LGALS3 promotes liver fibrosis by enhancing the expression and phosphorylation of ERK1/2

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Current therapeutic approaches predominantly focus on alleviating symptoms or addressing the underlying causes, with limited options available for reversing fibrosis. Existing antifibrotic drugs often exhibit low efficacy and carry significant side effects, underscoring the urgent need for novel therapeutic targets. LGALS3, a β-galactoside-binding lectin, participates in inflammation and fibrosis across multiple organs. However, its specific role in liver fibrosis remains poorly understood. This study endeavors to elucidate the cellular and molecular mechanisms by which LGALS3 regulates liver fibrosis and assess its potential as a therapeutic target. Subjects and Methods: To clarify the role of LGALS3 in liver fibrosis, a TGF-β1-induced liver fibrosis model was established, and the expression level of LGALS3 was analyzed. LGALS3 overexpression and knockdown cell lines were constructed in LX-2 cell line. RT-qPCR, western blot, CCK8 assay, and wound healing assay were employed to investigate the impact of LGALS3 on LX-2 cell activation, proliferation, migration, and the ERK1/2 pathway. In the LGALS3 overexpression cell model, PD98059 intervention was applied to mimic the therapeutic effect on liver fibrosis. Results: LGALS3 overexpression significantly promoted the proliferation and migration of LX-2 cells, along with the expression and phosphorylation of ERK1/2. Conversely, LGALS3 knockdown and treatment with the PD98059 inhibitor reduced LX-2 cell proliferation and migration, as well as the expression and phosphorylation of ERK1/2. Conclusions: This study has clarified the pivotal regulatory role of the LGALS3-ERK1/2 signaling axis in liver fibrosis, uncovering novel molecular mechanisms underlying the activation, proliferation, and migration of hepatic stellate cells. These findings enhance our understanding of the pathological process of liver fibrosis, suggesting that targeting the LGALS3-ERK1/2 axis may serve as a novel therapeutic strategy for intervening in liver fibrosis. It also provides potential targets and innovative directions for the development of anti-liver fibrosis drugs. LGALS3 liver fibrosis ERK1/2 PD98059 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Research Highlights 1. Liver fibrosis model induced by TGF-β1 was established, in which the expression of LGALS3 was increased. 2. LGALS3 overexpression induces the expression and phosphorylation of ERK1/2, thereby facilitating the progression of liver fibrosis. 3. The therapeutic effect of PD98059 targeting ERK1/2 on liver fibrosis. 1. Introduction Liver fibrosis, a common pathological endpoint of chronic liver diseases, represents a significant global health burden [ 1 ] . Approximately 1.75 million deaths worldwide are attributed to liver fibrosis and its complications annually, with an estimated 20–30% of patients progressing to cirrhosis within 5–10 years of disease onset. This condition is characterized by excessive extracellular matrix (ECM) deposition, primarily type I collagen, and disruption of liver lobular architecture, leading to impaired hepatic function and an increased risk of hepatocellular carcinoma [ 2 – 3 ] . Despite advancements in etiological treatments (e.g., antiviral therapy for hepatitis C), no specific anti-fibrotic drugs have been approved by regulatory agencies, highlighting the urgent need for novel therapeutic targets [ 4 – 5 ] .​ Transforming growth factor-β1 (TGF-β1), a prototypic profibrotic cytokine, plays a central role in the activation of hepatic stellate cells (HSCs). With its high tissue expression and bioactivity, TGF-β1 directly activates HSCs, inducing the expression of α-smooth muscle actin (α-SMA) and collagen type I (Collagen I) [ 6 – 8 ] . Currently, TGF-β1 is widely used to induce cellular fibrosis. TGF-β1 stimulates HSCs to produce type I collagen (CollagenI), thereby mediating the fibrosis process [ 9 ] . Preclinical studies have shown that blocking TGF-β1 signaling reduces fibrosis in animal models [ 10 ] . However, clinical trials targeting TGF-β1 have faced challenges due to off-target effects, underscoring the necessity to explore alternative pathways [ 11 ] .​ Galectin-3 (LGALS3) has emerged as a key regulator in fibrotic diseases across multiple organs [ 12 ] . In cardiac fibrosis, LGALS3 promotes myofibroblast activation by enhancing TGF-β1 signaling and facilitating the recruitment of inflammatory cells, ultimately leading to myocardial ECM remodeling [ 13 ] . In idiopathic pulmonary fibrosis (IPF), LGALS3 upregulation contributes to fibroblast accumulation and alveolar destruction [ 14 – 15 ] . LGALS3 secreted by hepatocellular carcinoma (HCC) cells promotes HCC bone metastasis via facilitating the establishment of a pre-metastatic niche [ 16 ] . However, the role of LGALS3 in liver fibrosis remains unknown. These findings suggest that LGALS3 may also orchestrate liver fibrosis through similar molecular mechanisms.​ TGF-β1 can induce the occurrence of liver fibrosis [ 17 – 18 ] . The current study utilized TGF-β1 to trigger the activation of LX-2 cells. α-SMA and Collagen1 are regarded as indicative markers of HSC activation [ 19 – 21 ] . The ERK signaling pathway exerts a critical function in maintaining cell survival by suppressing multiple steps of apoptotic signaling cascades. Additionally, ERK activation is observed in a variety of tumor types [ 25 – 27 ] .In breast cancer cells, LGALS3 enhances ERK1/2 phosphorylation through CD44, promoting cancer cell migration [ 28 ] . Therefore, we hypothesize that LGALS3 directly activates the phosphorylation of ERK1/2 in HSCs. PD98059 is a reversible MEK inhibitor, and studies have shown that PD98059 inhibits the activation of the ERK signaling pathway in prostate cancer, thereby inhibiting cancer development. The ERK1/2 inhibitor PD98059 was selected based on its high specificity and efficacy in blocking the MEK-ERK cascade [ 29 – 30 ] . This investigation seeks to illuminate the role of LGALS3 in human liver fibrosis and identify potential therapeutic targets within the LGALS3-ERK1/2 axis.​ 2. Materials and methods 2.1 Cell culture and treatment The human hepatic stellate cell line, sourced from procell in Wuhan, China, was cultured in DMEM medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% penicillin-streptomycin (HyClone). These cells were maintained in incubator at 37°C for 48 hours, either with or without 5 ng/mL TGF-β1 (MCE) treatment for 24h, 48h, 72h [ 31 ] . Using 50µmol/L PD98059 to treat LGALS3 overexpressed cell line for 24h [ 32 ] . 2.2 Cell transfection LGALS3 shRNA targeting sequences5'-ACCCAAACCCTCAAGGATATC-3’; were constructed using pLKO.1 vector. A non-silencing shRNA with the scrambled sequence 5'-GCAAGCTGACCCTGAAGTTCAT-3' was used as a control;were constructed using pLKO.1 vector.To achieve stable and durable LGALS3 knockdown in LX-2 cells, the pLKO.1-puro-shLGALS3 lentivirus was constructed and packaged. The lentivirus was packed using 293T cells with psPAX2 (4.5 µg), pMD2.G (3 µg) and pLKO.1-puro-shLGALS3 plasmid (6 µg) in 60mm dishs, and cells were transfected with plasmid using Lipofectamine 2000 (Invitrogen, USA) according to themanufacturer’s guidelines. After 72 h of infection, the cells were cultured in a medium containing puromycin(1mg/mL) for 1 month. For the construction of LGALS3-overexpressed cell line in LX-2, LX2 cells were plated in 6-well plates at the density of 2×10 6 cells/well. pcDNA3.1-LGALS3 vector, and pcDNA3.1 empty vector using Lipofectamine 2000(Invitrogen) as the manufacturer recommended. Successful knockdown and overexpression of LGALS3 was confirmed by RT-qPCR and western blot. 2.3 Quantitative real-time PCR (qRT-PCR) Total RNA was isolated from LX2 cells with TRIzol reagent (Beyotime). Subsequently, the extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Takara). This was followed by quantitative PCR amplification employing ChamQ Universal SYBR qPCR Master Mix (Vazyme). The qRT-PCR reactions were performed on Quantagene q225 Fluorescent Quantitative PCR Instrument. The relative expression levels were determined by normalizing to GAPDH, utilizing the 2−∆∆Ct method. The primers were listed as follows: Gene name Sequence Orientation α-SMA 5′-CCCTGGAGAAGAGCTACGAG-3′ forward α-SMA 5′-GTACGACCAGAGGCATACAG -3′ reverse Collagen1 5' - CAGCCGCTTCACCTACAGC forward Collagen1 5' - GGTCACCTTCACCGTTCTC − 3' reverse LGALS3 5′-CCATCTTCTGGACAGCCAAGTG-3′ forward LGALS3 5′-TATCAGCATGCGAGGCACCACT-3′ reverse GAPDH 5′-ACAACTTTGGTATCGTGGAAGG-3′ forward GAPDH 5′-GCCATCACGCCACAGTTTC-3′ reverse 2.4 Western blotting Cell lysates were generated using ice-cold RIPA buffer (Beyotime). The protein concentration of the lysates was measured by BCA Protein Concentration Assay Kit (Beyotime). The lysates were subjected to SDS-PAGE ,every lane was added 40 ug lysates.The resolved proteins were electrotransferred onto PVDF membranes. To minimize non-specific antibody binding, the membranes were blocked with a 5% BSA solution(Sigma).Thereafter, the membranes were incubated with the respective primary antibodies at 4°C overnight. The primary antibodies employed were as follows: α- SMA antibody (Abclonal; dilution ratio 1:1000), Collagen1 antibody (Abclonal; dilution ratio 1:500), LGALS3 antibody (Abclonal; dilution ratio 1:1000), and GAPDH antibody (Servicebio; dilution ratio 1:1000).Following the overnight incubation with primary antibodies, the membranes were incubated with HRP-conjugated secondary antibodies (Promega; dilution ratio 1:10000) for 1 hour at room temperature. Protein bands were visualized and detected using BeyoECL Moon (Beyotime). 2.5 Wound healing assay In the wound healing assay, cells were plated in 6-well plates, and scratches were created using a 1-ml pipette tip. Using an inverted microscope, representative images of cell migration into the wounds were acquired at 0, 48, and 72 hours in the same scratched area (10 fields per group). The scratch closure distance was quantified with Image J software. 2.6 CCK8 assay LX-2 cells were plated in 96-well plates at a concentration of 2×10^3 cells per well. The proliferation of cells was evaluated using the Cell Counting Kit from yeasen, following the protocol specified by the manufacturer. The absorbance was determined at 490 nm. 2.7 Statistical analyses Statistical analyses were conducted using GraphPad Prism 8 software, with the specific tests employed detailed in the respective figure legends. For multiple comparisons, t-tests were carried out with adjustments made via the Holm–Sidak method. In cases specified in the figure legends, two-tailed unpaired t-tests were utilized. A threshold of P < 0.05 was set to denote statistical significance, with precise P value notations provided in each figure legend. 3. Results 3.1 LGALS3 is highly expressed in TGF-β1-stimulated LX2 cell line-induced liver fibrosis Transforming growth factor beta (TGF-β) plays a crucial role in chronic inflammation-related diseases. TGF-β signaling generally inhibits the proliferation of normal epithelial, endothelial, and immune cells and promotes the differentiation of fibroblast cell lineages that deposit extracellular matrix (ECM) components. α-SMA, and Collagen1 were considered as biomarkers of HSC activation.We established an experimental liver fibrosis cell model by inducing liver fibrosis in the LX2 cell with TGF-β1. To assess the fibrotic levels of LX-2 cells after TGF-β1 stimulation, we examined the expression of α-SMA and Collagen1 in LX-2 cells at 0h, 24h, 48h, and 72h. The results of RT-qPCR and western blot indicated that after 24 and 48 hours of TGF-β1 stimulation of LX-2 cells, the expression levels of α-SMA and Collagen1 remained unchanged (Figs. 1 A-D). This suggested that 48 hours was insufficient to activate LX-2 cells. Therefore, the expression of α-SMA and Collagen1 was analyzed in LX-2 cells upon 72-hour TGF-β1 stimulation. The RT-qPCR results showed that compared with the control group, the expression of α-SMA and Collagen1 mRNA in the TGF-β1 treatment group increased by 2 fold (Figs. 1 A-B). The western blot indicated that the protein expression levels of α-SMA and Collagen1 increased by 2 fold at 72h, and the differences were statistically significant (Figs. 1 A-D).(Fig. 1 A-D). Meanwhile, the expression level of LGALS3 significantly increased at 72h by 2 fold compared to the control group (Fig. 1 E-F). This indicates that the expression of LGALS3 is increased after the activation of LX-2 cells. 3.2 LGALS3 overexpression promotes the activation and migratory of LX-2 cells Next, we wanted to determine whether LGALS3 overexpression promoted the activation and migration of LX-2 cells. We transfected LX-2 with LGALS3 to construct LGALS3 overexpressed cell line (LGALS3-OE) and transfected LX-2 with an empty vector as conrtol(LGALS3-EV). After transfection, we detected the expression of LGALS3 by performing RT-qPCR and western blot to verify the transfection efficiency. The results showed that the expression level of LGALS3 increases by 2.5-fold after 72 hours of LGALS3 overexpression (Fig. 2 A-B). The expression levels of α-SMA and Collagen I significantly increased by 2-fold and 3-fold separately after 72 hours of LGALS3 overexpression (Fig. 2 C-D). Wound healing assay indicated that LGALS3 overexpression enhanced the migration ability of LX-2 cells at 72h (Fig. 2 E-F). These results suggested that LGALS3 overexpression can promote the activation and migration of LX-2 cells.The results suggest that overexpression of LGALS3 enhances the activation and migration of LX-2 cells (Fig. 2 F). 3.3 Knockdown of LGALS3 suppresses the activation and migration of LX-2 cells To address whether LGALS3 knockdown inhibited the activation and migration of LX-2 cells, we transfected LX-2 with LGALS3 shRNA to construct LGALS3 knockdown cell line (LGALS3-KD) and transfected LX-2 with an empty vector as control(LGALS3-EV). After transfection, we performed RT-qPCR and western blot for LGALS3 to verify the transfection efficiency. The results showed that the expression level of LGALS3 was significantly reduced by 2-fold after 72 hours of LGALS3 knockdown (Fig. 3 A-B).Using RT-qPCR and western blot, we analyzed the expression of α-SMA and Collagen I in the cells 72 hours after transfection to detect the activation status of LX-2 cells. The expression levels of α-SMA and Collagen I significantly decreased by 3-fold after LGALS3 knockdown (Fig. 3 C-D). Wound healing assay indicated that LGALS3 knockdown inhibited the migration ability of LX-2 cells after 72 hours(Fig. 3 E-F). These results suggest that LGALS3 knockdown can inhibit the activation and migration of LX-2 cells. 3.4 LGALS3 promotes liver fibrosis through upregulating the level of p-ERK To determine whether the change of LGALS3 expression in liver are associated with the activation of the ERK, we detected the expression and phosphorylation status of ERK1/2 protein in the LGALS3 overexpression and LGALS3 knockdown cell lines. The results showed that the expression of ERK1/2 remained unchange(Fig. 4 A-B), while the phosphorylation levels of ERK1/2 after 72 hours of LGALS3 overexpression increased by 3-fold(Fig. 4 C). The expression of ERK1/2 remained unchange(Fig. 4 D-E), while the phosphorylation levels of ERK1/2 decreased by 2-fold after LGALS3 knockdown 72 hours(Fig. 4 D-F). 3.5 PD98059 reverses the effects of increased LGALS3 expression We wanted to know whether PD98059 can inhibit the level of p-ERK and the expression of LGALS3 in LX-2 cells. We added PD98059 to the LGALS3 overexpression cell line and detected the expression of α-SMA and Collagen1. The results showed that after the addition of PD98059, the expression of α-SMA and Collagen1 significantly decreased by 2-fold (Fig. 5 A-B). Next, we detected the expression of LGALS3, it significantly decreased by 2-fold after the addition of PD98059 (Fig. 5 C, E). Although the expression of ERK1/2 remained unchange after PD98059 treat, PD98059 significantly reduced the expression and phosphorylation levels of ERK1/2 by 2-fold (Fig. 5 D, F). These results indicate that PD98059 can reverse the effects of increased LGALS3 expression. 3.6 PD98059 alleviates TGF-β1-stimulated LX2 cell line-induced liver fibrosis Next, we wanted to explore whether PD98059 can be used to treat liver fibrosis in liver fibrosis cell models.The results showed that TGF-β1 stimulation of LX2 cells increased the expression of α-SMA and Collagen1, while the expression of α-SMA and Collagen1 significantly decreased after PD98059 treatment 72h(Fig. 6 A-B). PD98059 also significantly reducde the expression of LGALS3 by 2-fold(Fig. 6 C, E). Although the expression of ERK1/2 remained unchange after PD98059 treat, PD98059 reduced the phosphorylation levels of ERK1/2 after 72h of TGF-β1 stimulation(Fig. 6 D-F). These results indicate that PD98059 can alleviate TGF-β1-stimulated LX2 cell line-induced liver fibrosis. In liver fibrosis, the expression of LGALS3 increases, promoting the phosphorylation of ERK1/2. The ERK1/2 inhibitor PD98059 alleviates the phenotypes associated with liver fibrosis. 4. Discussion Liver fibrosis, a common outcome of various chronic hepatic disorders, is characterized by excessive extracellular matrix (ECM) accumulation and disruption of the liver lobular structure. Activated hepatic stellate cells (HSCs) undergo phenotypic transformations that are pivotal for the initiation and progression of liver fibrosis. In experimental models, TGF-β1 is commonly used to induce HSC activation. Although LX-2 shares similar genetic phenotypes with HSCs in normal human livers, a cell line cannot fully represent the complex in vivo liver microenvironment and the interactions among various cell types, such as hepatocytes, endothelial cells, and immune cells, during the process of liver fibrosis. Moreover, during in vitro culture, the state and response of LX-2 cells may be influenced by culture conditions, such as the composition of the culture medium, cell density, and the number of passages. Furthermore, this model primarily focuses on the TGF-β1 signaling pathway and falls short in studying the interactions and regulatory mechanisms of other related signaling pathways. This study reveals the key effect of LGALS3 in liver fibrosis and demonstrates its potential as a therapeutic target. Inhibition of LGALS3 not only alleviates the activation of hepatic stellate cells but also mitigates liver fibrosis by suppressing the expression and phosphorylation of ERK1/2. PD98059 has been reported to inhibit ERK1/2 [ 33 ] . The inhibition of ERK1/2 has been reported to inhibit liver fibrosis [ 34 ] . However, the mechanism by which PD98059 inhibits the expression and phosphorylation of ERK1/2 and how this inhibition leads to the reversal of liver fibrosis, requires further investigation. In this study, we used PD98059 to inhibit the occurrence of liver fibrosis and achieved good results. The ERK1/2 signaling cascade, with multiple inhibitors, is pivotal in governing cellular proliferation, differentiation, and survival. For example, U0126 works by blocking the activation of MEK1/2, which phosphorylate and activate ERK1/2. This inhibition of MEK1/2 activity prevents the subsequent activation of ERK1/2 and the downstream signaling events that they mediate [ 35 ] . GDC-0994, on the other hand, is a selective ERK inhibitor that directly targets the active site of ERK1/2, preventing their ability to phosphorylate target proteins and thus disrupting the signal transduction pathway [ 36 ] . These inhibitors have been widely used in research to investigate the specific functions of the ERK1/2 signaling pathway in different cellular contexts and to explore potential therapeutic applications in diseases where this pathway is dysregulated, such as cancer and fibrosis. It is worth further exploring whether these inhibitors can also treat liver fibrosis and whether their combination with PD98059 can exert better effects. Furthermore, this research has shown the effectiveness of PD98059 in treating liver fibrosis in vitro model. Nevertheless, investigating the chronic safety and efficacy of PD98059, as well as its possible toxicities, is essential for advancing its clinical application. This work primarily focuses on how LGALS3 affects liver fibrosis by modulating the ERK1/2 signaling pathway. However, the specific molecular mechanisms involved, such as how LGALS3 interacts with key molecules in the ERK1/2 signaling pathway, and how these interactions contribute to the progression of liver fibrosis, have not been elucidated.Meanwhile, multiple signaling pathways play a role in the process of liver fibrosis. For instance, the PI3K/AKT signaling pathway, JNK and NF-κB signaling pathwayare crucial in the inflammatory responsesassociated with liver fibrosis. The PI3K/AKT pathway, for example, is involved in cell survival and proliferation, and its activation can lead to the accumulation of inflammatory cells and the production of pro-fibrotic factors [ 37 ] . In the context of liver fibrosis, many inflammation pathways interact synergistically, promoting both inflammatory responses and HSC activation to exacerbate the extent of liver fibrosis. [ 38 ] . Elucidating the functional mechanisms of these molecular signaling pathways in liver fibrosis is critical for precision therapeutic interventions aimed at arresting fibrotic deterioration and restoring hepatic physiological integrity. Moreover, the role of LGALS3 in regulating liver fibrosis progression via these signaling pathways warrants further study. 5. Conclusion In conclusion, this study reveals that LGALS3 expression is upregulated in liver fibrosis, and its overexpression promotes the phosphorylation of ERK1/2, while LGALS3 knockdown inhibits the phosphorylation of ERK1/2. The ERK1/2 inhibitor PD98059 effectively treats liver fibrosis, highlighting the critical role of the LGALS3-ERK1/2 signaling axis in fibrotic progression. These findings underscore the therapeutic potential of targeting LGALS3 or ERK1/2. Future investigations should systematically explore the crosstalk between LGALS3 and other pivotal signaling pathways, expedite the development of LGALS3-targeted therapeutic modalities, and identify robust biomarkers (such as serum LGALS3 concentrations and ERK1/2 phosphorylation status) for predicting treatment responsiveness. These endeavors are expected to substantially advance the mechanistic elucidation of liver fibrosis and facilitate the translation of precision therapeutic strategies. Declarations Acknowledgments Funding : This work was supported by Basic Research Operating Funds for Undergraduate Colleges and Universities in Heilongjiang Province (2024-KYYWF-0363). Footnote Conflicts of Interest: The authors have no conflicts of interest to declare. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 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Cite Share Download PDF Status: Published Journal Publication published 14 Nov, 2025 Read the published version in European Journal of Medical Research → Version 1 posted Editorial decision: Revision requested 14 Aug, 2025 Reviews received at journal 14 Aug, 2025 Reviews received at journal 10 Aug, 2025 Reviewers agreed at journal 05 Aug, 2025 Reviewers agreed at journal 02 Aug, 2025 Reviewers invited by journal 01 Aug, 2025 Editor assigned by journal 31 Jul, 2025 Submission checks completed at journal 31 Jul, 2025 First submitted to journal 30 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. We do this by developing innovative software and high quality services for the global research community. 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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-7249117","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494583678,"identity":"9211a4a1-2220-4654-91cf-49b0cf7e84bc","order_by":0,"name":"Xin Zheng","email":"","orcid":"","institution":"The second affiliated hospital of Qiqihar medical university","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zheng","suffix":""},{"id":494583681,"identity":"61d2ba44-a225-46f0-841f-72e28dd77697","order_by":1,"name":"Lu Yang","email":"","orcid":"","institution":"The second affiliated hospital of Qiqihar medical university","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Yang","suffix":""},{"id":494583682,"identity":"efb63a84-9d26-4f52-b37b-88b7fab3b77b","order_by":2,"name":"Wenbin Wang","email":"","orcid":"","institution":"The second affiliated hospital of Qiqihar medical university","correspondingAuthor":false,"prefix":"","firstName":"Wenbin","middleName":"","lastName":"Wang","suffix":""},{"id":494583683,"identity":"31819e12-dfee-4fc1-b425-cda5045e9e43","order_by":3,"name":"Jingying Sun","email":"","orcid":"","institution":"Department of Brucellosis","correspondingAuthor":false,"prefix":"","firstName":"Jingying","middleName":"","lastName":"Sun","suffix":""},{"id":494583684,"identity":"7c260e3d-8c14-4899-a96f-755757d00cea","order_by":4,"name":"Yue Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYBACPmYQWQHhSBClhQ2s5QxJWkAEYxtJWth5zCQ+zrOONjjAfPA2D4NdHhEO4zGTnLktPXfDAbZkax6G5GKitEjzbjsM1AJk8DAcSGwgSsvfOSAt/N9I0MLYALaFjVgtbMWWPcfSc2ceZjO2nGOQTFgLP//hjTd+1Fjn9h1vfnjjTYUdYS0MDBwGQIIZjBgYDAirBwL2BwxQ9aNgFIyCUTAKsAMAY+ky1bMJu6IAAAAASUVORK5CYII=","orcid":"","institution":"The second affiliated hospital of Qiqihar medical university","correspondingAuthor":true,"prefix":"","firstName":"Yue","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-30 06:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7249117/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7249117/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40001-025-03331-7","type":"published","date":"2025-11-14T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88362950,"identity":"ee393e76-cab6-4ddc-b784-fc1208c7a648","added_by":"auto","created_at":"2025-08-05 16:49:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":69797,"visible":true,"origin":"","legend":"\u003cp\u003eIn the TGF-β1-stimulated liver fibrosis model, there is a notable upregulation of LGALS3 expression.\u003c/p\u003e\n\u003cp\u003eA-B: RT-qPCR results showed that the expression level of α-SMA(A) and Collagen1(B) significantly increased in the TGF-β1-stimulated LX2 cell line-induced liver fibrosis group.\u003c/p\u003e\n\u003cp\u003eC-D: Western blot results showed that the expression level of α-SMA(C) and Collagen1(D) significantly increased in the TGF-β1-stimulated LX2 cell line-induced liver fibrosis group.\u003c/p\u003e\n\u003cp\u003eE: RT-qPCR(E) and western blot(F) results showed that the expression level of LGALS3 increased in the TGF-β1-stimulated LX2 cell line-induced liver fibrosis group.\u003c/p\u003e\n\u003cp\u003en = 3 independent experiments, with each experiment's technique repeated three times. Statistical data were compared by two-tailed unpaired t-tests. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/c27fdb51a4313048b25a2eaf.png"},{"id":88362956,"identity":"667c4381-8c0d-465a-9a5a-d49769df720e","added_by":"auto","created_at":"2025-08-05 16:49:37","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":423561,"visible":true,"origin":"","legend":"\u003cp\u003eThe overexpression of LGALS3 facilitates the activation and migration of LX-2 cells\u003c/p\u003e\n\u003cp\u003eA: RT-qPCR detection of LGALS3 mRNA levels at 72 hours of LGALS3 overexpression.\u003c/p\u003e\n\u003cp\u003eB: Western blot detection of LGALS3 protein levels at 72 hours of LGALS3 overexpression.\u003c/p\u003e\n\u003cp\u003eC: RT-qPCR detection of α-SMA and Collagen I mRNA levels in cells at 72 hours after transfection.\u003c/p\u003e\n\u003cp\u003eD: Western blot detection of α-SMA and Collagen I protein levels in cells at 72 hours after transfection.\u003c/p\u003e\n\u003cp\u003eE: Wound healing assay after LGALS3 overexpression in LX-2 cells.\u003c/p\u003e\n\u003cp\u003eF: Wound healing assay statistics after LGALS3 overexpression in LX-2 cells.\u003c/p\u003e\n\u003cp\u003en = 3 independent experiments, with each experiment's technique repeated three times. Statistical data were compared by two-tailed unpaired t-tests. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/c2e1f178d41a02461adcc484.jpeg"},{"id":88363763,"identity":"67c2a7eb-1441-473e-8116-7d60d1d1c1d3","added_by":"auto","created_at":"2025-08-05 16:57:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80865,"visible":true,"origin":"","legend":"\u003cp\u003eDecreasing the expression of LGALS3 suppresses the activation and migration of LX-2 cells\u003c/p\u003e\n\u003cp\u003eA: RT-qPCR detection of LGALS3 mRNA levels at 72 hours of LGALS3 knockdown;\u003c/p\u003e\n\u003cp\u003eB: Western blot detection of LGALS3 protein levels at 72 hours of LGALS3 knockdown;\u003c/p\u003e\n\u003cp\u003eC: RT-qPCR detection of α-SMA and Collagen I mRNA levels in cells 72 hours after transfection;\u003c/p\u003e\n\u003cp\u003eD: Western blot detection of α-SMA and Collagen I protein levels in cells 72 hours after transfection;\u003c/p\u003e\n\u003cp\u003eE: Wound healing assay after LGALS3 knockdown in LX-2 cells.\u003c/p\u003e\n\u003cp\u003eF: Wound healing assay statistics after LGALS3 knockdown in LX-2 cells.\u003c/p\u003e\n\u003cp\u003en = 3 independent experiments, with each experiment's technique repeated three times. Statistical data were compared by two-tailed unpaired t-tests. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/1b9a54f034af819566c7e401.png"},{"id":88362955,"identity":"0d9b71c5-ae90-4b1f-aacf-476a199e84e9","added_by":"auto","created_at":"2025-08-05 16:49:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86970,"visible":true,"origin":"","legend":"\u003cp\u003eLGALS3 facilitates the level of p-ERK\u003c/p\u003e\n\u003cp\u003eA: RT-qPCR detection of ERK1/2 expression at 72 hours in LGALS3-EV and LGALS3-OE groups.\u003c/p\u003e\n\u003cp\u003eB: Western blot detection of ERK1/2 expression at 72 hours in LGALS3-EV and LGALS3-OE groups.\u003c/p\u003e\n\u003cp\u003eC: Western blot detection of ERK1/2 phosphorylation level at 72 hours in LGALS3-EV and LGALS3-OE groups.\u003c/p\u003e\n\u003cp\u003eD: RT-qPCR detection of ERK1/2 expression at 72 hours in LGALS3-EV and LGALS3-KD groups.\u003c/p\u003e\n\u003cp\u003eE: Western blot detection of ERK1/2 expression at 72 hoursin LGALS3-EV and LGALS3-KD groups.\u003c/p\u003e\n\u003cp\u003eF: Western blot detection of ERK1/2 phosphorylation level at 72 hours in LGALS3-EV and LGALS3-KD groups.\u003c/p\u003e\n\u003cp\u003en = 3 independent experiments, with each experiment's technique repeated three times. Statistical data were compared by two-tailed unpaired t-tests. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/46a7d1dc18e7a0757b7daf3e.png"},{"id":88363764,"identity":"dd891cc4-bb32-4465-991a-8f9fdae3405a","added_by":"auto","created_at":"2025-08-05 16:57:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59822,"visible":true,"origin":"","legend":"\u003cp\u003ePD98059 suppresses the level of p-ERK\u003c/p\u003e\n\u003cp\u003eA: RT-qPCR detection of α-SMA and Collagen1 expression.\u003c/p\u003e\n\u003cp\u003eB: Western blot detection of α-SMA and Collagen1 expression.\u003c/p\u003e\n\u003cp\u003eC: RT-qPCR detection of LGALS3 expression in LGALS3-EV, LGALS3-OE, and LGALS3-OE+PD98059 groups.\u003c/p\u003e\n\u003cp\u003eD: RT-qPCR detection of ERK1/2 expression in LGALS3-EV, LGALS3-OE, and LGALS3-OE+PD98059 groups.\u003c/p\u003e\n\u003cp\u003eE: Western blot detection of LGALS3 and ERK1/2 protein expression in LGALS3-EV, LGALS3-OE, and LGALS3-OE+PD98059 groups.\u003c/p\u003e\n\u003cp\u003eF: Western blot detection of ERK1/2 phosphorylation status in LGALS3-EV, LGALS3-OE, and LGALS3-OE+PD98059 groups.\u003c/p\u003e\n\u003cp\u003en = 3 independent experiments, with each experiment's technique repeated three times. Statistical data were compared by two-tailed unpaired t-tests. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/7506e51df8f0b6e08073b2b1.png"},{"id":88362958,"identity":"545c651f-3b96-4300-8a04-04a8fd07674b","added_by":"auto","created_at":"2025-08-05 16:49:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":101510,"visible":true,"origin":"","legend":"\u003cp\u003ePD98059 mitigates liver fibrosis induced by the TGF-β1-stimulated LX2 cell line.\u003c/p\u003e\n\u003cp\u003eA: RT-qPCR detection of α-SMA and Collagen1 mRNA expression in the control group (Control), TGF-β1-induced liver fibrosis group (TGF-β1), and TGF-β1-induced liver fibrosis group treated with PD98059 (TGF-β1+PD98059).\u003c/p\u003e\n\u003cp\u003eB: Western blot detection of α-SMA and Collagen1 protein expression..\u003c/p\u003e\n\u003cp\u003eC:RT-qPCR detection of LGALS3 mRNA expression.\u003c/p\u003e\n\u003cp\u003eD:RT-qPCR detection of ERK1/2 mRNA expression.\u003c/p\u003e\n\u003cp\u003eE: Western blot detection of LGALS3 and ERK1/2 protein expression.\u003c/p\u003e\n\u003cp\u003eF:Western blot detection of the phosphorylation status of ERK1/2.\u003c/p\u003e\n\u003cp\u003en = 3 independent experiments, with each experiment's technique repeated three times. Statistical data were compared by two-tailed unpaired t-tests. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. Data are presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/4052041f7fdf7b459de58e4f.png"},{"id":88362964,"identity":"b09e9dd1-bc84-40ac-b653-e5877b72bcb2","added_by":"auto","created_at":"2025-08-05 16:49:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":136108,"visible":true,"origin":"","legend":"\u003cp\u003eLGALS3 promotes liver fibrosis through upregulating the level of p-ERK.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/d567eb8230a305dbcf4aaac1.png"},{"id":96104968,"identity":"0bdd60d6-ccfb-4da3-a116-5da6cce51ac5","added_by":"auto","created_at":"2025-11-17 16:05:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1873687,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7249117/v1/b5e120ca-a840-4b0e-b41f-b894b0cb5967.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LGALS3 promotes liver fibrosis by enhancing the expression and phosphorylation of ERK1/2","fulltext":[{"header":"Research Highlights","content":"\u003cp\u003e1. Liver fibrosis model induced by TGF-\u0026beta;1 was established, in which the expression of LGALS3 was increased.\u003c/p\u003e\n\u003cp\u003e2. LGALS3 overexpression induces the expression and phosphorylation of ERK1/2, thereby facilitating the progression of liver fibrosis.\u003c/p\u003e\n\u003cp\u003e3. The therapeutic effect of PD98059 targeting ERK1/2 on liver fibrosis.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eLiver fibrosis, a common pathological endpoint of chronic liver diseases, represents a significant global health burden\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Approximately 1.75\u0026nbsp;million deaths worldwide are attributed to liver fibrosis and its complications annually, with an estimated 20\u0026ndash;30% of patients progressing to cirrhosis within 5\u0026ndash;10 years of disease onset. This condition is characterized by excessive extracellular matrix (ECM) deposition, primarily type I collagen, and disruption of liver lobular architecture, leading to impaired hepatic function and an increased risk of hepatocellular carcinoma\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Despite advancements in etiological treatments (e.g., antiviral therapy for hepatitis C), no specific anti-fibrotic drugs have been approved by regulatory agencies, highlighting the urgent need for novel therapeutic targets\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.​\u003c/p\u003e\u003cp\u003eTransforming growth factor-β1 (TGF-β1), a prototypic profibrotic cytokine, plays a central role in the activation of hepatic stellate cells (HSCs). With its high tissue expression and bioactivity, TGF-β1 directly activates HSCs, inducing the expression of α-smooth muscle actin (α-SMA) and collagen type I (Collagen I) \u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Currently, TGF-β1 is widely used to induce cellular fibrosis. TGF-β1 stimulates HSCs to produce type I collagen (CollagenI), thereby mediating the fibrosis process\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Preclinical studies have shown that blocking TGF-β1 signaling reduces fibrosis in animal models\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. However, clinical trials targeting TGF-β1 have faced challenges due to off-target effects, underscoring the necessity to explore alternative pathways\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.​\u003c/p\u003e\u003cp\u003eGalectin-3 (LGALS3) has emerged as a key regulator in fibrotic diseases across multiple organs\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In cardiac fibrosis, LGALS3 promotes myofibroblast activation by enhancing TGF-β1 signaling and facilitating the recruitment of inflammatory cells, ultimately leading to myocardial ECM remodeling\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In idiopathic pulmonary fibrosis (IPF), LGALS3 upregulation contributes to fibroblast accumulation and alveolar destruction\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. LGALS3 secreted by hepatocellular carcinoma (HCC) cells promotes HCC bone metastasis via facilitating the establishment of a pre-metastatic niche\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, the role of LGALS3 in liver fibrosis remains unknown. These findings suggest that LGALS3 may also orchestrate liver fibrosis through similar molecular mechanisms.​\u003c/p\u003e\u003cp\u003eTGF-β1 can induce the occurrence of liver fibrosis\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The current study utilized TGF-β1 to trigger the activation of LX-2 cells. α-SMA and Collagen1 are regarded as indicative markers of HSC activation\u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe ERK signaling pathway exerts a critical function in maintaining cell survival by suppressing multiple steps of apoptotic signaling cascades. Additionally, ERK activation is observed in a variety of tumor types\u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.In breast cancer cells, LGALS3 enhances ERK1/2 phosphorylation through CD44, promoting cancer cell migration\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Therefore, we hypothesize that LGALS3 directly activates the phosphorylation of ERK1/2 in HSCs.\u003c/p\u003e\u003cp\u003ePD98059 is a reversible MEK inhibitor, and studies have shown that PD98059 inhibits the activation of the ERK signaling pathway in prostate cancer, thereby inhibiting cancer development. The ERK1/2 inhibitor PD98059 was selected based on its high specificity and efficacy in blocking the MEK-ERK cascade\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. This investigation seeks to illuminate the role of LGALS3 in human liver fibrosis and identify potential therapeutic targets within the LGALS3-ERK1/2 axis.​\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Cell culture and treatment\u003c/h2\u003e\u003cp\u003eThe human hepatic stellate cell line, sourced from procell in Wuhan, China, was cultured in DMEM medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% penicillin-streptomycin (HyClone). These cells were maintained in incubator at 37\u0026deg;C for 48 hours, either with or without 5 ng/mL TGF-β1 (MCE) treatment for 24h, 48h, 72h\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Using 50\u0026micro;mol/L PD98059 to treat LGALS3 overexpressed cell line for 24h\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell transfection\u003c/h2\u003e\u003cp\u003eLGALS3 shRNA targeting sequences5'-ACCCAAACCCTCAAGGATATC-3\u0026rsquo;; were constructed using pLKO.1 vector.\u003c/p\u003e\u003cp\u003eA non-silencing shRNA with the scrambled sequence 5'-GCAAGCTGACCCTGAAGTTCAT-3' was used as a control;were constructed using pLKO.1 vector.To achieve stable and durable LGALS3 knockdown in LX-2 cells, the pLKO.1-puro-shLGALS3 lentivirus was constructed and packaged. The lentivirus was packed using 293T cells with psPAX2 (4.5 \u0026micro;g), pMD2.G (3 \u0026micro;g) and pLKO.1-puro-shLGALS3 plasmid (6 \u0026micro;g) in 60mm dishs, and cells were transfected with plasmid using Lipofectamine 2000 (Invitrogen, USA) according to themanufacturer\u0026rsquo;s guidelines. After 72 h of infection, the cells were cultured in a medium containing puromycin(1mg/mL) for 1 month.\u003c/p\u003e\u003cp\u003eFor the construction of LGALS3-overexpressed cell line in LX-2, LX2 cells were plated in 6-well plates at the density of 2\u0026times;10\u003csup\u003e6\u003c/sup\u003ecells/well. pcDNA3.1-LGALS3 vector, and pcDNA3.1 empty vector using Lipofectamine 2000(Invitrogen) as the manufacturer recommended.\u003c/p\u003e\u003cp\u003eSuccessful knockdown and overexpression of LGALS3 was confirmed by RT-qPCR and western blot.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated from LX2 cells with TRIzol reagent (Beyotime). Subsequently, the extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Takara). This was followed by quantitative PCR amplification employing ChamQ Universal SYBR qPCR Master Mix (Vazyme). The qRT-PCR reactions were performed on Quantagene q225 Fluorescent Quantitative PCR Instrument. The relative expression levels were determined by normalizing to GAPDH, utilizing the 2\u0026minus;∆∆Ct method.\u003c/p\u003e\u003cp\u003eThe primers were listed as follows:\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOrientation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eα-SMA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CCCTGGAGAAGAGCTACGAG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eα-SMA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-GTACGACCAGAGGCATACAG\u0026nbsp;-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCollagen1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5' - CAGCCGCTTCACCTACAGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCollagen1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5' - GGTCACCTTCACCGTTCTC \u0026minus;\u0026thinsp;3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLGALS3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CCATCTTCTGGACAGCCAAGTG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLGALS3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-TATCAGCATGCGAGGCACCACT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-ACAACTTTGGTATCGTGGAAGG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eforward\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-GCCATCACGCCACAGTTTC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ereverse\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Western blotting\u003c/h2\u003e\u003cp\u003eCell lysates were generated using ice-cold RIPA buffer (Beyotime). The protein concentration of the lysates was measured by BCA Protein Concentration Assay Kit (Beyotime). The lysates were subjected to SDS-PAGE ,every lane was added 40 ug lysates.The resolved proteins were electrotransferred onto PVDF membranes. To minimize non-specific antibody binding, the membranes were blocked with a 5% BSA solution(Sigma).Thereafter, the membranes were incubated with the respective primary antibodies at 4\u0026deg;C overnight. The primary antibodies employed were as follows: α- SMA antibody (Abclonal; dilution ratio 1:1000), Collagen1 antibody (Abclonal; dilution ratio 1:500), LGALS3 antibody (Abclonal; dilution ratio 1:1000), and GAPDH antibody (Servicebio; dilution ratio 1:1000).Following the overnight incubation with primary antibodies, the membranes were incubated with HRP-conjugated secondary antibodies (Promega; dilution ratio 1:10000) for 1 hour at room temperature. Protein bands were visualized and detected using BeyoECL Moon (Beyotime).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Wound healing assay\u003c/h2\u003e\u003cp\u003eIn the wound healing assay, cells were plated in 6-well plates, and scratches were created using a 1-ml pipette tip. Using an inverted microscope, representative images of cell migration into the wounds were acquired at 0, 48, and 72 hours in the same scratched area (10 fields per group). The scratch closure distance was quantified with Image J software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 CCK8 assay\u003c/h2\u003e\u003cp\u003eLX-2 cells were plated in 96-well plates at a concentration of 2\u0026times;10^3 cells per well. The proliferation of cells was evaluated using the Cell Counting Kit from yeasen, following the protocol specified by the manufacturer. The absorbance was determined at 490 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analyses\u003c/h2\u003e\u003cp\u003eStatistical analyses were conducted using GraphPad Prism 8 software, with the specific tests employed detailed in the respective figure legends. For multiple comparisons, t-tests were carried out with adjustments made via the Holm\u0026ndash;Sidak method. In cases specified in the figure legends, two-tailed unpaired t-tests were utilized. A threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was set to denote statistical significance, with precise P value notations provided in each figure legend.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 LGALS3 is highly expressed in TGF-\u0026beta;1-stimulated LX2 cell line-induced liver fibrosis\u003c/h2\u003e\n \u003cp\u003eTransforming growth factor beta (TGF-\u0026beta;) plays a crucial role in chronic inflammation-related diseases. TGF-\u0026beta; signaling generally inhibits the proliferation of normal epithelial, endothelial, and immune cells and promotes the differentiation of fibroblast cell lineages that deposit extracellular matrix (ECM) components. \u0026alpha;-SMA, and Collagen1 were considered as biomarkers of HSC activation.We established an experimental liver fibrosis cell model by inducing liver fibrosis in the LX2 cell with TGF-\u0026beta;1. To assess the fibrotic levels of LX-2 cells after TGF-\u0026beta;1 stimulation, we examined the expression of \u0026alpha;-SMA and Collagen1 in LX-2 cells at 0h, 24h, 48h, and 72h. The results of RT-qPCR and western blot indicated that after 24 and 48 hours of TGF-\u0026beta;1 stimulation of LX-2 cells, the expression levels of \u0026alpha;-SMA and Collagen1 remained unchanged (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). This suggested that 48 hours was insufficient to activate LX-2 cells. Therefore, the expression of \u0026alpha;-SMA and Collagen1 was analyzed in LX-2 cells upon 72-hour TGF-\u0026beta;1 stimulation. The RT-qPCR results showed that compared with the control group, the expression of \u0026alpha;-SMA and Collagen1 mRNA in the TGF-\u0026beta;1 treatment group increased by 2 fold (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). The western blot indicated that the protein expression levels of \u0026alpha;-SMA and Collagen1 increased by 2 fold at 72h, and the differences were statistically significant (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-D).(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). Meanwhile, the expression level of LGALS3 significantly increased at 72h by 2 fold compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). This indicates that the expression of LGALS3 is increased after the activation of LX-2 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 LGALS3 overexpression promotes the activation and migratory of LX-2 cells\u003c/h2\u003e\n \u003cp\u003eNext, we wanted to determine whether LGALS3 overexpression promoted the activation and migration of LX-2 cells. We transfected LX-2 with LGALS3 to construct LGALS3 overexpressed cell line (LGALS3-OE) and transfected LX-2 with an empty vector as conrtol(LGALS3-EV). After transfection, we detected the expression of LGALS3 by performing RT-qPCR and western blot to verify the transfection efficiency. The results showed that the expression level of LGALS3 increases by 2.5-fold after 72 hours of LGALS3 overexpression (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-B).\u003c/p\u003e\n \u003cp\u003eThe expression levels of \u0026alpha;-SMA and Collagen I significantly increased by 2-fold and 3-fold separately after 72 hours of LGALS3 overexpression (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). Wound healing assay indicated that LGALS3 overexpression enhanced the migration ability of LX-2 cells at 72h (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). These results suggested that LGALS3 overexpression can promote the activation and migration of LX-2 cells.The results suggest that overexpression of LGALS3 enhances the activation and migration of LX-2 cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Knockdown of LGALS3 suppresses the activation and migration of LX-2 cells\u003c/h2\u003e\n \u003cp\u003eTo address whether LGALS3 knockdown inhibited the activation and migration of LX-2 cells, we transfected LX-2 with LGALS3 shRNA to construct LGALS3 knockdown cell line (LGALS3-KD) and transfected LX-2 with an empty vector as control(LGALS3-EV). After transfection, we performed RT-qPCR and western blot for LGALS3 to verify the transfection efficiency. The results showed that the expression level of LGALS3 was significantly reduced by 2-fold after 72 hours of LGALS3 knockdown (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-B).Using RT-qPCR and western blot, we analyzed the expression of \u0026alpha;-SMA and Collagen I in the cells 72 hours after transfection to detect the activation status of LX-2 cells. The expression levels of \u0026alpha;-SMA and Collagen I significantly decreased by 3-fold after LGALS3 knockdown (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Wound healing assay indicated that LGALS3 knockdown inhibited the migration ability of LX-2 cells after 72 hours(Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). These results suggest that LGALS3 knockdown can inhibit the activation and migration of LX-2 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 LGALS3 promotes liver fibrosis through upregulating the level of p-ERK\u003c/h2\u003e\n \u003cp\u003eTo determine whether the change of LGALS3 expression in liver are associated with the activation of the ERK, we detected the expression and phosphorylation status of ERK1/2 protein in the LGALS3 overexpression and LGALS3 knockdown cell lines. The results showed that the expression of ERK1/2 remained unchange(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-B), while the phosphorylation levels of ERK1/2 after 72 hours of LGALS3 overexpression increased by 3-fold(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The expression of ERK1/2 remained unchange(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD-E), while the phosphorylation levels of ERK1/2 decreased by 2-fold after LGALS3 knockdown 72 hours(Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD-F).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 PD98059 reverses the effects of increased LGALS3 expression\u003c/h2\u003e\n \u003cp\u003eWe wanted to know whether PD98059 can inhibit the level of p-ERK and the expression of LGALS3 in LX-2 cells. We added PD98059 to the LGALS3 overexpression cell line and detected the expression of \u0026alpha;-SMA and Collagen1. The results showed that after the addition of PD98059, the expression of \u0026alpha;-SMA and Collagen1 significantly decreased by 2-fold (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Next, we detected the expression of LGALS3, it significantly decreased by 2-fold after the addition of PD98059 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, E). Although the expression of ERK1/2 remained unchange after PD98059 treat, PD98059 significantly reduced the expression and phosphorylation levels of ERK1/2 by 2-fold (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, F). These results indicate that PD98059 can reverse the effects of increased LGALS3 expression.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 PD98059 alleviates TGF-\u0026beta;1-stimulated LX2 cell line-induced liver fibrosis\u003c/h2\u003e\n \u003cp\u003eNext, we wanted to explore whether PD98059 can be used to treat liver fibrosis in liver fibrosis cell models.The results showed that TGF-\u0026beta;1 stimulation of LX2 cells increased the expression of \u0026alpha;-SMA and Collagen1, while the expression of \u0026alpha;-SMA and Collagen1 significantly decreased after PD98059 treatment 72h(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). PD98059 also significantly reducde the expression of LGALS3 by 2-fold(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, E). Although the expression of ERK1/2 remained unchange after PD98059 treat, PD98059 reduced the phosphorylation levels of ERK1/2 after 72h of TGF-\u0026beta;1 stimulation(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). These results indicate that PD98059 can alleviate TGF-\u0026beta;1-stimulated LX2 cell line-induced liver fibrosis.\u003c/p\u003e\n \u003cp\u003eIn liver fibrosis, the expression of LGALS3 increases, promoting the phosphorylation of ERK1/2. The ERK1/2 inhibitor PD98059 alleviates the phenotypes associated with liver fibrosis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLiver fibrosis, a common outcome of various chronic hepatic disorders, is characterized by excessive extracellular matrix (ECM) accumulation and disruption of the liver lobular structure. Activated hepatic stellate cells (HSCs) undergo phenotypic transformations that are pivotal for the initiation and progression of liver fibrosis. In experimental models, TGF-β1 is commonly used to induce HSC activation.\u003c/p\u003e\u003cp\u003eAlthough LX-2 shares similar genetic phenotypes with HSCs in normal human livers, a cell line cannot fully represent the complex in vivo liver microenvironment and the interactions among various cell types, such as hepatocytes, endothelial cells, and immune cells, during the process of liver fibrosis. Moreover, during in vitro culture, the state and response of LX-2 cells may be influenced by culture conditions, such as the composition of the culture medium, cell density, and the number of passages. Furthermore, this model primarily focuses on the TGF-β1 signaling pathway and falls short in studying the interactions and regulatory mechanisms of other related signaling pathways.\u003c/p\u003e\u003cp\u003eThis study reveals the key effect of LGALS3 in liver fibrosis and demonstrates its potential as a therapeutic target. Inhibition of LGALS3 not only alleviates the activation of hepatic stellate cells but also mitigates liver fibrosis by suppressing the expression and phosphorylation of ERK1/2. PD98059 has been reported to inhibit ERK1/2\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. The inhibition of ERK1/2 has been reported to inhibit liver fibrosis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. However, the mechanism by which PD98059 inhibits the expression and phosphorylation of ERK1/2 and how this inhibition leads to the reversal of liver fibrosis, requires further investigation. In this study, we used PD98059 to inhibit the occurrence of liver fibrosis and achieved good results. The ERK1/2 signaling cascade, with multiple inhibitors, is pivotal in governing cellular proliferation, differentiation, and survival. For example, U0126 works by blocking the activation of MEK1/2, which phosphorylate and activate ERK1/2. This inhibition of MEK1/2 activity prevents the subsequent activation of ERK1/2 and the downstream signaling events that they mediate\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. GDC-0994, on the other hand, is a selective ERK inhibitor that directly targets the active site of ERK1/2, preventing their ability to phosphorylate target proteins and thus disrupting the signal transduction pathway\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. These inhibitors have been widely used in research to investigate the specific functions of the ERK1/2 signaling pathway in different cellular contexts and to explore potential therapeutic applications in diseases where this pathway is dysregulated, such as cancer and fibrosis. It is worth further exploring whether these inhibitors can also treat liver fibrosis and whether their combination with PD98059 can exert better effects. Furthermore, this research has shown the effectiveness of PD98059 in treating liver fibrosis in vitro model. Nevertheless, investigating the chronic safety and efficacy of PD98059, as well as its possible toxicities, is essential for advancing its clinical application.\u003c/p\u003e\u003cp\u003eThis work primarily focuses on how LGALS3 affects liver fibrosis by modulating the ERK1/2 signaling pathway. However, the specific molecular mechanisms involved, such as how LGALS3 interacts with key molecules in the ERK1/2 signaling pathway, and how these interactions contribute to the progression of liver fibrosis, have not been elucidated.Meanwhile, multiple signaling pathways play a role in the process of liver fibrosis. For instance, the PI3K/AKT signaling pathway, JNK and NF-κB signaling pathwayare crucial in the inflammatory responsesassociated with liver fibrosis. The PI3K/AKT pathway, for example, is involved in cell survival and proliferation, and its activation can lead to the accumulation of inflammatory cells and the production of pro-fibrotic factors\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In the context of liver fibrosis, many inflammation pathways interact synergistically, promoting both inflammatory responses and HSC activation to exacerbate the extent of liver fibrosis.\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Elucidating the functional mechanisms of these molecular signaling pathways in liver fibrosis is critical for precision therapeutic interventions aimed at arresting fibrotic deterioration and restoring hepatic physiological integrity. Moreover, the role of LGALS3 in regulating liver fibrosis progression via these signaling pathways warrants further study.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, this study reveals that LGALS3 expression is upregulated in liver fibrosis, and its overexpression promotes the phosphorylation of ERK1/2, while LGALS3 knockdown inhibits the phosphorylation of ERK1/2. The ERK1/2 inhibitor PD98059 effectively treats liver fibrosis, highlighting the critical role of the LGALS3-ERK1/2 signaling axis in fibrotic progression. These findings underscore the therapeutic potential of targeting LGALS3 or ERK1/2. Future investigations should systematically explore the crosstalk between LGALS3 and other pivotal signaling pathways, expedite the development of LGALS3-targeted therapeutic modalities, and identify robust biomarkers (such as serum LGALS3 concentrations and ERK1/2 phosphorylation status) for predicting treatment responsiveness. These endeavors are expected to substantially advance the mechanistic elucidation of liver fibrosis and facilitate the translation of precision therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e: This work was supported by Basic Research Operating Funds for Undergraduate Colleges and Universities in Heilongjiang Province (2024-KYYWF-0363).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnote\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflicts of Interest: The authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003eEthical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003eContributions: (I) Conception and design: Xin Zheng ;(II) Administrative support: \u0026nbsp;Lu Yang ; (III) Provision of study materials or patients: Jingying Sun; (IV) Collection and assembly of data: Yue Li ; (V) Data analysis and interpretation: Wenbin Wang ; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKarsdal MA, Manon-Jensen T, Genovese F, et al. 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Mil Med Res, 2023,10(1):56.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LGALS3, liver fibrosis, ERK1/2, PD98059","lastPublishedDoi":"10.21203/rs.3.rs-7249117/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7249117/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground:\u003c/p\u003e\n\u003cp\u003eLiver fibrosis represents the common endpoint of numerous chronic liver diseases. Current therapeutic approaches predominantly focus on alleviating symptoms or addressing the underlying causes, with limited options available for reversing fibrosis. Existing antifibrotic drugs often exhibit low efficacy and carry significant side effects, underscoring the urgent need for novel therapeutic targets. LGALS3, a β-galactoside-binding lectin, participates in inflammation and fibrosis across multiple organs. However, its specific role in liver fibrosis remains poorly understood. This study endeavors to elucidate the cellular and molecular mechanisms by which LGALS3 regulates liver fibrosis and assess its potential as a therapeutic target.\u003c/p\u003e\n\u003cp\u003eSubjects and Methods:\u003c/p\u003e\n\u003cp\u003eTo clarify the role of LGALS3 in liver fibrosis, a TGF-β1-induced liver fibrosis model was established, and the expression level of LGALS3 was analyzed. LGALS3 overexpression and knockdown cell lines were constructed in LX-2 cell line. RT-qPCR, western blot, CCK8 assay, and wound healing assay were employed to investigate the impact of LGALS3 on LX-2 cell activation, proliferation, migration, and the ERK1/2 pathway. In the LGALS3 overexpression cell model, PD98059 intervention was applied to mimic the therapeutic effect on liver fibrosis.\u003c/p\u003e\n\u003cp\u003eResults:\u003c/p\u003e\n\u003cp\u003eLGALS3 overexpression significantly promoted the proliferation and migration of LX-2 cells, along with the expression and phosphorylation of ERK1/2. Conversely, LGALS3 knockdown and treatment with the PD98059 inhibitor reduced LX-2 cell proliferation and migration, as well as the expression and phosphorylation of ERK1/2.\u003c/p\u003e\n\u003cp\u003eConclusions:\u003c/p\u003e\n\u003cp\u003eThis study has clarified the pivotal regulatory role of the LGALS3-ERK1/2 signaling axis in liver fibrosis, uncovering novel molecular mechanisms underlying the activation, proliferation, and migration of hepatic stellate cells. These findings enhance our understanding of the pathological process of liver fibrosis, suggesting that targeting the LGALS3-ERK1/2 axis may serve as a novel therapeutic strategy for intervening in liver fibrosis. It also provides potential targets and innovative directions for the development of anti-liver fibrosis drugs.\u003c/p\u003e","manuscriptTitle":"LGALS3 promotes liver fibrosis by enhancing the expression and phosphorylation of ERK1/2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 16:49:32","doi":"10.21203/rs.3.rs-7249117/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-14T10:19:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T09:07:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T03:44:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99064552706582686183544944724002100911","date":"2025-08-05T04:00:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93571764847075650136157888773450214662","date":"2025-08-02T06:29:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-01T08:51:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-31T08:40:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-31T07:35:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Medical Research","date":"2025-07-30T06:15:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3ada669-fd2b-4da6-9788-9cbccc2c817c","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T15:59:49+00:00","versionOfRecord":{"articleIdentity":"rs-7249117","link":"https://doi.org/10.1186/s40001-025-03331-7","journal":{"identity":"european-journal-of-medical-research","isVorOnly":false,"title":"European Journal of Medical Research"},"publishedOn":"2025-11-14 15:56:57","publishedOnDateReadable":"November 14th, 2025"},"versionCreatedAt":"2025-08-05 16:49:32","video":"","vorDoi":"10.1186/s40001-025-03331-7","vorDoiUrl":"https://doi.org/10.1186/s40001-025-03331-7","workflowStages":[]},"version":"v1","identity":"rs-7249117","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7249117","identity":"rs-7249117","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}