Yuan, “Structural Characterization of Two Hericium Erinaceus Polysaccharides and Their Protective Effects on the Alcohol-induced Gastric Mucosal Injury,” Food Chemistry 375 (2022): 131896.

[59] K. Lin, T. Deng, H. Qu, et al., “Gastric Protective Effect of Alpinia officinarum Flavonoids: Mediating TLR4/NF-κB and TRPV1 Signalling Pathways and Gastric Mucosal Healing,” Pharmaceutical Biology 61, no. 1 (2023): 50–60.

[60] Y. Ke, L. Zhan, T. Lu, et al., “Polysaccharides of Dendrobium Officinale Kimura & Migo Leaves Protect against Ethanol-Induced Gastric Mucosal Injury via the AMPK/mTOR Signaling Pathway in Vitro and Vivo,” Frontiers in Pharmacology 11 (2020): 526349.

[61] M. Xie, H. Chen, S. Nie, W. Tong, J. Yin, and M. Xie, “Gastroprotective Effect of Gamma-aminobutyric Acid Against Ethanol-induced Gastric Mucosal Injury,” Chemico-Biological Interactions 272 (2017): 125–134.

[62] L. Liu, L. Zhang, S. Zhao, et al., “Non-Canonical Notch Signaling Regulates Actin Remodeling in Cell Migration by Activating PI3K/AKT/Cdc42 Pathway,” Frontiers in pharmacology 10 (2019): 370.

[63] B. Zhou, W. Lin, and Y. Long, “Notch Signaling Pathway: Architecture, Disease, and Therapeutics,” Signal Transduction and Targeted Therapy 7, no. 1 (2022): 95.

[64] B. Swaminathan, S. W. Youn, L. A. Naiche, et al., “Endothelial Notch Signaling Directly Regulates the Small GTPase RND1 to Facilitate Notch Suppression of Endothelial Migration,” Scientific Reports 12, no. 1 (2022): 1655.

[65] R. J. Playford, T. Marchbank, R. Chinery, et al., “Human Spasmolytic Polypeptide Is a Cytoprotective Agent That Stimulates Cell Migration,” Gastroenterology 108, no. 1 (1995): 108–116.

[66] T.-Y. Du, Y. Zhang, and Y. Zhang, “Trefoil Factor: From Laboratory to Clinic,” Zoological Research 31, no. 1 (2010): 17–26.

[67] J. A. Clara, C. Monge, Y. Yang, and N. Takebe, “Targeting Signalling Pathways and the Immune Microenvironment of Cancer Stem Cells—A Clinical Update,” Nature Reviews Clinical Oncology 17, no. 4 (2020): 204–232.

[68] N. Takebe, D. Nguyen, and S. X. Yang, “Targeting Notch Signaling Pathway in Cancer: Clinical Development Advances and Challenges,” Pharmacology & Therapeutics 141, no. 2 (2014): 140–149.

[69] W. Fischbach and P. Malfertheiner, “Helicobacter Pylori Infection,” Deutsches Ärzteblatt International 115, no. 25 (2018): 429–436.

[70] T. Xu, S. S. Park, B. D. Giaimo, et al., “RBPJ/CBF1 Interacts With L3MBTL3/MBT1 to Promote Repression of Notch Signaling via Histone Demethylase KDM1A/LSD1,” The EMBO Journal 36, no. 21 (2017): 3232–3249.

[71] L. Pan, P. Hoffmeister, A. Turkiewicz, et al., “Transcription Factor RBPJL Is Able to Repress Notch Target Gene Expression but Is Non-Responsive to Notch Activation,” Cancers (Basel) 13, no. 19 (2021): 5027.

[72] S. G. E. Shams and R. G. Eissa, “Amelioration of Ethanol-induced Gastric Ulcer in Rats by Quercetin: Implication of Nrf2/HO1 and HMGB1/TLR4/NF-κB Pathways,” Heliyon 8, no. 10 (2022): e11159.

[73] A. M. Jones, “Dietary Nitrate Supplementation and Exercise Performance,” Sports Medicine (Auckland, NZ) 44 Suppl 1, no. Suppl 1 (2014): S35–S45.

[74] A. K. Ganguly, “A Method for Quantitative Assessment of Experimentally Produced Ulcers in the Stomach of Albino Rats,” Experientia 25, no. 11 (1969): 1224.

[75] L. M. Hellman and M. G. Fried, “Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-nucleic Acid Interactions,” Nature Protocols 2, no. 8 (2007): 1849–1861.

RIGHTS & PERMISSIONS

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.