Inhibition of microRNA-155 regulates gastric mucosal barrier repair and inflammation by targeting SOCS1 for the treatment of acute gastritis

Inhibition of microRNA-155 regulates gastric mucosal barrier repair and inflammation by targeting SOCS1 for the treatment of acute gastritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Inhibition of microRNA-155 regulates gastric mucosal barrier repair and inflammation by targeting SOCS1 for the treatment of acute gastritis Lei Luo, Kai Zhang, Siyu Mu, Shuangyong Liu, Wenjing Xiao, Xiaolei Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6971173/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 5 You are reading this latest preprint version Abstract Acute gastritis is a common gastrointestinal disorder characterized by rapid onset of mucosal injury and infiltration of inflammatory cells, often induced by chemical irritants such as ethanol. Although inflammation is essential for mucosal defense and repair, excessive immune responses can lead to tissue damage and impaired healing. MicroRNAs (miRNAs), particularly those involved in immune regulation, have emerged as important modulators in various inflammatory conditions. However, their specific roles in the early phase of acute gastric inflammation remain unclear, and the mechanisms by which they influence mucosal damage are still not fully understood. In this study, we investigated the expression and functional significance of microRNA-155 (miR-155) in a mouse model of ethanol-induced acute gastritis. Our results showed that miR-155 expression was significantly upregulated in gastric mucosa following ethanol exposure, concomitant with elevated levels of pro-inflammatory cytokines including TNF-α and IL-6. Histological examination revealed that inhibition of miR-155 using a specific antagomir reduced inflammatory cell infiltration and attenuated mucosal injury. These findings suggest that miR-155 contributes to the exacerbation of acute gastric inflammation by promoting cytokine expression and epithelial damage. Importantly, this study highlights the role of miR-155 as an early-response regulator during acute gastric injury. Targeting miR-155 may offer a novel therapeutic strategy for the prevention or treatment of acute gastric mucosal damage. Furthermore, our findings provide a foundation for future exploration of miRNA-based interventions in inflammation-related gastrointestinal diseases. Biological sciences/Biochemistry/Cytokines Biological sciences/Biochemistry/Histocytochemistry miR-155 inflammation cytokines acute gastritis Ethanol-induced injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Acute gastritis is a common gastrointestinal disorder characterized by sudden onset of mucosal congestion, edema, and infiltration of inflammatory cells in the gastric lining (Mihaly et al., 2014 ). It typically develops rapidly in response to chemical irritants such as excessive alcohol consumption, nonsteroidal anti-inflammatory drugs (NSAIDs), or bacterial toxins. These triggers disrupt the gastric epithelial barrier, leading to mucosal hemorrhage and local immune activation, accompanied by a sharp increase in pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). While acute gastritis is often self-limiting, repeated or unresolved episodes may progress to chronic atrophic gastritis, peptic ulcers, or even gastric cancer (Taylor, 1969 ; Yang & Hu, 2022 ). Thus, understanding the early immunological events that underline mucosal inflammation is crucial for identifying novel targets for prevention and therapy. MicroRNAs (miRNAs) are short, non-coding RNA molecules that regulate gene expression at the post-transcriptional level, primarily through base-pairing with the 3′ untranslated regions (3′UTRs) of target mRNAs (Rupaimoole & Slack, 2017 ). Over the past decade, their roles in shaping gastrointestinal immune responses and preserving mucosal homeostasis have gained significant research interest (Bi et al., 2020 ; Rawat et al., 2025 ). Dysregulation of miRNA expression has been implicated in several inflammatory and neoplastic diseases (Hussen et al., 2021 ), including inflammatory bowel disease (IBD), Helicobacter pylori –associated gastritis, and colorectal cancer (Larabi et al., 2020 ; Mohamed et al., 2025 ). These regulatory RNAs exert their effects largely through modulating classic inflammatory pathways such as NF-κB, JAK/STAT, and MAPK (Hoesel & Schmid, 2013 ). Among them, miR-155 has emerged as a prototypical pro-inflammatory miRNA that is rapidly induced by TLR signaling, cytokine stimulation, or microbial infection (Elton et al., 2013 ; Leng et al., 2011 ; O'Connell et al., 2007 ). It promotes inflammatory responses by suppressing key negative regulators such as SOCS1 (Li et al., 2013 ; Prieto et al., 2023 ; Rao et al., 2014 ) and SHIP1(O'Connell et al., 2009 ), thereby contributing to macrophage activation, T cell differentiation, and cytokine amplification (Huang et al., 2013 ; O'Connell et al., 2009 ; Renrick et al., 2021 ). Although the role of miR-155 has been extensively studied in chronic inflammatory conditions (Jafarzadeh et al., 2021 ; Su et al., 2017 ), its involvement in acute gastric inflammation—particularly during the early phase of chemically induced injury—remains poorly defined (Wang et al., 2016 ; X. Yu et al., 2024 ). It is still unclear whether miR-155 participates in the initiation or amplification of mucosal inflammation in acute gastritis, and if so, through which downstream effectors. To address these questions, we employed a well-established HCl/ethanol-induced mouse model of acute gastritis, complemented by in vitro stimulation of human gastric epithelial cells, to investigate the expression dynamics, regulatory functions, and molecular targets of miR-155. Our study aims to elucidate the potential contribution of miR-155 in acute gastric injury and provide mechanistic insight that may guide future anti-inflammatory interventions. 2. Material and methods 2.1. Materials Human gastric epithelial GES-1 cells were obtained from ATCC (USA). Cell culture reagents, including RPMI-1640 medium, fetal bovine serum (FBS), Trypsin-EDTA, and penicillin-streptomycin, were sourced from commercial suppliers such as Gibco and Cytiva Hyclone. Phosphate-buffered saline (PBS) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. miR-155-related oligonucleotides (mimic, inhibitor, and negative controls) were synthesized by GenePharma (China). Antibodies against IKKα, IκBα, their phosphorylated forms, GAPDH, and HRP-conjugated secondary antibodies were provided by Cell Signaling Technology. Additional reagents used included TRIzol and Lipofectamine 3000 (Invitrogen), SYBR Green PCR Master Mix (Applied Biosystems), ELISA kits (R&D Systems), and the RNA immunoprecipitation kit along with chemiluminescent substrate (Millipore). 2.2. Cell culture GES-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic solution. Cells were maintained at 37°C in a humidified incubator containing 5% CO₂. 2.3. RNA Isolation and qRT-PCR Total RNA was extracted from gastric tissues and GES-1 cells using a standard phenol-based method. RNA concentration and purity were assessed using a microvolume spectrophotometer. cDNA synthesis was performed with a commercial reverse transcription kit, and quantitative PCR was carried out using SYBR Green dye on a real-time PCR system. Gene expression was normalized to internal controls (U6 or GAPDH), and relative levels were calculated using the 2^−ΔΔCt method. (All primer sequences used for qPCR are listed in Table 1.) 2.4. miRNA Mimics, Inhibitors, and Plasmid Transfection GES-1 cells were transfected with miR-155 mimic (5’-UUAAUGCUAAUCGUGAUAGGGGU-3’, 5’-ACCCCUAUCACGAUUAGCAUUA-3’), inhibitor (5'-ACCCCUAUCACGAUUAGCAUU-3'), or corresponding negative controls (5’-CAGUACUUUUUGUGUAGUACAA-3’, GenePharma) using a commercial lipid-based transfection reagent, according to the manufacturer's protocol. For rescue assays, cells were co-transfected with miR-155 mimic and a SOCS1-expressing plasmid (GenePharma), or with an empty vector. After 48 hours, cells were collected for subsequent analyses. 2.5. Cytokine Analysis (ELISA) Culture supernatants were collected, and in cytokine levels, including TNF-α, IL-1β, IL-6, and IL-10, were quantified by enzyme-linked immunosorbent assay using commercially available kits, following standard protocols. 2.6. Western Blot Analysis GES-1 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo Scientific). Protein concentrations were quantified using the BCA protein assay kit (Beyotime, China). Proteins (30 µg per lane) were separated by SDS-PAGE, transferred onto PVDF membranes, and blocked in 5% non-fat milk. Membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Protein bands were visualized using ECL substrate (Millipore). 2.7. RNA pulldown assay A biotin-labeled miR-155 probe (biotin-5’-ACCCCUAUCACGAUUAGCAUUA-3’) and a negative control probe (5’-UUCUCCGAACGUGUCACGUTT-3’, GenePharma). GES-1 cells were lysed in RNA binding buffer, and 1 mg of lysate was incubated with 1 µg of biotin-labeled RNA probe at 4°C overnight. Streptavidin magnetic beads (Thermo Fisher) were added to pull down RNA-protein complexes. The bound fractions were analyzed by Western blot to detect SOCS1 protein enrichment. 2.8. Animals and Gastritis Model Male C57BL/6 mice (8–10 weeks old) were housed under specific pathogen-free conditions with free access to food and water. To induce acute gastritis, mice were orally administered 0.2 mL of 150 mM HCl dissolved in 60% ethanol, while control animals received sterile saline. The study was approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Qingdao University (Approval No. ZYFYWZLL30239). All animal experiments, including euthanasia procedures, were performed in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020 Edition). For euthanasia, animals were exposed to carbon dioxide (CO₂) via a gradual displacement method in a closed chamber, with CO₂ delivered at a flow rate of 20–50% of the chamber volume per minute. This rate ensures unconsciousness occurs before CO₂ concentrations reach ~ 40% (the threshold associated with nociceptor activation), minimizing potential distress. Death was confirmed by the absence of respiratory movement, heartbeat (palpation), and pupillary reflexes; CO₂ flow was maintained for at least 1 minute after respiratory arrest to ensure euthanasia completion. Subsequent tissue collection (including stomach) was performed immediately post-confirmation of death. All methods in this study are reported in accordance with the ARRIVE guidelines (Boutron et al., 2020). 2.9. miR-155 Antagomir Administration miR-155 antagomir (5’-ACCCCUAUCACGAUUAGCAUU-3’, GenePharma) or scrambled control antagomir (5’-CAGUACUUUUUGUGUAGUACAA-3’, GenePharma) was dissolved in sterile saline and intravenously injected via tail vein at 45 mg/kg body weight (200 µL per mouse) on days 0, 2, and 4. Mice were sacrificed on day 5 for tissue analysis. 2.10. Histological and Macroscopic Evaluation Dissecting fresh gastric tissue, and gastric injury scores were evaluated based on hemorrhage, edema, epithelial exfoliation, and inflammatory cell infiltration. Macroscopic gastric lesions were photographed, visually scored and image J quantization. 2.11. Statistical Analysis Data are expressed as mean ± SEM from at least three independent experiments. Statistical analyses were performed using GraphPad Prism software (version 8.0). Differences between groups were evaluated by Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test, with a significance threshold set at P < 0.05. 3. Result 3.1 miR-155 is significantly upregulated in gastric inflammation both in vivo and in vitro miR-155 is one of the most extensively studied inflammation-related microRNAs and has been shown to be upregulated in various acute and chronic inflammatory models, where it modulates immune cell activation and cytokine production. However, its expression dynamics in acute gastric inflammation—particularly during the early epithelial response—remain poorly defined. In this study, we first examined miR-155 expression in a mouse model of acute gastritis induced by HCl/ethanol. qRT-PCR analysis revealed a significant elevation of miR-155 levels in the gastric mucosa 24 hours after induction compared to control mice (Fig. 1 A), suggesting a potential regulatory role in the inflammatory process. To further investigate whether similar changes occur in gastric epithelial cells, we established an in vitro inflammation model by treating human gastric epithelial GES-1 cells with lipopolysaccharide (LPS, 100 ng/mL) for 24hours. Consistent with our in vivo findings, LPS stimulation led to a marked increase in miR-155 expression (Fig. 1 B). Together, these results indicate that miR-155 is rapidly induced during the early phase of acute gastric inflammation and may play a critical role in initiating or amplifying epithelial immune responses. 3.2 miR-155 promotes pro-inflammatory cytokine expression in gastric epithelial cells The functional role of miR-155 in gastric epithelial inflammation was further explored by transfecting GES-1 cells with a miR-155 mimic or inhibitor. qRT-PCR analysis confirmed successful transfection, with miR-155 expression markedly increased in the mimic group and significantly reduced in the inhibitor group (Fig. 2 A). Functionally, overexpression of miR-155 significantly elevated the mRNA levels of several key pro-inflammatory cytokines and mediators, including tumor necrosis factor-alpha (TNF-α; Fig. 2 B), interleukin-1 beta (IL-1β; Fig. 2 C), interleukin-6 (IL-6; Fig. 2 D), interleukin-18 (IL-18; Fig. 2 E), and inducible nitric oxide synthase (iNOS; Fig. 2 F). Conversely, miR-155 inhibition led to a marked reduction in the expression of these genes compared with the respective negative control groups. These findings indicate that miR-155 functions as a positive regulator of inflammatory gene expression in gastric epithelial cells, potentially contributing to the amplification of inflammatory responses during acute gastritis. 3.3 miR-155 enhances LPS-induced cytokine production and activates NF-κB signaling in gastric epithelial cells To further investigate the mechanism by which miR-155 promotes inflammation in gastric epithelial cells, we assessed its effect on cytokine secretion and NF-κB signaling in LPS-stimulated GES-1 cells. ELISA analysis revealed that transfection with miR-155 mimic significantly enhanced the secretion of TNF-α, IL-1β, and IL-10 in response to LPS stimulation compared to the negative control group (Fig. 3 A–C). In contrast, the inhibition of miR-155 markedly reduced the levels of these cytokines following LPS exposure. We also examined the phosphorylation status of key components in the canonical NF-κB pathway. Western blot analysis showed that miR-155 overexpression increased the phosphorylation of IKKα and IκBα, whereas miR-155 inhibition suppressed these phosphorylation events (Fig. 3 D). Together, these results suggest that miR-155 enhances LPS-induced inflammatory responses in gastric epithelial cells, at least in part by activating the NF-κB signaling pathway. 3.4 SOCS1 is a direct functional target of miR-155 We next performed integrative bioinformatic analysis using the miRDB, TargetScan, and miRWalk databases to identify potential downstream targets of miR-155. A total of 154 overlapping candidate genes were identified across all three platforms (Fig. 4 A). Based on their known involvement in negative regulation of NF-κB signaling, SOCS1(J. Yu et al., 2024 ), FOSL2(Hu et al., 2014 ), XKR4, and HBP1 were selected for further validation (Li et al., 2021 ). qRT-PCR analysis revealed that miR-155 mimic transfection significantly reduced SOCS1 mRNA expression in GES-1 cells, while miR-155 inhibition restored SOCS1 levels (Fig. 4 B). In contrast, the expression of the other predicted targets showed no significant changes, supporting the specificity of the miR-155–SOCS1 interaction. Sequence alignment analysis further predicted a conserved binding site for miR-155 within the 3′ untranslated region (3′UTR) of SOCS1 (Fig. 4 C). To confirm the direct interaction between miR-155 and SOCS1 mRNA, an RNA pulldown assay was performed using a biotin-labeled miR-155 probe. GES-1 cells were lysed after co-transfection with either wild-type or mutant SOCS1 3′UTR constructs, and lysates were incubated with streptavidin-conjugated magnetic beads to capture the probe-associated complexes. SOCS1 protein was markedly enriched in the pulldown complex when co-transfected with wild-type 3′UTR, but not with the mutant lacking the predicted binding site (Fig. 4 D). These results provide strong evidence that SOCS1 is a direct and functional target of miR-155 in gastric epithelial cells. 3.5 miR-155 promotes inflammatory cytokine expression through SOCS1 suppression in GES-1 cells To evaluate whether the pro-inflammatory effects of miR-155 are mediated through suppression of SOCS1, we conducted a rescue experiment in GES-1 cells. Overexpression of miR-155 markedly increased mRNA levels of TNF-α, IL-1β, IL-6, and IL-8 (Fig. 5 B–E). However, these effects were substantially reversed by co-transfection with a SOCS1-expressing plasmid, which restored cytokine expression to near basal levels. qRT-PCR confirmed successful SOCS1 overexpression (Fig. 5 A). These findings indicate that miR-155 enhances inflammatory cytokine expression in gastric epithelial cells, at least in part, by downregulating SOCS1. The reversal of miR-155-mediated effects by SOCS1 restoration further supports the functional relevance of the miR-155/SOCS1 regulatory axis in epithelial inflammation. 3.6 Inhibition of miR-155 alleviates gastric inflammation in vivo In order to further evaluate the therapeutic potential of targeting miR-155 in vivo, we employed a well-established ethanol/HCl-induced acute gastritis mouse model. Mice administered a miR-155 antagomir via tail vein injection. Remarkably, systemic delivery of the antagomir significantly alleviated gastric mucosal hemorrhage and tissue injury, producing therapeutic effects comparable to those of ranitidine (Fig. 6 A). Gastric lesion scores were also markedly improved following treatment (Fig. 6 B). At the molecular level, gastric tissues from antagomir-treated mice exhibited a notable reduction in miR-155 expression along with restored SOCS1 levels (Fig. 6 C, D), confirming the protective effect of systemic miR-155 inhibition against acute gastric injury. 4. Conclusion In this study, we demonstrated that miR-155 expression was markedly elevated in ethanol-induced acute gastritis mice and LPS-stimulated gastric epithelial cells. Functional assays confirmed that miR-155 promotes inflammatory cytokine production, NF-κB pathway activation, and gastric mucosal injury, suggesting a critical pro-inflammatory role during acute gastric inflammation. Previous reports established miR-155 as a potent inflammatory mediator in chronic colitis and H. pylori–associated gastritis through targeting negative regulators such as SOCS1 (O'Connell et al., 2007 ). Consistent with these findings, we identified SOCS1 as a direct target of miR-155 in gastric epithelial cells. Suppression of SOCS1 by miR-155 likely amplifies NF-κB signaling, thus exacerbating the inflammatory response. Notably, while miR-155 has traditionally been studied in immune cells, our data highlights its functional importance in epithelial cells, indicating a broader role in gastric mucosal inflammation. In vivo, intravenous administration of miR-155 antagomir significantly alleviated gastric injury, underscoring its therapeutic potential. While intravenous delivery ensures antagomir stability, it might limit direct mucosal targeting, suggesting future studies could explore protective oral delivery systems. Our study has limitations, including potential undiscovered miR-155 targets, reliance on a single inflammatory stimulus, and absence of temporal profiling. Future research should address these aspects to clarify the comprehensive role of miR-155 in gastric inflammation. Collectively, these findings establish miR-155 as a key mediator in acute gastric inflammation and support miR-155 inhibition as a promising therapeutic strategy. 5. Discussion In summary, our study demonstrates that miR-155 is significantly upregulated in both in vivo and in vitro models of acute gastric inflammation, functioning as a key pro-inflammatory regulator. By promoting the expression of cytokines such as TNF-α and IL-6 and enhancing NF-κB pathway activation, miR-155 exacerbates gastric mucosal damage and inflammatory cell infiltration. Mechanistic investigations confirmed SOCS1 as a direct functional target of miR-155, whose suppression attenuates the negative regulation of inflammatory signaling, thus amplifying the inflammatory response. Importantly, intravenous administration of miR-155 antagomir effectively alleviated gastric mucosal injury in a mouse model of acute gastritis, supporting the translational potential of miR-155 inhibition as a therapeutic strategy (Raisch et al., 2013 ; X. Yu et al., 2024 ). These findings expand our understanding of molecular events regulating acute gastric inflammation and highlight miR-155 as a promising candidate for therapeutic intervention (Sampath et al., 2021 ). Future studies exploring the temporal dynamics and broader regulatory network of miR-155 may provide deeper insights into gastrointestinal immunity and epithelial repair mechanisms. Declarations Author Contributions L.L.: Writing – original draft, Investigation, Formal analysis, Visualization, Data curation, Conceptualization. K.Z.: Investigation, Formal analysis, Visualization, Data curation. S.M.: Investigation, Visualization, Data curation, Conceptualization. S.L.: Investigation Formal analysis, Visualization. W.X.: Writing – review & editing, Supervision, Formal analysis, Data curation, Conceptualization, Funding acquisition. X.L.: Writing – review & editing, Supervision, Project administration, Formal analysis, Data curation, Conceptualization. Acknowledgements This study was funded by the Medical and Health Technology Project of Shandong Provincial Health Commission (No. 202309031687). Funding Declaration This study was funded by the Medical and Health Technology Project of Shandong Provincial Health Commission (No. 202309031687). Conflicts of Interest The authors declare that they have no competing interests. Ethics Approval All animal experiments were approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Qingdao University (Approval No. ZYFYWZLL30239). Data Availability Statement The data used and/or analyzed during the current study are available from the corresponding authors upon reasonable request. 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Cancer . 23 (1), 170. https://doi.org/10.1186/s12943-024-02084-x (2024). Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Supplementary.pdf Cite Share Download PDF Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Reviewers invited by journal 24 Jul, 2025 Editor assigned by journal 18 Jul, 2025 Editor invited by journal 18 Jul, 2025 Submission checks completed at journal 17 Jul, 2025 First submitted to journal 17 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6971173","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":490332245,"identity":"acdd67da-36da-46a2-91f0-db986778a409","order_by":0,"name":"Lei Luo","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Luo","suffix":""},{"id":490332246,"identity":"1e5b1324-7636-43b2-9657-f7775b34e3cb","order_by":1,"name":"Kai Zhang","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zhang","suffix":""},{"id":490332247,"identity":"5f0f2a4e-1f62-4beb-bccb-cf3e56cbc874","order_by":2,"name":"Siyu Mu","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Siyu","middleName":"","lastName":"Mu","suffix":""},{"id":490332248,"identity":"2b39cf64-77a2-48f6-b5b3-1945cde89706","order_by":3,"name":"Shuangyong Liu","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Shuangyong","middleName":"","lastName":"Liu","suffix":""},{"id":490332249,"identity":"eb759e26-c437-44c2-9f26-8f326ee4dd63","order_by":4,"name":"Wenjing Xiao","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Xiao","suffix":""},{"id":490332250,"identity":"7e36a4d1-608e-4b6b-acce-6ad169b28487","order_by":5,"name":"Xiaolei Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACNv7+5x8/GNjw8DMcP/ggoaKGsBY+iTNszBIVaXKSjWeSDR6cOUZYixxDDhsDz5nDxgbNB8wkH7YwE+EwhrPHHki2HU7cwHYgrSKxgY2Bv707Ab8W5r50g8K29MTtPAeP3UjcIcMgcebsBgK2HDCQkGyzTtw540DajcQzbAwGErmEtCQYSPC2MSduuP/ArCCxjZkYLTlmEjxnnI0NDhwwYyBOi8SxZGNwIDecSZZIOHOMh6Bf5PubDz6EReXHHxU1cvztvfi1YAAe0pSPglEwCkbBKMAKAG5VT0TgOEzRAAAAAElFTkSuQmCC","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":true,"prefix":"","firstName":"Xiaolei","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-06-25 06:23:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6971173/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6971173/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-25642-9","type":"published","date":"2025-11-24T15:58:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87767153,"identity":"9ce0d55f-808c-41d5-97ed-9f8272a4a8d1","added_by":"auto","created_at":"2025-07-28 18:23:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":303353,"visible":true,"origin":"","legend":"\u003cp\u003emiR-155 is upregulated in acute gastric inflammation.\u003c/p\u003e\n\u003cp\u003e(A) miR-155 expression in gastric mucosa of mice with HCl/ethanol-induced gastritis, detected by qRT-PCR. (B) miR-155 levels in GES-1 cells following 24 h LPS stimulation. Data are presented as mean ± SEM (n = 3); *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. control.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/e22be5aae4dcfdbf0568c1c7.png"},{"id":87767130,"identity":"546ea79b-542f-4d26-95cf-85cc9967ec83","added_by":"auto","created_at":"2025-07-28 18:23:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2208483,"visible":true,"origin":"","legend":"\u003cp\u003emiR-155 modulates pro-inflammatory gene expression in GES-1 cells.\u003c/p\u003e\n\u003cp\u003e(A) miR-155 levels following mimic or inhibitor transfection. (B–F) Relative mRNA expression of TNF-α (B), IL-1β (C), IL-6 (D), IL-18 (E), and iNOS (F). Data are presented as mean ± SEM (n = 3). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 compared with negative control (NC) groups.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/3987136db1f2a0787dcc7835.png"},{"id":87767120,"identity":"c0d42a14-9c71-4be3-b175-af4639dd2569","added_by":"auto","created_at":"2025-07-28 18:23:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2428528,"visible":true,"origin":"","legend":"\u003cp\u003emiR-155 enhances LPS-induced cytokine secretion and NF-κB signaling.\u003c/p\u003e\n\u003cp\u003e(A–C) ELISA analysis of TNF-α, IL-1β, and IL-10 levels in GES-1 cells. (D) Western blot showing phosphorylation of IKKα and IκBα. Data are presented as mean ± SEM (n = 3), *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. control.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/def31e2a717aa29aa4f5a032.png"},{"id":87767145,"identity":"ca156ace-15f7-4592-b48e-c1e5e4a0d38b","added_by":"auto","created_at":"2025-07-28 18:23:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5157006,"visible":true,"origin":"","legend":"\u003cp\u003eSOCS1 is a direct target of miR-155.\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram of predicted targets from three databases. (B) qRT-PCR of SOCS1 mRNA after miR-155 modulation. (C) Predicted binding site between miR-155 and SOCS1 3′UTR. (D) RNA pulldown assay showing SOCS1 enrichment with wild type but not mutant 3′UTR. Data are presented as mean ± SEM (n = 3). **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/25420ecd4ca926b0acc25351.png"},{"id":87767099,"identity":"7f7cfbc3-3da8-4bd1-a091-adfebda91fce","added_by":"auto","created_at":"2025-07-28 18:23:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1642892,"visible":true,"origin":"","legend":"\u003cp\u003eSOCS1 overexpression reverses miR-155-induced cytokine expression.\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR validating SOCS1 plasmid overexpression. (B–E) Rescue of TNF-α, IL-1β, IL-6, and IL-8 expression by SOCS1 in GES-1 cells co-transfected with miR-155 mimic. Data are presented as mean ± SEM (n = 3), *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/f12d39ba62d676e84f796784.png"},{"id":87767563,"identity":"3b3fafe8-ec1b-4756-99fc-cdb5fd19786c","added_by":"auto","created_at":"2025-07-28 18:31:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11483371,"visible":true,"origin":"","legend":"\u003cp\u003emiR-155 antagomir alleviates gastric injury in vivo.\u003c/p\u003e\n\u003cp\u003e(A) Gross gastric morphology in mice treated with miR-155 antagomir. (B) Quantification of gastric lesion scores. (C, D) Relative expression of miR-155 and SOCS1 in gastric tissues as measured by qRT-PCR. Data are presented as mean ± SEM (n = 6), *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. NC.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/6216cd01db5efbf7de8aa5e0.png"},{"id":97179344,"identity":"4035c088-81bb-47c6-a20b-4add2d2b6e40","added_by":"auto","created_at":"2025-12-01 16:14:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19740582,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/2be73524-cdac-4bf0-922b-e6a1b99ee195.pdf"},{"id":87767094,"identity":"a8c9f2c4-6bfc-452c-8f84-c886b8f1e108","added_by":"auto","created_at":"2025-07-28 18:23:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18920,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/ef15b7ab44c9d8c670164214.docx"},{"id":87768182,"identity":"cec349a8-b796-4615-8a2e-2389f036fd40","added_by":"auto","created_at":"2025-07-28 18:47:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":227685,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6971173/v1/2bf93a7ea7516f46f589dca8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of microRNA-155 regulates gastric mucosal barrier repair and inflammation by targeting SOCS1 for the treatment of acute gastritis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAcute gastritis is a common gastrointestinal disorder characterized by sudden onset of mucosal congestion, edema, and infiltration of inflammatory cells in the gastric lining (Mihaly et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It typically develops rapidly in response to chemical irritants such as excessive alcohol consumption, nonsteroidal anti-inflammatory drugs (NSAIDs), or bacterial toxins. These triggers disrupt the gastric epithelial barrier, leading to mucosal hemorrhage and local immune activation, accompanied by a sharp increase in pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). While acute gastritis is often self-limiting, repeated or unresolved episodes may progress to chronic atrophic gastritis, peptic ulcers, or even gastric cancer (Taylor, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Yang \u0026amp; Hu, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, understanding the early immunological events that underline mucosal inflammation is crucial for identifying novel targets for prevention and therapy.\u003c/p\u003e\u003cp\u003eMicroRNAs (miRNAs) are short, non-coding RNA molecules that regulate gene expression at the post-transcriptional level, primarily through base-pairing with the 3\u0026prime; untranslated regions (3\u0026prime;UTRs) of target mRNAs (Rupaimoole \u0026amp; Slack, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Over the past decade, their roles in shaping gastrointestinal immune responses and preserving mucosal homeostasis have gained significant research interest (Bi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rawat et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Dysregulation of miRNA expression has been implicated in several inflammatory and neoplastic diseases (Hussen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), including inflammatory bowel disease (IBD), \u003cem\u003eHelicobacter pylori\u003c/em\u003e\u0026ndash;associated gastritis, and colorectal cancer (Larabi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mohamed et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These regulatory RNAs exert their effects largely through modulating classic inflammatory pathways such as NF-κB, JAK/STAT, and MAPK (Hoesel \u0026amp; Schmid, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Among them, miR-155 has emerged as a prototypical pro-inflammatory miRNA that is rapidly induced by TLR signaling, cytokine stimulation, or microbial infection (Elton et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Leng et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; O'Connell et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It promotes inflammatory responses by suppressing key negative regulators such as SOCS1 (Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Prieto et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and SHIP1(O'Connell et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), thereby contributing to macrophage activation, T cell differentiation, and cytokine amplification (Huang et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; O'Connell et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Renrick et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough the role of miR-155 has been extensively studied in chronic inflammatory conditions (Jafarzadeh et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Su et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), its involvement in acute gastric inflammation\u0026mdash;particularly during the early phase of chemically induced injury\u0026mdash;remains poorly defined (Wang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; X. Yu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It is still unclear whether miR-155 participates in the initiation or amplification of mucosal inflammation in acute gastritis, and if so, through which downstream effectors. To address these questions, we employed a well-established HCl/ethanol-induced mouse model of acute gastritis, complemented by in vitro stimulation of human gastric epithelial cells, to investigate the expression dynamics, regulatory functions, and molecular targets of miR-155. Our study aims to elucidate the potential contribution of miR-155 in acute gastric injury and provide mechanistic insight that may guide future anti-inflammatory interventions.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Materials\u003c/h2\u003e\n \u003cp\u003eHuman gastric epithelial GES-1 cells were obtained from ATCC (USA). Cell culture reagents, including RPMI-1640 medium, fetal bovine serum (FBS), Trypsin-EDTA, and penicillin-streptomycin, were sourced from commercial suppliers such as Gibco and Cytiva Hyclone. Phosphate-buffered saline (PBS) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. miR-155-related oligonucleotides (mimic, inhibitor, and negative controls) were synthesized by GenePharma (China). Antibodies against IKK\u0026alpha;, I\u0026kappa;B\u0026alpha;, their phosphorylated forms, GAPDH, and HRP-conjugated secondary antibodies were provided by Cell Signaling Technology. Additional reagents used included TRIzol and Lipofectamine 3000 (Invitrogen), SYBR Green PCR Master Mix (Applied Biosystems), ELISA kits (R\u0026amp;D Systems), and the RNA immunoprecipitation kit along with chemiluminescent substrate (Millipore).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Cell culture\u003c/h2\u003e\n \u003cp\u003eGES-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic solution. Cells were maintained at 37\u0026deg;C in a humidified incubator containing 5% CO₂.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. RNA Isolation and qRT-PCR\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted from gastric tissues and GES-1 cells using a standard phenol-based method. RNA concentration and purity were assessed using a microvolume spectrophotometer. cDNA synthesis was performed with a commercial reverse transcription kit, and quantitative PCR was carried out using SYBR Green dye on a real-time PCR system. Gene expression was normalized to internal controls (U6 or GAPDH), and relative levels were calculated using the 2^\u0026minus;\u0026Delta;\u0026Delta;Ct method. (All primer sequences used for qPCR are listed in Table 1.)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. miRNA Mimics, Inhibitors, and Plasmid Transfection\u003c/h2\u003e\n \u003cp\u003eGES-1 cells were transfected with miR-155 mimic (5\u0026rsquo;-UUAAUGCUAAUCGUGAUAGGGGU-3\u0026rsquo;, 5\u0026rsquo;-ACCCCUAUCACGAUUAGCAUUA-3\u0026rsquo;), inhibitor (5\u0026apos;-ACCCCUAUCACGAUUAGCAUU-3\u0026apos;), or corresponding negative controls (5\u0026rsquo;-CAGUACUUUUUGUGUAGUACAA-3\u0026rsquo;, GenePharma) using a commercial lipid-based transfection reagent, according to the manufacturer\u0026apos;s protocol. For rescue assays, cells were co-transfected with miR-155 mimic and a SOCS1-expressing plasmid (GenePharma), or with an empty vector. After 48 hours, cells were collected for subsequent analyses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Cytokine Analysis (ELISA)\u003c/h2\u003e\n \u003cp\u003eCulture supernatants were collected, and in cytokine levels, including TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and IL-10, were quantified by enzyme-linked immunosorbent assay using commercially available kits, following standard protocols.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e2.6. Western Blot Analysis\u003c/h2\u003e\n \u003cp\u003eGES-1 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo Scientific). Protein concentrations were quantified using the BCA protein assay kit (Beyotime, China). Proteins (30 \u0026micro;g per lane) were separated by SDS-PAGE, transferred onto PVDF membranes, and blocked in 5% non-fat milk. Membranes were incubated overnight at 4\u0026deg;C with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Protein bands were visualized using ECL substrate (Millipore).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e2.7. RNA pulldown assay\u003c/h2\u003e\n \u003cp\u003eA biotin-labeled miR-155 probe (biotin-5\u0026rsquo;-ACCCCUAUCACGAUUAGCAUUA-3\u0026rsquo;) and a negative control probe (5\u0026rsquo;-UUCUCCGAACGUGUCACGUTT-3\u0026rsquo;, GenePharma). GES-1 cells were lysed in RNA binding buffer, and 1 mg of lysate was incubated with 1 \u0026micro;g of biotin-labeled RNA probe at 4\u0026deg;C overnight. Streptavidin magnetic beads (Thermo Fisher) were added to pull down RNA-protein complexes. The bound fractions were analyzed by Western blot to detect SOCS1 protein enrichment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e2.8. Animals and Gastritis Model\u003c/h2\u003e\n \u003cp\u003eMale C57BL/6 mice (8\u0026ndash;10 weeks old) were housed under specific pathogen-free conditions with free access to food and water. To induce acute gastritis, mice were orally administered 0.2 mL of 150 mM HCl dissolved in 60% ethanol, while control animals received sterile saline. The study was approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Qingdao University (Approval No. ZYFYWZLL30239). All animal experiments, including euthanasia procedures, were performed in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020 Edition). For euthanasia, animals were exposed to carbon dioxide (CO₂) via a gradual displacement method in a closed chamber, with CO₂ delivered at a flow rate of 20\u0026ndash;50% of the chamber volume per minute. This rate ensures unconsciousness occurs before CO₂ concentrations reach\u0026thinsp;~\u0026thinsp;40% (the threshold associated with nociceptor activation), minimizing potential distress. Death was confirmed by the absence of respiratory movement, heartbeat (palpation), and pupillary reflexes; CO₂ flow was maintained for at least 1 minute after respiratory arrest to ensure euthanasia completion. Subsequent tissue collection (including stomach) was performed immediately post-confirmation of death.\u003c/p\u003e\n \u003cp\u003eAll methods in this study are reported in accordance with the ARRIVE guidelines (Boutron et al., 2020).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e2.9. miR-155 Antagomir Administration\u003c/h2\u003e\n \u003cp\u003emiR-155 antagomir (5\u0026rsquo;-ACCCCUAUCACGAUUAGCAUU-3\u0026rsquo;, GenePharma) or scrambled control antagomir (5\u0026rsquo;-CAGUACUUUUUGUGUAGUACAA-3\u0026rsquo;, GenePharma) was dissolved in sterile saline and intravenously injected via tail vein at 45 mg/kg body weight (200 \u0026micro;L per mouse) on days 0, 2, and 4. Mice were sacrificed on day 5 for tissue analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e2.10. Histological and Macroscopic Evaluation\u003c/h2\u003e\n \u003cp\u003eDissecting fresh gastric tissue, and gastric injury scores were evaluated based on hemorrhage, edema, epithelial exfoliation, and inflammatory cell infiltration. Macroscopic gastric lesions were photographed, visually scored and image J quantization.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e2.11. Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three independent experiments. Statistical analyses were performed using GraphPad Prism software (version 8.0). Differences between groups were evaluated by Student\u0026rsquo;s t-test or one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test, with a significance threshold set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 miR-155 is significantly upregulated in gastric inflammation both in vivo and in vitro\u003c/h2\u003e\u003cp\u003emiR-155 is one of the most extensively studied inflammation-related microRNAs and has been shown to be upregulated in various acute and chronic inflammatory models, where it modulates immune cell activation and cytokine production. However, its expression dynamics in acute gastric inflammation\u0026mdash;particularly during the early epithelial response\u0026mdash;remain poorly defined. In this study, we first examined miR-155 expression in a mouse model of acute gastritis induced by HCl/ethanol. qRT-PCR analysis revealed a significant elevation of miR-155 levels in the gastric mucosa 24 hours after induction compared to control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting a potential regulatory role in the inflammatory process.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate whether similar changes occur in gastric epithelial cells, we established an in vitro inflammation model by treating human gastric epithelial GES-1 cells with lipopolysaccharide (LPS, 100 ng/mL) for 24hours. Consistent with our in vivo findings, LPS stimulation led to a marked increase in miR-155 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Together, these results indicate that miR-155 is rapidly induced during the early phase of acute gastric inflammation and may play a critical role in initiating or amplifying epithelial immune responses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 miR-155 promotes pro-inflammatory cytokine expression in gastric epithelial cells\u003c/h2\u003e\u003cp\u003eThe functional role of miR-155 in gastric epithelial inflammation was further explored by transfecting GES-1 cells with a miR-155 mimic or inhibitor. qRT-PCR analysis confirmed successful transfection, with miR-155 expression markedly increased in the mimic group and significantly reduced in the inhibitor group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Functionally, overexpression of miR-155 significantly elevated the mRNA levels of several key pro-inflammatory cytokines and mediators, including tumor necrosis factor-alpha (TNF-α; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), interleukin-1 beta (IL-1β; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), interleukin-6 (IL-6; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), interleukin-18 (IL-18; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), and inducible nitric oxide synthase (iNOS; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Conversely, miR-155 inhibition led to a marked reduction in the expression of these genes compared with the respective negative control groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings indicate that miR-155 functions as a positive regulator of inflammatory gene expression in gastric epithelial cells, potentially contributing to the amplification of inflammatory responses during acute gastritis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 miR-155 enhances LPS-induced cytokine production and activates NF-κB signaling in gastric epithelial cells\u003c/h2\u003e\u003cp\u003eTo further investigate the mechanism by which miR-155 promotes inflammation in gastric epithelial cells, we assessed its effect on cytokine secretion and NF-κB signaling in LPS-stimulated GES-1 cells. ELISA analysis revealed that transfection with miR-155 mimic significantly enhanced the secretion of TNF-α, IL-1β, and IL-10 in response to LPS stimulation compared to the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C). In contrast, the inhibition of miR-155 markedly reduced the levels of these cytokines following LPS exposure. We also examined the phosphorylation status of key components in the canonical NF-κB pathway. Western blot analysis showed that miR-155 overexpression increased the phosphorylation of IKKα and IκBα, whereas miR-155 inhibition suppressed these phosphorylation events (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Together, these results suggest that miR-155 enhances LPS-induced inflammatory responses in gastric epithelial cells, at least in part by activating the NF-κB signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 SOCS1 is a direct functional target of miR-155\u003c/h2\u003e\u003cp\u003eWe next performed integrative bioinformatic analysis using the miRDB, TargetScan, and miRWalk databases to identify potential downstream targets of miR-155. A total of 154 overlapping candidate genes were identified across all three platforms (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Based on their known involvement in negative regulation of NF-κB signaling, SOCS1(J. Yu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), FOSL2(Hu et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), XKR4, and HBP1 were selected for further validation (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eqRT-PCR analysis revealed that miR-155 mimic transfection significantly reduced SOCS1 mRNA expression in GES-1 cells, while miR-155 inhibition restored SOCS1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, the expression of the other predicted targets showed no significant changes, supporting the specificity of the miR-155\u0026ndash;SOCS1 interaction. Sequence alignment analysis further predicted a conserved binding site for miR-155 within the 3\u0026prime; untranslated region (3\u0026prime;UTR) of SOCS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To confirm the direct interaction between miR-155 and SOCS1 mRNA, an RNA pulldown assay was performed using a biotin-labeled miR-155 probe. GES-1 cells were lysed after co-transfection with either wild-type or mutant SOCS1 3\u0026prime;UTR constructs, and lysates were incubated with streptavidin-conjugated magnetic beads to capture the probe-associated complexes. SOCS1 protein was markedly enriched in the pulldown complex when co-transfected with wild-type 3\u0026prime;UTR, but not with the mutant lacking the predicted binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results provide strong evidence that SOCS1 is a direct and functional target of miR-155 in gastric epithelial cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 miR-155 promotes inflammatory cytokine expression through SOCS1 suppression in GES-1 cells\u003c/h2\u003e\u003cp\u003eTo evaluate whether the pro-inflammatory effects of miR-155 are mediated through suppression of SOCS1, we conducted a rescue experiment in GES-1 cells. Overexpression of miR-155 markedly increased mRNA levels of TNF-α, IL-1β, IL-6, and IL-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;E). However, these effects were substantially reversed by co-transfection with a SOCS1-expressing plasmid, which restored cytokine expression to near basal levels. qRT-PCR confirmed successful SOCS1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These findings indicate that miR-155 enhances inflammatory cytokine expression in gastric epithelial cells, at least in part, by downregulating SOCS1. The reversal of miR-155-mediated effects by SOCS1 restoration further supports the functional relevance of the miR-155/SOCS1 regulatory axis in epithelial inflammation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Inhibition of miR-155 alleviates gastric inflammation in vivo\u003c/h2\u003e\u003cp\u003eIn order to further evaluate the therapeutic potential of targeting miR-155 in vivo, we employed a well-established ethanol/HCl-induced acute gastritis mouse model. Mice administered a miR-155 antagomir via tail vein injection. Remarkably, systemic delivery of the antagomir significantly alleviated gastric mucosal hemorrhage and tissue injury, producing therapeutic effects comparable to those of ranitidine (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Gastric lesion scores were also markedly improved following treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). At the molecular level, gastric tissues from antagomir-treated mice exhibited a notable reduction in miR-155 expression along with restored SOCS1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D), confirming the protective effect of systemic miR-155 inhibition against acute gastric injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we demonstrated that miR-155 expression was markedly elevated in ethanol-induced acute gastritis mice and LPS-stimulated gastric epithelial cells. Functional assays confirmed that miR-155 promotes inflammatory cytokine production, NF-κB pathway activation, and gastric mucosal injury, suggesting a critical pro-inflammatory role during acute gastric inflammation.\u003c/p\u003e\u003cp\u003ePrevious reports established miR-155 as a potent inflammatory mediator in chronic colitis and H. pylori\u0026ndash;associated gastritis through targeting negative regulators such as SOCS1 (O'Connell et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Consistent with these findings, we identified SOCS1 as a direct target of miR-155 in gastric epithelial cells. Suppression of SOCS1 by miR-155 likely amplifies NF-κB signaling, thus exacerbating the inflammatory response. Notably, while miR-155 has traditionally been studied in immune cells, our data highlights its functional importance in epithelial cells, indicating a broader role in gastric mucosal inflammation. In vivo, intravenous administration of miR-155 antagomir significantly alleviated gastric injury, underscoring its therapeutic potential. While intravenous delivery ensures antagomir stability, it might limit direct mucosal targeting, suggesting future studies could explore protective oral delivery systems.\u003c/p\u003e\u003cp\u003eOur study has limitations, including potential undiscovered miR-155 targets, reliance on a single inflammatory stimulus, and absence of temporal profiling. Future research should address these aspects to clarify the comprehensive role of miR-155 in gastric inflammation. Collectively, these findings establish miR-155 as a key mediator in acute gastric inflammation and support miR-155 inhibition as a promising therapeutic strategy.\u003c/p\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eIn summary, our study demonstrates that miR-155 is significantly upregulated in both in vivo and in vitro models of acute gastric inflammation, functioning as a key pro-inflammatory regulator. By promoting the expression of cytokines such as TNF-α and IL-6 and enhancing NF-κB pathway activation, miR-155 exacerbates gastric mucosal damage and inflammatory cell infiltration. Mechanistic investigations confirmed SOCS1 as a direct functional target of miR-155, whose suppression attenuates the negative regulation of inflammatory signaling, thus amplifying the inflammatory response.\u003c/p\u003e\u003cp\u003eImportantly, intravenous administration of miR-155 antagomir effectively alleviated gastric mucosal injury in a mouse model of acute gastritis, supporting the translational potential of miR-155 inhibition as a therapeutic strategy (Raisch et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; X. Yu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These findings expand our understanding of molecular events regulating acute gastric inflammation and highlight miR-155 as a promising candidate for therapeutic intervention (Sampath et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Future studies exploring the temporal dynamics and broader regulatory network of miR-155 may provide deeper insights into gastrointestinal immunity and epithelial repair mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.L.: Writing – original draft, Investigation, Formal analysis, Visualization, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003eK.Z.: Investigation, Formal analysis, Visualization, Data curation.\u003c/p\u003e\n\u003cp\u003eS.M.: Investigation, Visualization, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003eS.L.: Investigation Formal analysis, Visualization.\u003c/p\u003e\n\u003cp\u003eW.X.: Writing – review \u0026amp; editing, Supervision, Formal analysis, Data curation, Conceptualization, Funding acquisition.\u003c/p\u003e\n\u003cp\u003eX.L.: Writing – review \u0026amp; editing, Supervision, Project administration, Formal analysis, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e This study was funded by the Medical and Health Technology Project of Shandong Provincial Health Commission (No. 202309031687).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e This study was funded by the Medical and Health Technology Project of Shandong Provincial Health Commission (No. 202309031687).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of the Affiliated Hospital of Qingdao University (Approval No. ZYFYWZLL30239).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e The data used and/or analyzed during the current study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBi, K., Zhang, X., Chen, W. \u0026amp; Diao, H. 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Cancer\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e (1), 170. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12943-024-02084-x\u003c/span\u003e\u003cspan address=\"10.1186/s12943-024-02084-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"miR-155, inflammation, cytokines, acute gastritis, Ethanol-induced injury","lastPublishedDoi":"10.21203/rs.3.rs-6971173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6971173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute gastritis is a common gastrointestinal disorder characterized by rapid onset of mucosal injury and infiltration of inflammatory cells, often induced by chemical irritants such as ethanol. Although inflammation is essential for mucosal defense and repair, excessive immune responses can lead to tissue damage and impaired healing. MicroRNAs (miRNAs), particularly those involved in immune regulation, have emerged as important modulators in various inflammatory conditions. However, their specific roles in the early phase of acute gastric inflammation remain unclear, and the mechanisms by which they influence mucosal damage are still not fully understood.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the expression and functional significance of microRNA-155 (miR-155) in a mouse model of ethanol-induced acute gastritis. Our results showed that miR-155 expression was significantly upregulated in gastric mucosa following ethanol exposure, concomitant with elevated levels of pro-inflammatory cytokines including TNF-α and IL-6. Histological examination revealed that inhibition of miR-155 using a specific antagomir reduced inflammatory cell infiltration and attenuated mucosal injury. These findings suggest that miR-155 contributes to the exacerbation of acute gastric inflammation by promoting cytokine expression and epithelial damage.\u003c/p\u003e\u003cp\u003eImportantly, this study highlights the role of miR-155 as an early-response regulator during acute gastric injury. Targeting miR-155 may offer a novel therapeutic strategy for the prevention or treatment of acute gastric mucosal damage. Furthermore, our findings provide a foundation for future exploration of miRNA-based interventions in inflammation-related gastrointestinal diseases.\u003c/p\u003e","manuscriptTitle":"Inhibition of microRNA-155 regulates gastric mucosal barrier repair and inflammation by targeting SOCS1 for the treatment of acute gastritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 18:23:13","doi":"10.21203/rs.3.rs-6971173/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-07-24T12:38:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-18T19:56:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-18T19:53:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-17T08:41:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-17T07:20:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b198695a-0500-4627-af93-a004c180a8d6","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52064001,"name":"Biological sciences/Biochemistry/Cytokines"},{"id":52064002,"name":"Biological sciences/Biochemistry/Histocytochemistry"}],"tags":[],"updatedAt":"2025-12-01T16:08:35+00:00","versionOfRecord":{"articleIdentity":"rs-6971173","link":"https://doi.org/10.1038/s41598-025-25642-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-24 15:58:39","publishedOnDateReadable":"November 24th, 2025"},"versionCreatedAt":"2025-07-28 18:23:13","video":"","vorDoi":"10.1038/s41598-025-25642-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-25642-9","workflowStages":[]},"version":"v1","identity":"rs-6971173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6971173","identity":"rs-6971173","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}