Development of β-Catenin/BCL9 Targeted Inhibitors and Investigation of Their Anti-Fibrotic Mechanism in Liver Fibrosis

Development of β-Catenin/BCL9 Targeted Inhibitors and Investigation of Their Anti-Fibrotic Mechanism in Liver Fibrosis | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 22 July 2025 V1 Latest version Share on Development of β-Catenin/BCL9 Targeted Inhibitors and Investigation of Their Anti-Fibrotic Mechanism in Liver Fibrosis Authors : Gang Chen , Yangbo , Huiyu Li , Yunlu Li , Cuiting Liu , Anqi Li , Yanfang Xian 0000-0002-5032-0366 , … Show All … , Xianjing Meng , Mei Feng , Wei lu , Daizhou Zhang , Chonggang Duan , and Di Zhu [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.175318772.20367004/v1 163 views 94 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Targeting β-catenin directly is challenging, but the β-catenin/BCL9 interaction shows promise for treating hepatic fibrosis. Pro-fibrotic M2 macrophages drive fibrosis progression via aberrant Wnt/β-catenin signaling. We developed novel peptide inhibitors based on hsBCL9 CT-24 to block β-catenin/BCL9 and alleviate fibrosis. Experimental Approach: Structure-activity relationship (SAR) optimization through biochemical assays yielded the lead peptide hsBCL9Z96. Pharmacological (hsBCL9 Z96 ) and genetic BCL9 inhibition were evaluated in fibrotic mice. Mechanisms were analyzed via flow cytometry and PCR. Key Results: SAR-optimized hsBCL9Z96 potently inhibited β-catenin/BCL9 in vitro (IC 50 = 0.02 μM). Both pharmacological and genetic BCL9 inhibition attenuated fibrosis in vivo, effectively blocking M2 macrophage polarization and TGF-β secretion; modulating Treg proportions and their IFN-γ secretion to suppress M2 conversion; and activating MMP13-mediated extracellular matrix (ECM) degradation. This triple mechanism synergistically reduced hepatic stellate cell activation and fibrogenesis. Conclusion and Implications: The β-catenin/BCL9 axis is a validated therapeutic target. hsBCL9 Z96 demonstrates favorable in vivo properties and alleviates fibrosis through dual mechanisms: inhibiting M2 polarization and promoting fibro lysis. It holds translational potential pending further immunomodulatory studies. Development of β -Catenin/BCL9 Targeted Inhibitors and Investigation of Their Anti-Fibrotic Mechanism in Liver Fibrosis Gang Chen 1，3# ；Yangbo He 1，3# ；Huiyu Li 2# ；Yunlu Li 2# ；Cuiting Liu 1，9 ；Anqi Li 2 ；Yan-Fang Xian 10 ；Xianjing Meng 5 ；Mei Feng 2 ；Wei lu 2 ；Daizhou Zhang 5 ；Chonggang Duan 5 ；Di Zhu 4，6，7，8* # Indicates that these authors’ contributions are consistent 1. Anhui University of Chinese Medicine, Hefei Anhui 230012； 2. Fudan University, Shanghai 200433； 3. Drug Research Institute of Yangtze Delta, Nantong Jiangsu 226133； 4. Department of Pharmacology, School of Basic Medical Sciences of Fudan University, Shanghai，201100 5. Shandong Academy Of Pharmaceutical Sciences, Jinan Shandong，250101； 6. Minhang Hospital, Fudan University, Shanghai 201100； 7. Key Laboratory of Tumor Immunology and Microenvironmental Regulation, Guilin Medical University, Guilin, Guangxi, 541004； 8. Department of Oncology, Second Affiliated Hospital of Guilin Medical University, Guilin, Guangxi, 541004； 9. Nantong Jutai Biotechnology Co., Ltd., Nantong Jiangsu 226133. 10. School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, P.R. China； Background and Purpose: Targeting β-catenin directly is challenging, but the β-catenin/BCL9 interaction shows promise for treating hepatic fibrosis. Pro-fibrotic M2 macrophages drive fibrosis progression via aberrant Wnt/β-catenin signaling. We developed novel peptide inhibitors based on hsBCL9 CT-24 to block β-catenin/BCL9 and alleviate fibrosis. Experimental Approach: Structure-activity relationship (SAR) optimization through biochemical assays yielded the lead peptide hsBCL9Z96. Pharmacological (hsBCL9 Z96 ) and genetic BCL9 inhibition were evaluated in fibrotic mice. Mechanisms were analyzed via flow cytometry and PCR. Key Results: SAR-optimized hsBCL9Z96 potently inhibited β-catenin/BCL9 in vitro (IC₅₀ = 0.02 μM). Both pharmacological and genetic BCL9 inhibition attenuated fibrosis in vivo, effectively blocking M2 macrophage polarization and TGF-β secretion; modulating Treg proportions and their IFN-γ secretion to suppress M2 conversion; and activating MMP13-mediated extracellular matrix (ECM) degradation. This triple mechanism synergistically reduced hepatic stellate cell activation and fibrogenesis. Conclusion and Implications: The β-catenin/BCL9 axis is a validated therapeutic target. hsBCL9 Z96 demonstrates favorable in vivo properties and alleviates fibrosis through dual mechanisms: inhibiting M2 polarization and promoting fibro lysis. It holds translational potential pending further immunomodulatory studies. Keywords: Hepatic fibrosis; β-Catenin/BCL9; Kupffer cells; MMP13; Immunology; Stapled peptide 1. Introduction The global burden of liver disease continues to escalate, with epidemiological evidence indicating that approximately 1.5 billion people worldwide suffer from chronic liver conditions, resulting in nearly 1.2 million annual fatalities[1]. Preventing hepatic disease progression remains a major therapeutic challenge. Liver pathologies are clinically categorized into acute and chronic injuries, with acute injury management being relatively straightforward compared to the complex treatment of chronic liver damage, where clinical outcomes critically depend on the extent of fibrosis and pathological deterioration. Hepatic fibrosis represents a convergent pathological pathway in progressive chronic liver diseases [2]. This condition is characterized by excessive extracellular matrix (ECM) accumulation and scar tissue formation following persistent hepatic injury [3]. During fibrogenesis, inflammatory signaling activates hepatic stellate cells (HSCs), driving their transdifferentiating into myofibroblasts that deposit ECM proteins [3]. Macrophages—key regulators of innate immunity—play dual roles in this process through their polarized phenotypes: classically activated (M1) and alternatively activated (M2) macrophages [4]. While M1 macrophages exacerbate post-inflammatory fibrosis, M2 macrophages paradoxically promote fibrogenesis via TGF- β secretion despite their anti-inflammatory functions in acute hepatitis [5, 6]. Notably, suppressing M2 polarization effectively attenuates fibrosis development [7]. Current antifibrotic strategies focus on inhibiting fibrotic progression, promoting hepatocyte regeneration, and mitigating inflammation. Although existing therapeutics (e.g., antivirals, anti-inflammatories, antifibrotics) show partial efficacy, the complexity of fibrotic mechanisms and chronic disease course necessitate novel targets and more effective treatments [8]. Within the hepatic microenvironment, Wnt/ β -Catenin signaling regulates liver homeostasis, development, and repair. β -Catenin critically drives fibrogenesis by activating HSC proliferation [9] and promoting M2 macrophage polarization [10]. Dysregulation of this pathway contributes to various liver pathologies including fibrosis, hepatoblastoma, and hepatocellular carcinoma [11]. While physiologically involved in cell adhesion and signaling, pathological β -Catenin activation triggers HSC transdifferentiating into collagen-secreting myofibroblasts, accelerating ECM deposition [12]. Furthermore, β -Catenin enhances proinflammatory cytokine secretion, establishing a feed-forward loop that perpetuates inflammation and fibrosis. Thus, β -Catenin represents both a key fibrotic regulator and promising therapeutic target. The β -Catenin/BCL9 protein-protein interaction (PPI) constitutes a validated target for inhibiting Wnt signal transduction [13]. However, the mechanistic role of BCL9-driven Wnt signaling in fibrogenesis remains incompletely understood. To address this, we previously developed hsBCL9 CT-24 —a stabilized α-helical stapled peptide inhibitor that effectively disrupts β -Catenin/BCL9 complex formation and selectively suppresses β -Catenin activity [14]. Building upon this foundation, we conducted extensive mutagenesis studies to design novel β -Catenin/BCL9 antagonists. Through systematic amino acid sequence optimization, we enhanced their PPI-inhibitory potency. Surface plasmon resonance (SPR) screening identified hsBCL9 Z96 as a high-affinity candidate (IC 50 = 0.02 μM) that potently blocks β -Catenin/BCL9 interaction and suppresses downstream signaling in vitro . Using hsBCL9 Z96 as a pharmacological tool, we investigated the mechanistic role of β -Catenin/BCL9 in fibrogenesis. Our findings demonstrate that this complex promotes HSC activation and upregulates profibrotic factors. hsBCL9 Z96 intervention effectively suppressed HSC hyperactivation and reduced hepatic fibrotic markers, attenuating fibrosis progression. The clinical relevance of BCL9 in fibrogenesis was further established through both genetic ablation studies and pharmacological inhibition with hsBCL9 Z96 , which protected against CCl 4 -induced fibrosis in murine models. Integrated single-cell RNA sequencing (scRNA-seq) and in vivo analyses revealed that BCL9 inhibition modulates macrophage M2 polarization via IFN-γ signaling and activates fibro lytic pathways (e.g., MMP13 -mediated ECM remodeling), collectively ameliorating hepatic fibrosis. 2. Materials and Methods 2.1 Stapled Peptide Synthesis Stapled peptides were synthesized via Fmoc solid-phase peptide synthesis (Fmoc-SPPS) using Rink amide resin (0.8 g, 0.52 mmol/g) as the solid support. The resin was swollen overnight in dichloromethane (DCM, 5 mL), filtered, and washed three times with N,N-dimethylformamide (DMF, 3×2 mL). Deprotection was performed by treating the resin with 20% (v/v) piperidine in DMF for 20 min. For amino acid coupling, Fmoc-2Nal-OH (87 mg, 0.2 mmol) and N,N-diisopropylethylamine (DIEA, 0.07 mL) in anhydrous DMF (5 mL) were added to the resin, followed by nitrogen bubbling at 25°C for 40 min. S5 and R8 residues required extended coupling (1 h). Sequential deprotection and coupling steps were repeated until the target linear peptide sequence was assembled. 2.1.1 Peptide Cyclization The fully protected linear peptide-resin was treated with DMF. Ring-closing metathesis (RCM) was initiated by adding a DCM solution containing second-generation Grubbs catalyst (0.125 g) to the resin. The reaction proceeded at 30°C for 12h under nitrogen. Post-reaction, the resin was drained and washed with DCM (4 × 10 mL). 2.1.2 Cleavage of Cyclized Peptides The cyclized peptide-resin was treated with cleavage cocktail (TFA/TIS/H₂O/phenol = 87.5:2.5:5:5, V/V/V/V, 5 mL) in a solid-phase reactor for 12 h at 25°C. The cleaved peptide was precipitated in pre-cooled diethyl ether, pelleted by centrifugation, washed, and vacuum-dried to yield crude products for LC-MS analysis. 2.1.3 Peptide Purification The reverse-phase high-performance liquid chromatography (RP-HPLC) system employed in this study was a NU3000C preparative chromatograph (Hanbang Technology Co., Ltd.). Preparative separations were performed using an Athena C18 semi-preparative column (20 mm × 250 mm, 10 μm) at a flow rate of 12 mL/min. The mobile phase consisted of ultrapure water (solvent A) supplemented with 0.1 vol% trifluoroacetic acid (TFA) and pure acetonitrile (ACN, solvent B), with a gradient elution method applied throughout the procedure. 2.2 Protein Purification The full-length β -Catenin fragment ( β -Catenin FL, residues 1–781) with a His-tag was cloned into the plasmid vector pET-28a, and recombinant plasmids were expressed in Escherichia coli (E. coli) strain DE3. The E. coli cells were cultured in LB medium containing 30 μg/mL kanamycin at 37°C and 200 rpm. When the optical density at 600 nm (OD600) reached 0.8, the culture was shifted to 18°C. Protein expression was induced by the addition of 0.2 mM IPTG (Isopropyl β -D-1-thiogalactopyranoside) for 18 hours. After induction, E. coli cells were harvested by centrifugation at 3500 rpm for 30 minutes at 4°C. The His-tagged β -Catenin FL protein was purified using a Ni-NTA affinity column. The purified β -Catenin FL protein was concentrated using a 10 kDa centrifugal concentrator and stored at −80°C. The protein was preserved in a buffer containing 25 mM Tris-HCl (pH 7.4), 200 mM NaCl, and 5% glycerol. 2.3 Fluorescence Polarization (FP) Assay The N-terminal FAM-labeled human BCL9 peptide (residues 350–375) was synthesized by Chinese Peptide (Hangzhou, China, purity with 180 μL of fluorescence polarization (FP) buffer, containing 25 mM HEPES, 100 mM NaCl, 0.01% Triton X-100, 0.1% BSA, and 20 nM BCL9-FAM tracer. At the beginning of the assay, 300 nM β -Catenin protein and varying concentrations of test compounds (0.625, 1.25, 2.5, 5, and 10 μM) were added to separate wells in triplicate. Negative control wells contained only the tracer without inhibitor, while positive control wells included 100% inhibition of the tracer and β -Catenin protein. The plate was shaken horizontally at room temperature for 2 hours to allow complete interaction between the compound and β -Catenin and competitive binding with BCL9-FAM. After shaking, fluorescence polarization was immediately measured using a microplate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively. The percentage inhibition was calculated using the formula: % inhibition = 100[1 − (mP − mP_positive) / (mP_negative − mP_positive)]. The results were analyzed using GraphPad software to determine the IC 50 value. 2.4 Animal Model Experiments This study was approved by the Fudan University Institutional Animal Care and Use Committee, and all experiments were conducted in accordance with the approved guidelines. Healthy male C57BL/6J mice (6–8 weeks, weighing 20–30 g) were purchased from the Fudan University Animal Center and housed in a pathogen-free environment with controlled temperature (24 ± 4°C) and a 12-hour light/dark cycle. After a 3-day acclimatization period, the mice were intraperitoneally injected with CCl 4 twice weekly to induce liver fibrosis. Two weeks post-induction, treatment with hsBCL9 Z96 was initiated by intraperitoneal injection every other day. CCl 4 injections continued twice weekly, and the entire experimental protocol lasted for six weeks. 2.5 Histological Analysis and Staining Mice were euthanized under deep anesthesia with 3% sodium pentobarbital (w/v). The liver was immediately excised, weighed, and prepared for further analysis. A portion of the liver tissue was fixed in 10% neutral buffered formalin (Jinyibai Biotechnology Co., Nanjing, China) at room temperature for 24–48 h. After fixation, the tissue was processed through dehydration, embedding, and sectioning. Masson’s trichrome staining was performed to assess collagen fiber deposition in the liver, followed by Sirius Red staining to further evaluate liver fibrosis. All tissue sections were examined and photographed under an optical microscope. Quantitative analysis of images was conducted using ImageJ software (National Institutes of Health, USA) to assess collagen content and fibrosis area. 2.6 Flow Cytometry Analysis To analyze liver macrophage phenotypes, particularly changes in the proportion of anti-fibrotic subsets, we performed flow cytometry on livers from alb-cre control and alb-cre BCL9 group mice. The specific steps were as follows: Livers were first digested via in situ two-step portal vein perfusion (sequentially using a calcium/magnesium-free buffer containing EGTA, followed by a digestion buffer containing collagenase IV/DNase I). The digested liver tissue was then homogenized and filtered to obtain a single-cell suspension. After low-speed centrifugation to remove parenchymal hepatocytes, the supernatant enriched in non-parenchymal cells (including macrophages) was collected. Subsequently, liver non-parenchymal cells were further enriched using Percoll density gradient centrifugation. The resulting cells were subjected to red blood cell lysis and resuspended in PBS containing 2% fetal bovine serum (FBS) (FACS buffer). Cells were treated with an Fc receptor blocking agent and then surface-stained with fluorochrome-conjugated antibodies (adding 1 µL each of CD45, CD25, CD11b, and CD206 antibodies per 100 µL of PBS buffer). After staining, cells were washed, resuspended in FACS buffer, and data acquisition was performed using a Beckman CytoFLEX S flow cytometer. Subsequent data analysis was conducted using FlowJo software (RRID: SCR_008520). 2.7 Real-Time Quantitative PCR Total RNA was extracted from mouse liver tissues according to the manufacturer’s instructions using TRIzol reagent (Invitrogen, USA). RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). cDNA was synthesized from 1 μg of total RNA using the PrimeScript™ RT reagent kit (with gDNA removal step, Takara, Japan). Real-time quantitative PCR was performed using the TB Green® Premix Ex Taq™ II (Takara, Japan) on the CFX96 Real-Time PCR system (Bio-Rad, USA). The amplification program consisted of an initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. All samples were analyzed in triplicate. The expression levels of inflammatory cytokines (IL-1 β , IL-17, TNF-α), pro-fibrotic factors (TGF- β 1, CTGF, PDGF), fibrosis markers (ACTA2, Fibronectin, COL1A1), and matrix metalloproteinases (MMP7, MMP12, MMP13) were measured. GAPDH was used as an internal reference gene, and relative expression levels were calculated using the 2^–ΔΔCt method. 2.8 Molecular Docking Studies Molecular docking was performed using MOE 2022 software. The process was divided into three main stages: molecular preparation, protein preparation, and analysis of docking results. First, the target molecules were modeled in 3D based on their sequences. Non-natural amino acids were incorporated into the molecular library for 3D modeling. The polypeptide library was then constructed, and the α-helix structure of the target molecule was inserted into the library. The molecular preparation stage was completed. Next, the β -Catenin protein was downloaded from the open-source PDB database and imported into MOE for residue repair and error correction. After ensuring the completeness of the protein’s residues, the protein preparation stage was completed. Finally, docking between the β -Catenin protein and the target molecules was carried out using MOE software. The docking results were exported and further processed with PyMOL software. 2.9 Statistical Analysis Statistical analyses were performed using GraphPad Prism 8 software. Data are expressed as mean ± standard deviation (SD). Differences between groups were analyzed using Student’s t-test or one-way or two-way analysis of variance (ANOVA), as appropriate. A P-value < 0.05 was considered statistically significant. 3. Structure Optimization and Biological Evaluation The hsBCL9 CT-24 peptide was designed and synthesized based on the critical α-helix region of the C-terminal domain of human BCL9, which binds to β -Catenin. This peptide was stabilized in its active conformation through the incorporation of non-natural amino acids and cyclization (”stapling”), making it an effective inhibitor of the β -Catenin/BCL9 PPI [14]. Molecular docking simulations were performed to predict the binding model of hsBCL9 CT-24 with β -Catenin, with the aim of further optimizing its inhibitory activity (Fig. 2A-B). The docking results revealed that residues L363, L366, I369, L373, and (2Nal)374 are involved in key hydrophobic interactions, while the charged residues R367 and R371 do not directly contribute to the binding. This observation prompted an investigation into the potential impact of charged amino acids on inhibitory activity. To assess the effects of charge, we substituted R367 and R371 with lysine (K) and glutamic acid (E), respectively, to generate mutants S1-S4 (Table 1). Activity assays demonstrated a significant reduction in the inhibitory activity of all mutants, with the negatively charged mutants S3 and S4 showing the most pronounced decreases in activity, approximately 17-fold and 28-fold, respectively. These results confirm that the overall charge state of the peptide significantly influences its inhibitory activity against β -Catenin, suggesting that the introduction of negatively charged amino acids should be carefully controlled in subsequent peptide design strategies. Table 1 Sequences of S1-S4 and their β -Catenin inhibitory activity hsBCL9 CT-24 LQTLRXIQRXL-(2Nal)-NH 2 2.7 S1 LQTLKXIQRXL-(2Nal)-NH 2 14.38 S2 LQTLRXIQKXL-(2Nal)-NH 2 10.61 S3 LQTLEXIQRXL-(2Nal)-NH 2 47.10 S4 LQTLRXIQEXL-(2Nal)-NH 2 50.00 In the previous analysis of the docking mode between hsBCL9 CT-24 and β -Catenin (Fig. 2A), we focused on the hydrophobic interactions mediated by residues L363, L366, I369, L373, and (2Nal)374. To optimize these interactions, we introduced amino acids with non-natural hydrophobic side chains at key positions in the hsBCL9 CT-24 backbone (Fig. 1). A series of M1-M14 mutants (Table 2) were designed and synthesized to evaluate their inhibitory activity. Fig. 1 Structure diagram of noncanonical amino acids side chains Systematic mutation of L373, replacing it with noncanonical amino acids such as allylglycine (Allyl Gly), cyclobutylalanine (Cba), and cyclohexylalanine (Cha), yielded mutants M1-M3. AlphaScreen assays showed that the M2 exhibited an approximately 8-fold increase in inhibitory activity, while M3 showed a 3-fold reduction in activity. These results suggest that amino acids with non-natural aliphatic side chains may improve hydrophobic interactions, with four-membered rings (Cba) offering superior enhancement compared to six-membered rings (Cha). Further substitutions at positions L363 and L366 with Cba were attempted to improve inhibitory activity, but these modifications did not yield the expected improvements. Additionally, to enhance receptor selectivity, N-methylation of Q362 was performed in the M2 to yield M6. This modification did not significantly alter activity, as the activity of M6 was comparable to M2, confirming the feasibility of the N-methylation strategy. Based on these findings, additional noncanonical amino acid substitutions were introduced at critical hydrophobic sites to create mutants M8-M14. However, none of these modifications improved inhibitory activity; some mutants even exhibited reduced activity. Molecular docking analysis (Fig. 2C-2G and 3A-3D) revealed that while all mutants retained the ability to form hydrophobic interactions with β -Catenin, spatial topological differences within the binding pocket led to varying levels of activity. Notably, the four-membered ring structure in M2 (Cba373) optimized the hydrophobic cavity, thereby enhancing interaction, whereas modifications at other sites failed to adequately fill the binding pocket or caused spatial displacements of key residues (e.g., 2Nal374), ultimately reducing activity. The choice of n noncanonical amino acid side chain conformation must align precisely with the local topology of the binding pocket to optimize hydrophobic interactions. Table 2 Sequences of M1-M14 and their β -Catenin inhibitory activity hsBCL9 CT-24 Ac-LQTLRXIQRXL(2Nal)-NH 2 2.70 M1 Ac-LQTLRXIQRX(Allyl G)(2-Nal)NH 2 9.71 M2 Ac-LQTLRXIQRX(Cba)(2-Nal)-NH 2 0.32 M3 Ac-LQTLRXIQRX(Cha)(2-Nal)-NH 2 10.08 M4 Ac-LQT(Cba)RXIQRX(Cba)(2-Nal)-NH 2 4.53 M5 Ac-(Cba)QTLRXIQRX(Cba)(2-Nal)-NH 2 6.99 M6 Ac-L(N-MeQ)TLRXIQRX(Cba)(2-Nal) -NH 2 0.42 M7 Ac-LQTLRXIQRXL(4’I-Phe)-NH 2 6.79 M8 Ac-L(N-MeQ)TLRX(Cba)QRX(Cba)(2-Nal)-NH 2 2.06 M9 Ac-L(N-MeQ)T(Cha)RXIQRX(Cba)(2-Nal)-NH 2 3.63 M10 Ac-L(N-MeQ)T(N-MeL)RXIQRX(Cba)(2-Nal)-NH 2 7.26 M11 Ac-(Cba)(N-MeQ)TLRXIQRX(Cba)(2-Nal)-NH 2 4.90 M12 Ac-L(N-MeQ)TLRXIQRX(N-MeCha)(2-Nal)-NH 2 6.88 M13 Ac-L(N-MeQ)TLRXIQRX(4-ClPhe)(2-Nal)-NH 2 13.00 M14 Ac-L(N-MeQ)TLRXIQRX(Cpa)(2-Nal)-NH 2 15.84 Fig. 2 A-B) Docking models of hsBCL9 CT-24 (green) and β -Catenin (pink) (PDB: 2GL7), left and right views. C) Docking model of M1 (brown-yellow) and hsBCL9 CT-24 (green) with β -Catenin (PDB: 2GL7). D) Docking model of M2 (gray) and hsBCL9 CT-24 (green) with β -Catenin (PDB: 2GL7). E) Docking model of M3 (blue) and hsBCL9 CT-24 (green) with β -Catenin (PDB: 2GL7). F) Docking model of M4 (magenta) and hsBCL9 CT-24 (green) with β -Catenin (PDB: 2GL7). G) Docking model of M5 (gold) and hsBCL9 CT-24 (green) with β -Catenin (PDB: 2GL7). Fig. 3 A) Docking binding model of M6 (green), M8 (cyan), and β -Catenin (PDB: 2GL7). B) Docking binding model of M6 (green), M9 (blue), and β -Catenin (PDB: 2GL7). C) Computational docking binding model of M6 (green), M13 (cyan), and β -Catenin (PDB: 2GL7). D) Docking binding model of M6 (green), M14 (gray), and β -Catenin (PDB: 2GL7). D-amino acids have been shown to improve enzymatic stability [15], and we hypothesized that their incorporation might enhance the inhibitory activity of our stapled peptides. Leucine (L) in the staple was replaced with D-amino acids to produce mutants M15-M17 (Table 3), which were evaluated for inhibitory activity by AlphaScreen. Results indicated that the M16 mutant exhibited inhibitory activity comparable to the parent peptide (hsBCL9 CT24 ), while M15 and M17 showed reduced activity, with M17 exhibiting the lowest activity at 13.2 μM. These findings suggest that D-amino acids do not significantly enhance inhibitory potency in this context. Table 3 Sequences of M15-M17 and their β -Catenin inhibitory activity hsBCL9 CT-24 LQTLR XIQR XL-2Nal-NH 2 2.7 M15 (D-L)QTLR XIQR XL-2Nal-NH 2 6.84 M16 LQT(D-L)R XIQR XL-2Nal-NH 2 2.86 M17 LQTLR XIQR X(D-L)-2Nal-NH 2 13.2 Palmitoylation modification, a classical strategy in peptide drug development, was employed to enhance cell membrane permeability and potentially facilitate target interaction. The peptides M6, M3, and M2 were conjugated to a palmitic acid moiety at the C-terminus and to β -alanine ( β -Ala) at the N-terminus as a flexible linker. The resulting palmitoylated peptides (M18-M22) exhibited significantly enhanced inhibitory activity, particularly M20, which demonstrated an IC 50 of 0.02 μM, approximately 20 times greater than the parent peptide. This effect surpassed the enhancements observed with key hydrophobic site mutations, indicating that palmitoylation’s contribution to cellular uptake and target engagement may play a more significant role in enhancing activity than subtle hydrophobic modifications. Given its exceptional inhibitory potency, M20 was designated hsBCL9 Z96 and subjected to pharmacological studies for liver fibrosis, focusing on its specific inhibition of the β -Catenin/BCL9 complex and its antifibrotic efficacy in vivo models. Table 4 Sequences of M18-M23 and their β -Catenin inhibitory activity hsBCL9 CT-24 LQTLRXIQRXL-2Nal-NH 2 2.7 M18 Ac-L(N-MeQ)TLRXIQRX(Cba)(2-Nal)- β Ala- β Ala-NH 2 0.41 M19 Pal-PEG4-L(N-MeQ) TLRXIQRX (Cba) (2-Nal)-NH 2 0.47 M20 （hsBCL9 Z96 ） Ac-LQTLRXIQRXL(2-Nal)- β -Ala- β -Ala GRKKRRQRRRPQK(PEG4-pal)-NH 2 0.02 M21 Ac-LQTLRXIQRX (Cha) (2-Nal)- β Ala- β Ala GRKKRRQRRRPQK(PEG4-pal)-NH 2 0.06 M22 Ac-LQTLRXIQRX (Cba) (2-Nal)- β Ala- β Ala GRKKRRQRRRPQK(PEG4-pal)-NH 2 0.03 M23 Pal-PEG4-LQTLRXIQRX (Cba) (2-Nal)- β Ala- β Ala GRKKRRQRRRPQK(PEG4-pal)-NH 2 0.1 4. Research on the pharmacological mechanism of hsBCL9 Z96 4.1. hsBCL9 Z96 Alleviates Liver Fibrosis The therapeutic effect of hsBCL9 Z96 on the CCl 4 -induced mouse liver fibrosis model was assessed by Masson’s trichrome staining, as shown in Fig. 4 A-B under optical microscopy, the liver tissue of the normal group displayed intact structure, with clear liver lobule architecture and orderly arrangement of hepatocytes. A small amount of blue collagen fibers was observed only around the bile ducts, with no significant fibrotic changes, indicating that the liver was in a normal state. In contrast, the model group mice, after induction with CCl 4 , showed a substantial increase in blue collagen fibers, which were widely distributed around the liver lobules. These fibers encircled and separated the lobules, forming pseudo-lobules of varying sizes, indicative of significant liver fibrosis. In the hsBCL9 Z96 treatment group, blue collagen fibers were also observed near the bile duct structures, but these fibers were finer, and the degree of fibrosis was reduced compared to the model group. Specifically, collagen fiber proliferation was significantly alleviated in the hsBCL9 Z96 group, with the fibrosis level being lower than in the model group, suggesting that hsBCL9 Z96 has a marked inhibitory effect on CCl 4 -induced liver fibrosis. These results demonstrate that hsBCL9 Z96 can effectively slow the progression of liver fibrosis by reducing collagen fiber proliferation and the formation of pseudo-lobules, thereby significantly improving liver structural changes. As shown in Fig. 4, Sirius Red staining was used to assess collagen deposition in liver tissue sections. The blank control group showed no significant collagen deposition, and the liver tissue maintained normal architecture, indicating no fibrotic damage. In contrast, the model group, following CCl 4 induction, exhibited considerable collagen deposition, confirming successful induction of liver fibrosis and causing noticeable liver tissue damage and fibrosis. However, in the hsBCL9 Z96 treatment group, the amount of collagen deposition was significantly reduced, demonstrating the positive effect of hsBCL9 Z96 in inhibiting liver fibrosis. This suggests that hsBCL9 Z96 effectively alleviates CCl 4 -induced liver fibrosis damage, reduces the accumulation of fibrosis markers in the liver, and improves the integrity of liver structure. Compared to the model group, the liver tissue in the hsBCL9 Z96 group showed clear improvements, indicating that hsBCL9 Z96 may slow or reverse the progression of liver fibrosis by modulating fibrosis-related molecular mechanisms. These results further support the potential application of hsBCL9 Z96 as an anti-fibrotic drug, suggesting its significant pharmacological activity in treating liver fibrosis by effectively alleviating liver damage and promoting tissue repair. Fig. 4. hsBCL9 Z96 reduces the degree of liver fibrosis. A) Mouse Sirius red staining section. B) Mouse Masson staining section （Scale bar：200μm，** P ＜0.01） 4.3 Inhibition of β -Catenin/BCL9 Promotes Macrophage Polarization Towards the M1 Phenotype Macrophages play a pivotal role in liver diseases [17], and they are generally classified into two phenotypes: classical activated phenotype (M1) and alternative activated phenotype (M2). M2 macrophages, upon activation, secrete TGF- β , which further activates hepatic stellate cells (HSC) [18], significantly contributing to fibrosis formation. Previous studies have shown that promoting the polarization of macrophages from the M2 phenotype towards the M1 phenotype can effectively improve liver function and alleviate liver fibrosis [19]. Therefore, regulating macrophage phenotype transition in liver fibrosis is of considerable importance. Using flow cytometry, we assessed the polarization status of macrophages in the liver of mice from different treatment groups (Fig. 5). The results demonstrated a significant reduction in M2 macrophages and a substantial increase in M1 macrophages in the hsBCL9 Z96 treatment group. This observation suggests that hsBCL9 Z96 may influence the polarization of macrophages towards the M1 phenotype. Based on this, we hypothesize that the inhibition of the Wnt/ β -Catenin signaling pathway may impact the polarization process of macrophages in the liver. Fig.5. hsBCL9 Z96 promotes macrophage M2 polarization（** P ＜0.01；*0.01＜ P ＜0.05） 4.4 hsBCL9 Z96 Reduces Liver Fibrosis Biomarkers and Inflammatory Response We aimed to explore the molecular mechanisms through which hsBCL9 Z96 affects liver fibrosis. qPCR sequencing was performed on liver tissues from both hsBCL9 Z96 -treated and control mice. The results showed that hsBCL9 Z96 significantly reduced key liver fibrosis biomarkers, including Acta2 , Fibronectin , and Col1a1 (Fig. 6A). Acta2 , Fibronectin , and Col1a1 are critical molecules in the progression of liver fibrosis and are commonly used as indicators of HSC activation and fibrosis severity. By modulating the Wnt/ β -Catenin pathway, hsBCL9 Z96 reduced the expression of fibrosis-associated genes, thereby significantly lowering the levels of these biomarkers. TGF- β 1, the most crucial fibrosis-promoting factor, activates HSCs and facilitates their transformation into myofibroblasts, leading to collagen and extracellular matrix accumulation. CTGF interacts with TGF- β 1, further enhancing the fibrosis process, while PDGF promotes HSC proliferation and migration, thereby exacerbating liver fibrosis. In the hsBCL9 Z96 treatment group, the expression levels of TGF- β 1, CTGF, and PDGF were significantly reduced, indicating that hsBCL9 Z96 may effectively block HSC activation by suppressing the expression of these critical factors (Fig. 6B). IL-1 β and TNF-α promote liver inflammation by activating NF-κB and other signaling pathways, which further enhances HSC activation and the release of fibrosis-promoting factors. IL-17 exacerbates chronic inflammation by promoting immune cell infiltration and the secretion of inflammatory mediators, thus accelerating the fibrosis process. In the Z96 group, the expression of these cytokines was significantly downregulated (Fig. 6C), suggesting that hsBCL9 Z96 exerts a therapeutic effect on CCl 4 -induced liver fibrosis. This also implies that Wnt/ β -Catenin inhibitors may offer therapeutic potential for liver fibrosis treatment. Furthermore, hsBCL9 Z96 may alleviate liver inflammation by modulating immune responses and inhibiting the production of inflammatory cytokines, thus reducing the inflammatory burden on the liver. By suppressing the release of these pro-inflammatory factors, hsBCL9 Z96 effectively mitigates HSC activation and collagen deposition, thereby slowing the progression of liver fibrosis. This result suggests that hsBCL9 Z96 , by alleviating inflammation and inhibiting inflammation-related fibrosis factors, could provide a promising therapeutic strategy for liver fibrosis, indicating its potential application in anti-fibrotic treatments. Matrix metalloproteinases (MMPs), such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, and MMP-13, play crucial roles in the formation and resolution of liver fibrosis by regulating extracellular matrix (ECM) degradation [20]. MMP-7 promotes ECM degradation by modulating HSC activation and apoptosis, thus facilitating the reversal of liver fibrosis. MMP-12 primarily alleviates fibrosis and inflammation by activating macrophages and promoting ECM degradation. MMP-13 promotes fibrosis regression by degrading collagen, inhibiting ECM accumulation, and affecting HSC activity. In this study, we observed a significant upregulation in the expression of MMP-7, MMP-12, and MMP-13 in the hsBCL9 Z96 treatment group (Fig. 6D). These results suggest that hsBCL9 Z96 promotes the expression of these three MMPs, which play critical roles in reversing liver fibrosis, offering potential therapeutic benefits for liver fibrosis treatment. This finding indicates that the inhibition of the Wnt/ β -Catenin signaling pathway leads to the upregulation of MMP-7, MMP-12, and MMP-13, which may contribute to liver fibrosis resolution. Fig.6. hsBCL9 Z96 impacts liver fibrosis biomarker secretion and alleviates inflammation (** P ＜0.01；*0.01＜ P ＜0.05.) A) Expression of fibrosis-related structural proteins Acta2, Fibronectin, and Col1a1. B) Expression of fibrosis-promoting factors TGF- β , CTGF, PDGF. C) Expression of inflammatory cytokines IL-1 β , TNF-α, IL-17. D) Expression of matrix metalloproteinases MMP-7, MMP-12, MMP-13. 4.5 bcl9 Deficiency Alleviates Liver Fibrosis in Mice In the aforementioned study, we pharmacologically inhibited the β -Catenin signaling pathway and observed a suppression of liver fibrosis in mice. Furthermore, the pharmacological inhibition tool hsBCL9 Z96 targeting the β -Catenin/BCL9 complex was shown to affect macrophage polarization, consequently influencing liver fibrosis. Our experimental results indicate that the hsBCL9 Z96 -treated group exhibited an increase in M2-type macrophages in the liver, accompanied by a reduction in the secretion of pro-inflammatory cytokines such as IL-1 β , IL-17, and TNF-α. Additionally, expression of ECM metalloproteinases (MMP7, MMP12, MMP13) was upregulated. These findings suggest that the β -Catenin/BCL9 signaling pathway may modulate liver fibrosis through macrophage polarization. Further investigation into bcl9 -deficient mice (using a liver-specific bcl9 knockout model) injected with CCl 4 for liver fibrosis induction showed significantly attenuated liver fibrosis. Sirius red staining revealed a reduction in fibrosis severity, while expression of the fibrosis-related gene MMP13 was upregulated. Notably, macrophage repolarization in the liver mirrored previous results, showing an increase in M1-type macrophage differentiation (Fig7 A-B). Additionally, qPCR analysis demonstrated that collagen markers, such as Col1a1 , Col3a1 , and Acta2 , were significantly decreased in bcl9 -deficient mice compared to wild-type controls (Fig7 C). Flow cytometry analysis of macrophages in liver tissue confirmed an increased proportion of anti-fibrotic M1 macrophages in the alb-cre bcl9 knockout mice (Fig7 D). Taken together, these results indicate that BCL9 plays a role in promoting liver fibrosis and that its deletion can inhibit the expression of critical fibrosis genes. Fig.7. Effects of bcl9 Knockout on Liver Fibrosis. A) Knockout of bcl9 promotes M1 macrophage expression of MMP13 and MMP2. B) Sirius red staining of liver tissue from alb-cre bcl9 knockout and model mice. C) qPCR results for fibrosis markers Acta2 , COL1A1 , and COL3A1 in alb-cre bcl9 knockout and model mice. D) Flow cytometric analysis of macrophages from the livers of alb-cre bcl9 knockout and model mice. 4.6 β -Catenin Pathway Inhibition Modulates Immune Response to Alleviate Liver Fibrosis Immune cells play dual roles in both protecting the body and promoting liver damage and fibrosis. Interestingly, regulatory T cells (Tregs) can suppress inflammation and fibrosis. Previous studies have shown that CCl 4 -induced chronic liver inflammation preferentially expands Treg cells, preventing fibrosis. Rapamycin has been shown to enhance the function of CD4+CD25+ Tregs and their ability to inhibit HSC activation [21, 22]. Additionally, Th1 cells, which secrete IFN-γ, and Th2 cells, which produce IL-4, play contrasting roles in fibrosis, with Tregs being key modulators of these subsets. To explore the regulatory mechanisms underlying macrophage polarization, we first performed flow cytometry on liver cells (Fig.8 A). bcl9 -deficient mice exhibited a marked expansion of Tregs and increased IFN-γ secretion. Furthermore, expression of the T cell chemokine CXCL9 was elevated, suggesting a role in Treg-mediated suppression of inflammation and promotion of liver repair [23]. This indicates that BCL9 modulates immune cell differentiation and the progression of liver fibrosis. qPCR analysis revealed that in bcl9 -deficient mice, CCl 4 -induced CD40 expression in the liver was significantly reduced, indicating a decrease in liver inflammation (Fig.8 C). Additionally, CXCL9 and IFN-γ expression levels were significantly higher in the knockout group. CXCL9 plays a critical role in T cell migration and is implicated in liver immune responses, likely facilitating Treg-mediated inhibition of inflammation and promoting liver tissue repair. We hypothesize that BCL9 inhibition enhances the suppressive function of Tregs on immune responses, potentially through modulation of Th1 immune responses and pro-inflammatory chemokine signaling. This could alter the hepatic immune microenvironment, favoring IFN-γ production, which further promotes M1 macrophage polarization. The activation of M1 macrophages increases the secretion of matrix metalloproteinase (MMP13), thereby facilitating liver tissue repair and slowing fibrosis progression (Fig.8 B). Fig.8. Impact of bcl9 Knockout on Immune Cells in Liver Fibrosis. A) Flow cytometric analysis of Treg cells in liver tissue from alb-cre bcl9 knockout and model mice. B) qPCR results for ECM remodeling factors Timp1 , Timp2 , and Mmp13 in the livers of alb-cre bcl9 knockout and wild-type mice. C) qPCR results for inflammatory regulators CD40 , CXCL9 , and IFN-γ in the livers of alb-cre bcl9 knockout and wild-type mice. 5. Discussion Liver fibrosis is a common pathological feature of various chronic liver diseases, and macrophages play a crucial role in its development. Macrophages can be classified into M1 and M2 types, where M1 macrophages primarily promote inflammatory responses through the secretion of pro-inflammatory cytokines, while M2 macrophages secrete anti-inflammatory cytokines that alleviate liver inflammation. However, M2 macrophages also secrete TGF- β , which can exacerbate liver fibrosis. TGF- β activates hepatic stellate cells, promoting their transformation into fibroblast-like cells, thereby further accelerating the development of liver fibrosis. Therefore, inhibiting the function of M2 macrophages may be an effective strategy for treating liver fibrosis. However, the regulatory mechanisms of macrophages are complex and multifaceted. In existing studies, the Wnt/ β -Catenin signaling pathway has been implicated in the functional polarization of macrophages. The Wnt/ β -Catenin signaling pathway is a critical regulator of cell proliferation, differentiation, and survival, playing an essential role in liver development, regeneration, and homeostasis maintenance [24]. Particularly, it is significant in the proliferation, survival, and differentiation of hepatocytes. Dysregulated activation of this pathway is closely associated with the onset and progression of various liver diseases, including liver fibrosis, steatohepatitis, hepatocellular carcinoma, cholestasis, and liver cysts [12, 25]. Thus, targeting the Wnt/ β -Catenin signaling pathway holds promise as a potential strategy for the treatment of liver fibrosis, especially in CCl 4 -induced mouse models of liver fibrosis. In this study, we designed and synthesized a series of novel peptides targeting the Wnt/ β -Catenin pathway, based on the known Wnt/ β -Catenin inhibitor hsBCL9 CT-24 . These peptides were screened for their inhibitory activity against the Wnt/ β -Catenin pathway using Alphascreen technology. The results revealed that peptides with hydrophobic side-chain structures, such as R367/R371 and R373, combined with β -alanine and TAT tags, significantly enhanced the inhibition of β -Catenin. Through further optimization, we identified hsBCL9 Z96 (IC 50 = 0.02 μM) as the most potent inhibitor. Encouragingly, our findings demonstrate that inhibition of BCL9 significantly suppresses the polarization of macrophages toward the M2 phenotype, thereby attenuating the progression of liver fibrosis. Specifically, we found a negative correlation between BCL9 levels and liver fibrosis progression. BCL9 inhibition, either through pharmacological means or genetic knockout, effectively suppressed the M2 polarization of macrophages. This effect may be mediated by an increase in regulatory T cells (Tregs), leading to an imbalance in Th1/Th17 cell populations, and by the secretion of IFN-γ, which suppresses M2 macrophage function. Additionally, BCL9 inhibition also activated fibrosis-related repair genes, such as MMP13, which further mitigated liver fibrosis. In conclusion, this study for the first time reveals the potential role of the BCL9/ β -Catenin signaling pathway in combating liver fibrosis. By designing and synthesizing a series of peptides with anti-fibrotic activity, we demonstrate the potential of hsBCL9 Z96 as a therapeutic agent for liver fibrosis. Future studies will further investigate the mechanism of action of this molecule, aiming to provide new therapeutic strategies for the treatment of liver fibrosis. 6. Author Contributions Gang Chen and Yangbo He performed compound synthesis, data analysis, and manuscript writing. Huiyu Li and Yunlu Li designed the pharmacological experiments and conducted the main experimental procedures. Di Zhu provided guidance in experimental design, revised the manuscript, and oversaw the project. All authors reviewed and approved the final manuscript. 7. Conflict of Interest Statement The authors declare no conflicts of interest. 8. Acknowledgments We would like to thank the Jinan Science and Technology Bureau’s Tumor Immunotherapy New Drug and Evaluation Innovation Team (2020GXRC041 to D.Z.). References [1] A.M. Moon, A.G. Singal, E.B. Tapper, Contemporary Epidemiology of Chronic Liver Disease and Cirrhosis, Clin Gastroenterol Hepatol 18(12) (2020) 2650-2666.[2] D. Ezhilarasan, Oxidative stress is bane in chronic liver diseases: Clinical and experimental perspective, Arab J Gastroenterol 19(2) (2018) 56-64.[3] S.L. Friedman, Liver fibrosis – from bench to bedside, J Hepatol 38 Suppl 1 (2003) S38-53.[4] S. Gordon, Alternative activation of macrophages, Nat Rev Immunol 3(1) (2003) 23-35.[5] Z. Wang, K. Du, N. Jin, B. Tang, W. Zhang, Macrophage in liver Fibrosis: Identities and mechanisms, Int Immunopharmacol 120 (2023) 110357.[6] B. Dewidar, C. Meyer, S. Dooley, N. Meindl-Beinker, TGF-β in hepatic stellate cell activation and liver fibrogenesis—updated 2019, Cells 8(11) (2019) 1419.[7] Y. Zhang, L. Zhang, Y. Zhao, J. He, Y. Zhang, X. Zhang, PGC-1α inhibits M2 macrophage polarization and alleviates liver fibrosis following hepatic ischemia reperfusion injury, Cell Death Discovery 9(1) (2023) 337.[8] C. Trautwein, S.L. Friedman, D. Schuppan, M. Pinzani, Hepatic fibrosis: Concept to treatment, J Hepatol 62(1 Suppl) (2015) S15-24.[9] J. Hu, Y. Liu, Z. Pan, X. Huang, J. Wang, W. Cao, Z. Chen, Eupatilin Ameliorates Hepatic Fibrosis and Hepatic Stellate Cell Activation by Suppressing β-catenin/PAI-1 Pathway, Int J Mol Sci 24(6) (2023).[10] Y. Yang, Y.-C. Ye, Y. Chen, J.-L. Zhao, C.-C. Gao, H. Han, W.-C. Liu, H.-Y. Qin, Crosstalk between hepatic tumor cells and macrophages via Wnt/β-catenin signaling promotes M2-like macrophage polarization and reinforces tumor malignant behaviors, Cell Death & Disease 9(8) (2018) 793.[11] K. Takada, D. Zhu, G.H. Bird, K. Sukhdeo, J.J. Zhao, M. Mani, M. Lemieux, D.E. Carrasco, J. Ryan, D. Horst, M. Fulciniti, N.C. Munshi, W. Xu, A.L. Kung, R.A. Shivdasani, L.D. Walensky, D.R. Carrasco, Targeted disruption of the BCL9/β-catenin complex inhibits oncogenic Wnt signaling, Sci Transl Med 4(148) (2012) 148ra117.[12] S.P. Monga, β-Catenin Signaling and Roles in Liver Homeostasis, Injury, and Tumorigenesis, Gastroenterology 148(7) (2015) 1294-310.[13] H. Zhang, Y. Bao, C. Liu, J. Li, D. Zhu, Q. Zhang, Recent advances in β-catenin/BCL9 protein-protein interaction inhibitors, Future Med Chem 13(10) (2021) 927-940.[14] M. Feng, J.Q. Jin, L. Xia, T. Xiao, S. Mei, X. Wang, X. Huang, J. Chen, M. Liu, C. Chen, S. Rafi, A.X. Zhu, Y.X. Feng, D. Zhu, Pharmacological inhibition of β-catenin/BCL9 interaction overcomes resistance to immune checkpoint blockades by modulating T(reg) cells, Sci Adv 5(5) (2019) eaau5240.[15] A.W. Partridge, H.Y.K. Kaan, Y.C. Juang, A. Sadruddin, S. Lim, C.J. Brown, S. Ng, D. Thean, F. Ferrer, C. Johannes, T.Y. Yuen, S. Kannan, P. Aronica, Y.S. Tan, M.R. Pradhan, C.S. Verma, J. Hochman, S. Chen, H. Wan, S. Ha, B. Sherborne, D.P. Lane, T.K. Sawyer, Incorporation of Putative Helix-Breaking Amino Acids in the Design of Novel Stapled Peptides: Exploring Biophysical and Cellular Permeability Properties, Molecules 24(12) (2019).[16] F. He, Z. Wu, C. Liu, Y. Zhu, Y. Zhou, E. Tian, R. Rosin-Arbesfeld, D. Yang, M.W. Wang, D. Zhu, Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration, Signal Transduct Target Ther 9(1) (2024) 139.[17] P. Ramachandran, J.P. Iredale, Macrophages: central regulators of hepatic fibrogenesis and fibrosis resolution, J Hepatol 56(6) (2012) 1417-9.[18] X. Hu, R.K. Leak, Y. Shi, J. Suenaga, Y. Gao, P. Zheng, J. Chen, Microglial and macrophage polarization—new prospects for brain repair, Nat Rev Neurol 11(1) (2015) 56-64.[19] Y. Wang, S. Huang, W. Kong, C. Wu, T. Zeng, S. Xie, Q. Chen, S. Kuang, R. Zheng, F. Wang, C. Zhou, Y. Chen, S. Huang, Z. Lv, Corilagin alleviates liver fibrosis in zebrafish and mice by repressing IDO1-mediated M2 macrophage repolarization, Phytomedicine 119 (2023) 155016.[20] N. Cui, M. Hu, R.A. Khalil, Biochemical and Biological Attributes of Matrix Metalloproteinases, Prog Mol Biol Transl Sci 147 (2017) 1-73.[21] Y. Ikeno, D. Ohara, Y. Takeuchi, H. Watanabe, G. Kondoh, K. Taura, S. Uemoto, K. Hirota, Foxp3+ Regulatory T Cells Inhibit CCl(4)-Induced Liver Inflammation and Fibrosis by Regulating Tissue Cellular Immunity, Front Immunol 11 (2020) 584048.[22] L. Gu, W.S. Deng, X.F. Sun, H. Zhou, Q. Xu, Rapamycin ameliorates CCl4-induced liver fibrosis in mice through reciprocal regulation of the Th17/Treg cell balance, Mol Med Rep 14(2) (2016) 1153-61.[23] H.E. Wasmuth, F. Lammert, M.M. Zaldivar, R. Weiskirchen, C. Hellerbrand, D. Scholten, M.L. Berres, H. Zimmermann, K.L. Streetz, F. Tacke, S. Hillebrandt, P. Schmitz, H. Keppeler, T. Berg, E. Dahl, N. Gassler, S.L. Friedman, C. Trautwein, Antifibrotic effects of CXCL9 and its receptor CXCR3 in livers of mice and humans, Gastroenterology 137(1) (2009) 309-19, 319.e1-3.[24] X. Tan, Y. Yuan, G. Zeng, U. Apte, M.D. Thompson, B. Cieply, D.B. Stolz, G.K. Michalopoulos, K.H. Kaestner, S.P. Monga, β‐Catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development, Hepatology 47(5) (2008) 1667-1679.[25] M.D. Thompson, S.P. Monga, WNT/beta-catenin signaling in liver health and disease, Hepatology 45(5) (2007) 1298-305. Information & Authors Information Version history V1 Version 1 22 July 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Gang Chen Anhui University of Chinese Medicine School of Pharmacy View all articles by this author Yangbo Anhui University of Chinese Medicine School of Pharmacy View all articles by this author Huiyu Li Fudan University View all articles by this author Yunlu Li Fudan University View all articles by this author Cuiting Liu Nantong Jutai Biotechnology Co Ltd View all articles by this author Anqi Li Fudan University View all articles by this author Yanfang Xian 0000-0002-5032-0366 Chinese University of Hong Kong View all articles by this author Xianjing Meng Shandong Academy of Pharmaceutical Sciences View all articles by this author Mei Feng Fudan University View all articles by this author Wei lu Fudan University View all articles by this author Daizhou Zhang Shandong Academy of Pharmaceutical Sciences View all articles by this author Chonggang Duan Shandong Academy of Pharmaceutical Sciences View all articles by this author Di Zhu [email protected] Department of Pharmacology School of Basic Medical Sciences of Fudan University View all articles by this author Metrics & Citations Metrics Article Usage 163 views 94 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Gang Chen, Yangbo, Huiyu Li, et al. Development of β-Catenin/BCL9 Targeted Inhibitors and Investigation of Their Anti-Fibrotic Mechanism in Liver Fibrosis. Authorea . 22 July 2025. DOI: https://doi.org/10.22541/au.175318772.20367004/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.175318772.20367004/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9ffd8af33b5ddf88',t:'MTc3OTQ3MDk2Mw=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();