BSP-Diol restores PKM2 and facilitates β-catenin–dependent epithelial repair in experimental colitis

BSP-Diol restores PKM2 and facilitates β-catenin–dependent epithelial repair in experimental colitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article BSP-Diol restores PKM2 and facilitates β-catenin–dependent epithelial repair in experimental colitis Houshu Tu, Menglin Chen, Cui Xu¹, Chengming Yang¹, Sai Li¹, Jing Hong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9086575/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Background Impaired mucosal healing is a major determinant of persistent inflammation and relapse in inflammatory bowel disease (IBD). Although repair-associated signaling pathways such as β-catenin can remain active during inflammation, epithelial regeneration is frequently insufficient. The mechanisms limiting proliferation-dependent repair in the context of inflammatory colitis remain incompletely defined. Methods The therapeutic effects of the Huanglian–Baiji herb pair were evaluated in a dextran sulfate sodium (DSS)–induced colitis model. An integrated strategy combining component prioritization with experimental validation was applied to identify active constituents and regulatory nodes. The effects of BSP-Diol on epithelial repair were examined in vivo and in vitro, focusing on epithelial proliferation, goblet cell recovery, β-catenin nuclear localization, and PKM2 regulation. The requirement of PKM2 was assessed using protein profiling and loss-of-function approaches. Results Huanglian–Baiji treatment significantly promoted epithelial proliferation and goblet cell restoration in DSS colitis. BSP-Diol was identified as a key component that restored β-catenin nuclear localization in inflamed epithelium. Inflammatory conditions markedly reduced PKM2 protein abundance, coinciding with impaired proliferative responses. Restoration of PKM2 by BSP-Diol facilitated β-catenin–dependent epithelial proliferation. In contrast, transcriptional regulators of mitochondrial biogenesis exhibited only modest changes, indicating predominant regulation at the protein and subcellular levels. PKM2 knockdown significantly attenuated BSP-Diol–induced β-catenin nuclear localization, demonstrating that PKM2 is required for effective repair signaling. Conclusions These findings identify PKM2 as a critical metabolic regulator of β-catenin–dependent epithelial repair in experimental colitis. Restoration of PKM2 relieves inflammation-associated metabolic constraints and promotes mucosal regeneration. Targeting metabolic regulation may represent a complementary strategy to enhance epithelial repair in IBD. inflammatory bowel disease epithelial repair PKM2 β-catenin colitis metabolic regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Inflammatory bowel disease (IBD), encompassing Crohn’s disease and ulcerative colitis, is a chronic relapsing inflammatory disorder of the gastrointestinal tract. The global incidence and prevalence of IBD continue to rise, particularly in newly industrialized regions, imposing a substantial healthcare burden [ 1 , 2 ] . Persistent disruption of the intestinal epithelial barrier and defective mucosal repair are central pathophysiological features of IBD, contributing to sustained inflammation and progressive tissue injury [ 3 , 4 ] . Although current therapies primarily target immune-mediated inflammation [ 5 ] , durable remission is closely associated with effective mucosal healing, which depends on the ability of intestinal epithelial cells to re-enter and sustain proliferative programs. Wnt/β-catenin signaling plays a central role in intestinal epithelial regeneration. Nuclear translocation of β-catenin activates transcriptional programs necessary for epithelial proliferation and barrier restoration. However, under inflammatory stress, epithelial repair frequently remains insufficient despite the presence of repair-associated signaling activity. This raises an important question: does repair failure reflect absence of regenerative signals, or does inflammatory stress impose metabolic constraints that limit the effective execution of these signaling programs? Emerging evidence indicates that chronic inflammation induces mitochondrial dysfunction, altered metabolic reprogramming, and impaired biosynthetic capacity in intestinal epithelial cells [ 6 , 7 ] . Such metabolic disturbances may restrict cellular proliferation, migration, and barrier reconstruction even when regenerative signaling pathways are intactt [ 8 , 9 ] . These findings suggest that metabolic state may critically modulate signaling-dependent epithelial repair. Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme that also participates in metabolic regulation of proliferative responses. In intestinal epithelium, PKM2 has been implicated in maintaining epithelial integrity and facilitating β-catenin–dependent repair processes [ 14 , 15 ] , Epithelial-specific deletion of PKM2 exacerbates experimental colitis and impairs regenerative responses, highlighting a potential role for PKM2 in supporting signaling-dependent mucosal healing. These observations position PKM2 as a candidate metabolic regulator of epithelial repair under inflammatory stress. The Huanglian–Baiji herb pair is traditionally used to promote tissue regeneration. Bletilla striata (Baiji) contains bioactive constituents including polysaccharides and small molecules such as BSP-Diol, which exhibit anti-inflammatory and wound-healing properties [ 12 , 13 ] . However, whether Baiji-derived components modulate epithelial repair through metabolic regulation of signaling pathways remains unclear. In the present study, we employed an integrated strategy combining in vivo validation, network pharmacology–guided component prioritization, and mechanistic analyses to investigate whether BSP-Diol promotes epithelial repair through modulation of PKM2-associated metabolic regulation. Using a DSS-induced colitis model, we examined epithelial proliferation, goblet cell restoration, β-catenin nuclear localization, and PKM2 expression under inflammatory stress. Our findings reveal a PKM2–β-catenin regulatory axis that links metabolic state to signaling-dependent epithelial regeneration in IBD. 2. Materials and Methods 2.1 Animals and experimental colitis model Specific pathogen–free (SPF) male C57BL/6J mice (8 weeks old, 20–23 g) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China; production license No. SCXK (Beijing) 2024-0001). Animals were housed under standard SPF conditions with a 12 h light/dark cycle, controlled temperature (22–25°C), and relative humidity (50–65%), with free access to food and water. All experimental procedures were approved by the Animal Ethics Committee of Jiangxi Hengqing Technical Testing Co., Ltd. (approval No. JXHQ2025002). Experimental colitis was induced by administering 3% (w/v) dextran sulfate sodium (DSS; MP Biomedicals, molecular weight 36–50 kDa, Cat. No. YD05012) in drinking water for 7 consecutive days. After a 5-day acclimatization period, a total of 48 mice were randomly assigned to six groups (n = 8 per group): (1) normal control group; (2) DSS model group; (3) mesalazine-treated positive control group (200 mg/kg/day); and (4–6) Huanglian–Baiji herb pair low-, medium-, and high-dose groups (100, 200, and 400 mg/kg/day, respectively). All pharmacological interventions were administered by oral gavage once daily concomitantly with DSS exposure throughout the 7-day induction period, corresponding to a preventive intervention protocol. 2.2 Drugs, reagents, and administration Huanglian granules (China National Pharmaceutical Group Zhonglian Pharmaceutical Co., Ltd., batch No. 240041; 1 g granules equivalent to 4.5 g crude drug) and Baiji granules (Beijing Kangrentang Pharmaceutical Co., Ltd., batch No. 24012071; 1 g granules equivalent to 4.3 g crude drug) were dissolved in normal saline prior to use. The high-dose Huanglian–Baiji group (CB-H) received a total granule dose of 400 mg/kg/day. Mesalazine enteric-coated tablets (Sunflower Pharmaceutical Group, batch No. 240807) were suspended in normal saline at a concentration of 20 mg/mL and administered at 200 mg/kg/day. All gavage volumes were fixed at 0.25 mL per mouse per day. BSP-Diol (YuanYe Bio, Cat. No. B22600) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution and diluted with culture medium to the desired working concentrations for in vitro experiments. 2.3 Tissue collection and histopathological evaluation On day 7, mice were anesthetized and euthanized. The entire colon was excised, measured for length, and photographed. Proximal colon segments were fixed in 4% paraformaldehyde for paraffin embedding, sectioning (5 µm), and histological analysis. Hematoxylin and eosin (H&E)–stained sections were evaluated using blinded pathological scoring. Remaining tissues were snap-frozen in liquid nitrogen and stored at − 80°C for subsequent molecular analyses. 2.4 Immunohistochemistry Paraffin-embedded sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% H₂O₂, followed by blocking with 5% bovine serum albumin (BSA) at room temperature for 1 h. Sections were incubated overnight at 4°C with the following primary antibodies: rabbit anti-Ki67 (Proteintech, Cat. No. 27309-1-AP, 1:4000) and rabbit anti-Muc2 (Proteintech, Cat. No. 27675-1-AP, 1:2000). After washing, sections were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (Servicebio, Cat. No. GB23204, 1:500) for 1 h at room temperature. Signal detection was performed using a DAB substrate kit (Wuhan Boster Bioengineering, Cat. No. B0053), followed by hematoxylin counterstaining. Slides were scanned using a digital slide scanner (3DHISTECH Pannoramic MIDI/DESK). Quantitative analysis was conducted using Image-Pro Plus 6.0 software on at least five randomly selected non-overlapping fields per section (200×). The percentage of Ki67-positive cells at the crypt base and the area fraction of Muc2-positive staining within the epithelial region were calculated. 2.5 RNA extraction and quantitative real-time PCR Total RNA was extracted from colon tissues using TRIzol reagent (TAKARA) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop™ One spectrophotometer (Thermo Fisher Scientific), with A260/A280 ratios between 1.8 and 2.0. RNA integrity was confirmed by agarose gel electrophoresis. Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, TAKARA) with 1 µg total RNA per sample. Quantitative PCR was conducted on an FQD-96A real-time PCR system (Hangzhou Bioer Technology, China) using SYBR® Green Premix Pro Taq HS (Xiamen Lifeint, Cat. No. A4004M). Actb (β-actin) was used as the internal reference gene, and relative gene expression levels were calculated using the 2^−ΔΔCt method. Primer sequences are listed in Supplementary Table S1 . 2.6 Cell culture and treatments Human colorectal adenocarcinoma Caco-2 cells (iCell, Cat. No. iCell-h032) were cultured in MEM supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% non-essential amino acids (NEAA) at 37°C in a humidified incubator with 5% CO₂. To establish an in vitro inflammatory model, cells were stimulated with lipopolysaccharide (LPS; 1 µg/mL; Solarbio, Cat. No. L8880) for 24 h. BSP-Diol was administered as a pretreatment prior to LPS stimulation where indicated. 2.7 Cell viability assay Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; GLPBIO, Cat. No. GK10001). Caco-2 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and treated with various concentrations of BSP-Diol (0–100 µM) for 24 h. After incubation with CCK-8 reagent for 2 h, absorbance at 450 nm was measured using a microplate reader (BK-EL10A, Shandong Biogain). Based on preliminary dose-screening experiments (Supplementary Figure S2 ), BSP-Diol at 1 µM, which did not affect cell viability, was selected for subsequent in vitro experiments. 2.8 siRNA transfection Small interfering RNAs targeting human PKM2 and a negative control siRNA (si-NC) were synthesized by General Biosystems (China). Caco-2 cells were transfected at 70–80% confluence using Lipo6000™ transfection reagent (Beyotime, Cat. No. C0526) according to the manufacturer’s protocol. After 4–6 h, the transfection medium was replaced with complete culture medium, and cells were incubated for an additional 48 h before further analysis. 2.9 Immunofluorescence staining Cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% goat serum. Cells were incubated overnight at 4°C with rabbit anti-β-catenin antibody (Proteintech, Cat. No. 51067-2-AP, 1:2000), followed by incubation with Cy3-conjugated goat anti-rabbit IgG secondary antibody (Solarbio, Cat. No. SF134, 1:200) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI. Images were captured using a confocal microscope (ZEISS LSM880) or fluorescence microscope (Nikon Eclipse CI). Nuclear regions were defined based on DAPI staining, and cytoplasmic regions were defined as DAPI-negative areas within the cell boundary. β-catenin nuclear translocation was quantified as the ratio of nuclear to cytoplasmic fluorescence intensity using Image-Pro Plus 6.0 software. 2.10 Western blot analysis Protein extracts from colon tissues or cultured cells were prepared using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were incubated overnight at 4°C with primary antibodies against PKM2 (Proteintech, Cat. No. 15822-1-AP, 1:10,000) and β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:50,000). After incubation with HRP-conjugated secondary antibodies, signals were detected using enhanced chemiluminescence reagents and imaged with a Tanon-5200 system. Band intensities were quantified using ImageJ software and normalized to β-actin. 2.11 Statistical analysis All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 9.0. Data distribution was assessed for normality prior to parametric testing. Comparisons between two groups were performed using Student’s t-test, and multiple-group comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant. 3. Results To investigate how epithelial repair is regulated under inflammatory stress, we designed an integrated in vivo and in vitro study, as outlined in Fig. 1 . 3.1 Huanglian–Baiji treatment restores epithelial proliferative capacity and goblet cell integrity in DSS-induced colitis To evaluate the effects of the Huanglian–Baiji herb pair on mucosal renewal and barrier repair, immunohistochemical staining was performed to assess the expression of the epithelial proliferation marker Ki67 and the goblet cell marker Muc2 in colonic tissues. Based on dose–response evaluation of the Huanglian–Baiji formulation in DSS-induced colitis (Supplementary Figure S1 ), the high-dose treatment group (CB-H) exhibiting the most robust therapeutic efficacy was selected for subsequent mechanistic analyses. As shown in Fig. 2 , compared with the normal control group, DSS-induced colitis markedly reduced the proportion of Ki67-positive cells at the crypt base (p < 0.01), indicating impaired epithelial proliferative capacity under inflammatory conditions. In parallel, the number of Muc2-positive goblet cells was significantly decreased (p < 0.001), suggesting concomitant disruption of mucus secretion and mucosal barrier integrity. Following high-dose Huanglian–Baiji treatment (CB-H), the proportion of Ki67-positive epithelial cells was significantly increased compared with the DSS model group (p < 0.05), indicating restoration of epithelial proliferative activity. Consistently, Muc2 immunoreactivity was markedly enhanced after CB-H intervention (p < 0.001), with goblet cells exhibiting a more continuous and organized distribution along the crypt–surface axis. Collectively, these results demonstrate that Huanglian–Baiji treatment simultaneously promotes epithelial proliferation and goblet cell recovery in DSS-induced colitis, supporting mucosal regeneration at both structural and functional levels. While these in vivo findings establish the reparative efficacy of the Huanglian–Baiji herb pair at the tissue level, the molecular determinants underlying this effect remain unclear. To enable mechanistic dissection under controlled conditions, we therefore next focused on BSP-Diol, a representative bioactive constituent identified through network-based screening of the Huanglian–Baiji formulation(Supplementary Figure S3 ). 3.2 BSP-Diol restores β-catenin–dependent proliferative signaling under inflammatory stress To investigate the downstream mechanisms by which BSP-Diol regulates epithelial repair, immunofluorescence staining was performed to assess β-catenin expression and subcellular localization under inflammatory stress. As illustrated in Fig. 3 , inflammatory stimulation markedly attenuated β-catenin signaling, as evidenced by reduced fluorescence intensity and predominant cytoplasmic localization in the model group compared with control cells. This distribution pattern suggests impaired β-catenin nuclear translocation under inflammatory stress. Upon BSP-Diol treatment, β-catenin fluorescence intensity was substantially enhanced, with a significant accumulation in the nucleus. Quantitative analysis of β-catenin nuclear-positive areas further confirmed a significant increase in nuclear localization in the BSP-Diol–treated group compared with the model group (p < 0.05). Further validation was performed using a loss-of-function approach. siRNA-mediated knockdown of PKM2 significantly diminished BSP-Diol–induced β-catenin nuclear translocation (Supplementary Figure S5 ), reinforcing the role of PKM2 as a metabolic checkpoint required for the effective execution of repair programs. Taken together, these findings demonstrate that BSP-Diol effectively restores β-catenin nuclear translocation under inflammatory conditions, laying the foundation for the activation of transcriptional programs associated with epithelial repair. 3.3 BSP-Diol restores PKM2 expression to support proliferation-associated metabolic competence Given the requirement for adequate metabolic support during epithelial regeneration, we next examined the expression of pyruvate kinase M2 (PKM2), a key regulator of glycolytic flux and metabolic plasticity, by Western blot analysis. As shown in Fig. 4 , inflammatory stress markedly reduced PKM2 protein expression compared with control conditions (p < 0.01), indicating compromised metabolic capacity in injured epithelial cells. Upon BSP-Diol treatment, PKM2 protein levels were significantly restored relative to the model group (p < 0.01). Densitometric quantification normalized to β-actin confirmed this recovery. These findings indicate that BSP-Diol reverses inflammation-induced suppression of PKM2 expression, thereby re-establishing a level of metabolic competence required for the effective execution of epithelial repair programs under inflammatory stress. 3.4 Limited transcriptional modulation of MAM-associated metabolic regulators Given the close functional interplay between mitochondria-associated endoplasmic reticulum membranes (MAMs), mitochondrial activity, and cellular energy metabolism, we further assessed the transcriptional expression of selected metabolic regulators, including Ppargc1a, Sirt1, and Sirt3, using RT-qPCR. As shown in Fig. 5 , DSS-induced inflammatory stress significantly downregulated Ppargc1a expression compared with the control group (p < 0.05), consistent with suppression of mitochondrial biogenesis–associated transcriptional programs under inflammatory conditions. Following high-dose Huanglian–Baiji treatment, Ppargc1a expression exhibited a trend toward recovery, although this change did not reach statistical significance. Similarly, Sirt1 and Sirt3 displayed modest alterations in the DSS model group, suggesting disturbance of mitochondrial deacetylation–associated regulatory networks during inflammation. Huanglian–Baiji intervention tended to normalize the expression of these genes; however, no statistically significant differences were observed between groups. Collectively, these results indicate that Huanglian–Baiji treatment does not induce broad transcriptional reprogramming of MAM-associated metabolic regulators. Instead, the reparative effects are more likely mediated through post-transcriptional mechanisms, protein-level regulation, or changes in subcellular organization, consistent with a metabolic licensing rather than a metabolic reprogramming model. 3.5 Proposed model of BSP-Diol–mediated epithelial proliferation and repair through PKM2-dependent metabolic licensing Based on the findings described above, we propose a working model illustrating how BSP-Diol facilitates epithelial repair under inflammatory stress (Fig. 6 ). Under inflammatory conditions, epithelial cells exhibit compromised metabolic capacity, characterized by reduced PKM2 expression, which limits the effective execution of repair-associated programs. BSP-Diol treatment restores PKM2-dependent metabolic competence, thereby establishing a permissive metabolic state that enables β-catenin nuclear translocation and the activation of downstream epithelial repair processes, including epithelial proliferation and mucus barrier restoration. This dependency on PKM2 is further supported by loss-of-function analyses, in which PKM2 silencing markedly impaired BSP-Diol–induced β-catenin nuclear localization (Supplementary Figure S5 ), underscoring the essential role of PKM2 in establishing the repair-associated molecular state. Collectively, this model positions PKM2-dependent metabolic licensing as a prerequisite for effective mucosal regeneration under inflammatory stress, rather than a consequence of forced activation of proliferative signaling pathways. 4. Discussion Effective epithelial repair, driven by coordinated epithelial proliferation, is a critical determinant of mucosal healing in inflammatory bowel disease (IBD). However, the molecular requirements that enable epithelial cells to execute repair programs under inflammatory stress remain incompletely defined. While substantial efforts have focused on proliferative and inflammatory signaling pathways, comparatively less attention has been paid to how metabolic status modulates the effectiveness of repair-associated signaling once such pathways are activated. In the present study, we identify PKM2 as a metabolic regulator that constrains β-catenin–dependent epithelial repair under inflammatory stress. Rather than directly activating proliferative signaling, BSP-Diol restores PKM2 protein abundance suppressed by inflammation, thereby enabling effective β-catenin nuclear localization and downstream regenerative responses. These findings indicate that restoration of PKM2-dependent metabolic regulation is required for productive β-catenin signaling during epithelial repair and provides a mechanistic explanation for the reparative phenotype observed following Huanglian–Baiji treatment. 4.1 PKM2 modulates β-catenin signaling under inflammatory stress To elucidate the regulatory logic underlying epithelial repair, it is necessary to consider interactions between metabolic regulation and canonical signaling pathways. PKM2 has been widely characterized as a rate-limiting enzyme in glycolysis and a key mediator of metabolic reprogramming in proliferative tissues [ 16 , 17 ] , whereas β-catenin functions as a central transcriptional regulator driving epithelial proliferation and regeneration [ 18 ] . Within conventional paradigms, β-catenin activation is typically viewed as upstream of metabolic adaptation. However, our findings suggest that metabolic regulation can instead determine the efficiency of β-catenin signaling under inflammatory stress. Achieving mucosal healing, defined as restoration of epithelial barrier integrity, is a primary therapeutic goal in ulcerative colitis [ 19 ] . Under sustained inflammatory conditions, PKM2 protein levels were markedly reduced, coinciding with impaired β-catenin nuclear localization and restricted epithelial proliferation. Restoration of PKM2 by BSP-Diol did not act as a direct upstream activator of β-catenin; rather, it relieved a metabolic constraint that limited β-catenin–dependent proliferative responses. Loss-of-function experiments further demonstrated that PKM2 knockdown markedly attenuated BSP-Diol–induced β-catenin nuclear localization, indicating that intact PKM2 expression is required for efficient repair-associated signaling under inflammatory stress. This interpretation is consistent with prior observations that PKM2 structural status influences its subcellular localization and regulatory interactions [ 20 ] . Furthermore, context-dependent coupling between PKM2 expression and β-catenin signaling has been reported in other regenerative and pathological settings [ 21 ] . supporting the existence of a functional PKM2–β-catenin axis. By preferentially restoring PKM2 protein abundance, BSP-Diol re-establishes the metabolic capacity necessary to sustain β-catenin–dependent proliferative responses and to meet the bioenergetic and biosynthetic demands of epithelial repair, including cytoskeletal remodeling, macromolecule synthesis, and energy supply. This metabolic regulatory role may facilitate the canonical nuclear functions of PKM2 described in other regenerative contexts, where PKM2 directly interacts with β-catenin–dependent transcriptional machinery [ 22 ] . Loss-of-function experiments further demonstrated that PKM2 knockdown markedly attenuated BSP-Diol–induced β-catenin nuclear localization, indicating that intact PKM2 expression is required for efficient repair signaling. Collectively, these data suggest that PKM2-dependent metabolic regulation enables effective translation of β-catenin signaling into regenerative outcomes under inflammatory conditions. In vivo evidence from colitis models further supports this regulatory relationship, as PKM2 overexpression has been shown to sustain regenerative responses in the intestinal epithelium [ 23 ] . Importantly, these findings indicate that metabolic state is not merely a downstream consequence of epithelial repair, but an active regulator of signaling-dependent regeneration. By defining a PKM2–β-catenin regulatory axis under inflammatory stress, our study adds a mechanistic layer to current paradigms of mucosal healing in IBD, complementing established immune-mediated and stromal regulatory mechanisms [ 19 ] . 4.2 PKM2 restoration is associated with protein-level regulation rather than extensive transcriptional remodeling In the present study, key transcriptional regulators associated with mitochondrial biogenesis and function, including Ppargc1a, as well as deacetylases such as Sirt1 and Sirt3, did not exhibit statistically significant changes in mRNA expression following BSP-Diol treatment. This observation suggests that BSP-Diol–mediated metabolic regulation occurs primarily at the protein level rather than through broad transcriptional reprogramming. Under sustained inflammatory stress, extensive transcriptional remodeling is energetically demanding and may further exacerbate metabolic strain in compromised epithelial cells. In this context, rapid and reversible regulatory mechanisms at the protein and structural levels may represent a more efficient strategy to support repair-associated signaling. Such regulation enables epithelial cells to adjust metabolic capacity without incurring the high energetic cost associated with large-scale transcriptional changes [ 24 ] . Consistent with this notion, restoration of PKM2 protein abundance represents a central protein-level regulatory event that provides immediate metabolic support for β-catenin–dependent proliferative responses. PKM2 is known to exert localization-dependent functions, including modulation of mitochondrial and cytoplasmic signaling processes [ 25 ] . In addition, mitochondria-associated endoplasmic reticulum membranes (MAMs) have been recognized as critical platforms for metabolic coordination and stress adaptation [ 26 ] . Regulation of MAM integrity predominantly involves post-translational modifications and structural reorganization [ 27 , 28 ] , facilitating rapid metabolic adaptation to environmental challenges [ 29 , 30 ] . Collectively, these findings indicate that BSP-Diol–mediated epithelial repair does not require extensive transcriptional reprogramming. Instead, restoration of PKM2 at the protein level appears sufficient to re-establish metabolic capacity and support effective repair signaling under inflammatory stress. This protein-centered regulatory mode may be particularly advantageous in inflammatory microenvironments, where epithelial cells must initiate regenerative responses under conditions of limited metabolic resources [ 31 ] . 4.3 From herb pair to mechanistic node: implications and perspectives In this study, we employed an integrated strategy combining in vivo validation of therapeutic efficacy, network pharmacology–guided identification of bioactive constituents, and in vitro interrogation of specific regulatory nodes [ 32 ] .Through this approach, BSP-Diol was identified as a principal contributor to the mucosal repair–promoting effects of the Huanglian–Baiji herb pair, and a PKM2–β-catenin regulatory axis under inflammatory stress was delineated. This workflow provides a practical framework for mechanistic dissection of complex herbal formulations and enables translation of empirically defined therapeutic effects into experimentally tractable molecular pathways [ 33 ] . From a translational perspective, our findings suggest that restoration of metabolic regulation may represent a viable approach to support epithelial repair. Unlike strategies that directly amplify proliferative signaling, modulation of metabolic capacity may enhance the efficiency of endogenous repair pathways without excessive pathway activation. In chronic inflammatory diseases such as IBD, persistent stimulation of proliferative signaling carries the risk of disrupting tissue homeostasis. By contrast, targeting metabolic regulation may provide a more balanced means to support regenerative responses within physiologically constrained environments. Similar principles have been observed in metabolic modulators such as metformin, which exert protective effects through coordinated regulation of metabolic and signaling networks [ 34 ][ 32 , 35 ] . Several limitations should be acknowledged. BSP-Diol represents a single bioactive component and does not fully recapitulate the compositional complexity of the Huanglian–Baiji formulation. In addition, complete mucosal healing in vivo requires coordinated interactions between epithelial cells, immune regulation [ 36 , 37 ] , and the intestinal microbiota [ 38 – 40 ] . PKM2 may also exert context-dependent functions in epithelial and immune compartments [ 41 ] , raising the possibility that BSP-Diol may influence repair indirectly through immunometabolic pathways. Future studies incorporating multi-cellular and microbiota-integrated systems will be required to further define these interactions [ 42 ] . In summary, our study demonstrates that BSP-Diol promotes epithelial repair by restoring PKM2 expression and enabling β-catenin–dependent regenerative signaling under inflammatory stress. These findings identify metabolic regulation as an important modulator of signaling-dependent mucosal healing and provide mechanistic insight into how restoration of metabolic capacity may complement existing anti-inflammatory therapies in IBD. 5 Conclusion In conclusion, this study demonstrates that BSP-Diol promotes proliferation-dependent epithelial repair under inflammatory stress by restoring PKM2 expression and enabling efficient β-catenin–dependent signaling. Rather than directly activating proliferative pathways, BSP-Diol alleviates metabolic constraints that limit repair-associated signaling execution. By defining a PKM2–β-catenin regulatory axis in inflamed epithelium, our findings provide mechanistic insight into how metabolic state modulates signal-dependent epithelial regeneration. This work offers a molecular interpretation of the traditional concept of “promoting regeneration” and underscores the importance of metabolic regulation in mucosal healing. Collectively, these results suggest that therapeutic strategies aimed at restoring metabolic capacity—rather than excessively amplifying proliferative signaling—may represent a balanced and physiologically aligned approach for enhancing mucosal repair in inflammatory bowel disease. Declarations Funding This work was supported by the following grants: (1) Superior Specialized Department of Spleen and Stomach Diseases, State Administration of Traditional Chinese Medicine (Guozhongyi Yizheng Han [2024] No. 90); (2) The Fifth Batch of National Outstanding Chinese Medicine Clinical Talent Training Program, State Administration of Traditional Chinese Medicine (Guozhongyi Renjiao Han [2022] No. 1). The funding bodies had no role in the study design; data collection, analysis, or interpretation; or manuscript preparation. Ethics Approval and Consent to Participate All animal experiments were approved by the Institutional Animal Care and Use Committee of Jiangxi University of Traditional Chinese Medicine (Approval No.: JXHQ2025002; Animal Experimentation Facility License No.: SYXK[Jiangxi]2025-0005) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011). No human participants were involved in this study. Consent for Publication Not applicable. Availability of Data and Materials All data supporting the findings of this study are included within the article and its supplementary materials. Uncropped Western blot images corresponding to all immunoblotting results are provided in Supplementary File 1. Additional datasets generated during the current study are available from the corresponding authors upon reasonable request. Competing Interests The authors declare that they have no competing interests. Author Contributions Hou-Shu Tu: Conceptualization, Investigation, Data Curation, Writing – Original Draft. Meng-Lin Chen: Molecular Docking, Data Analysis, Visualization. Cui Xu: Animal Experiments, Data Collection. Chengming Yang: Cell-based Experiments, Data Analysis. Sai Li: Statistical Analysis, Figure Preparation. Jing Hong: Supervision, Writing – Review & Editing. Ling He: Supervision, Project Administration, Writing – Review & Editing. All authors read and approved the final manuscript. Acknowledgements The authors sincerely thank the State Administration of Traditional Chinese Medicine and the affiliated institutions for their support. We also acknowledge our colleagues for technical assistance and valuable discussions. References Ng, S. C., Shi, H. Y., Hamidi, N., Underwood, F. E., Tang, W., Benchimol, E. I.,... Kaplan, G. G. (2017). Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. 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Opening the doors of precision medicine: novel tools to assess intestinal barrier in inflammatory bowel disease and colitis-associated neoplasia. Gut , 73(10), 1749-1762. doi: 10.1136/gutjnl-2023-331579 Alves-Filho, J. C., & Pålsson-McDermott, E. M. (2016). Pyruvate kinase M2: a potential target for regulating inflammation. Frontiers in immunology , 7, 145 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile1UncroppedWesternBlots.pptx SupplementaryFiguerS1.tif Supplementary Figure S1. Dose-dependent therapeutic effects of Huanglian–Baiji herb pair in DSS-induced colitis. Compared with the DSS model group, treatment with the Huanglian–Baiji herb pair significantly attenuated body weight loss, diarrhea, and rectal bleeding. Dynamic analysis of disease activity index (DAI) revealed that all treatment groups exhibited lower DAI scores throughout the intervention period, with a clear dose-dependent improvement. Among them, the high-dose group (CB-H) showed the most pronounced therapeutic effect (A). Macroscopic examination demonstrated that DSS-induced colonic shortening and edema were markedly alleviated following Huanglian–Baiji treatment. Representative colon images and quantitative analysis of colon length showed significant recovery in treated groups, with the CB-H group displaying the greatest improvement (B). Histological assessment by H&E staining revealed severe epithelial disruption, crypt distortion, and inflammatory cell infiltration in the DSS model group. In contrast, Huanglian–Baiji treatment markedly ameliorated mucosal damage, reduced inflammatory infiltration, and restored crypt architecture, with the CB-H group showing the most prominent histological improvement (C). Based on these dose–response comparisons, the high-dose Huanglian–Baiji formulation (CB-H) was selected for subsequent mechanistic studies. SupplementaryFiguerS2.tif Supplementary Figure S2. In vitro dose screening and experimental grouping design. (A) LPS-induced IL-6 production at different concentrations and stimulation durations (6 h, 12 h, and 24 h). (B) Effects of palmatine at indicated concentrations on cell viability assessed by CCK-8 assay. (C) Effects of BSP-Diol at indicated concentrations on cell viability assessed by CCK-8 assay. (D) Schematic illustration of experimental grouping and treatment design, including Control, LPS model, Palmatine, BSP-Diol, and combination groups. Data are presented as mean ± SEM. *P < 0.05 compared with the control group. Based on these results, palmatine (5 μM) and BSP-Diol (1 μM) were selected for subsequent in vitro experiments. SupplementaryFiguerS3.tif Supplementary Figure S3. Molecular docking analysis supporting compound selection. (A) Docking pose of LIMK1 with palmatine. (B) Docking pose of PKM with BSP-Diol. Molecular docking analysis suggested favorable binding energies for PKM–BSP-Diol (−9.8 kcal/mol) and LIMK1–palmatine (−8.4 kcal/mol) (Supplementary Table S2). These results support the structural plausibility of the predicted interactions and provide a computational basis for selecting BSP-Diol and palmatine for subsequent in vitro mechanistic analyses. SupplementaryFiguerS4.tif Supplementary Figure S4. Molecular dynamics simulations of docking complexes. Molecular dynamics simulations were performed to evaluate the dynamic stability of the docking complexes. Two independent 200-ns simulations were conducted for each system. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), total energy profiles, hydrogen bond numbers, and ligand–protein distance analyses indicated that both complexes maintained stable interaction patterns throughout the simulation period. Detailed trajectory analyses are provided in the Supplementary Materials. SupplementaryFiguerS5.tif Supplementary Figure S5. PKM2 is required for combination-enabled β-catenin nuclear localization. (A) Screening and validation of PKM2 siRNA efficiency by Western blot analysis. Cells were transfected with three independent siRNA sequences targeting PKM2 (si-1, si-2, si-3) or negative control siRNA (si-NC). Total PKM2 protein levels were assessed, with β-actin used as a loading control. Quantitative analysis confirmed a significant reduction of PKM2 protein levels in all PKM2 siRNA groups compared with si-NC (***P < 0.001), while no significant difference was observed between the control and si-NC groups (ns). (B) Immunofluorescence analysis of β-catenin expression and subcellular localization under PKM2 knockdown conditions following combination treatment. Compared with the si-NC + Combination group, PKM2 silencing markedly reduced β-catenin fluorescence intensity and impaired its nuclear localization in the si-PKM2 + Combination group. Quantification of β-catenin nuclear-positive cells is shown on the right (***P < 0.001). Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001; ns, not significant. SupplementaryTableS1.docx SupplementaryTableS2.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 11 May, 2026 Reviews received at journal 08 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 10 Mar, 2026 Submission checks completed at journal 10 Mar, 2026 First submitted to journal 10 Mar, 2026 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. 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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-9086575","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607414555,"identity":"dacf4626-eda6-4e70-a879-b77513253ba9","order_by":0,"name":"Houshu Tu","email":"","orcid":"","institution":"Jiangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Houshu","middleName":"","lastName":"Tu","suffix":""},{"id":607414558,"identity":"beef9c96-9d47-48ee-b024-23e729fe9699","order_by":1,"name":"Menglin Chen","email":"","orcid":"","institution":"Jiangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Menglin","middleName":"","lastName":"Chen","suffix":""},{"id":607414560,"identity":"80fb0655-b0e0-4c3f-ad4f-c751392a52e7","order_by":2,"name":"Cui Xu¹","email":"","orcid":"","institution":"Jiangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cui","middleName":"","lastName":"Xu¹","suffix":""},{"id":607414561,"identity":"d4ca480f-76fd-468e-a75f-7dc6c32d481d","order_by":3,"name":"Chengming Yang¹","email":"","orcid":"","institution":"Jiangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chengming","middleName":"","lastName":"Yang¹","suffix":""},{"id":607414562,"identity":"5f76b347-9a3c-4d31-abd5-d00d611a9f87","order_by":4,"name":"Sai Li¹","email":"","orcid":"","institution":"Jiangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sai","middleName":"","lastName":"Li¹","suffix":""},{"id":607414563,"identity":"069d2cad-2bb3-4524-9753-9a6779581d44","order_by":5,"name":"Jing Hong","email":"","orcid":"","institution":"Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Hong","suffix":""},{"id":607414564,"identity":"55625f36-c004-464f-a241-a5644f9f2992","order_by":6,"name":"Ling He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACAwbmhgMMDBI8/OyNjQ8+EKeFEaTFQkay53Cz4QxitQCpChuDG+lt0hzEaDEHuudwwS8JHsmZDxukGRjs5HQbCGix7DnYcHhmH9Av0okNxgUMycZmBwg57EZiw2HeHqAtsxMbkmcwHEjcRlDL/YcQLQY3gdbxEKXlBiNQ5Q+glhuMjc3EaTkDclgD0GE9ic2MMwyI8cvxw4c/8/yps+dnP/78x4cKOzmCWsCAsQ1uAjHKweAP0SpHwSgYBaNgJAIAHJlIbUy5FKoAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ling","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2026-03-10 17:09:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9086575/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9086575/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105004662,"identity":"ab3c2b9c-59ab-4c84-a1e6-7ee709331eb8","added_by":"auto","created_at":"2026-03-19 18:04:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10103032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy design and hypothesis-driven strategy for dissecting epithelial repair under inflammatory stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDSS-induced colitis leads to epithelial injury and impaired mucosal repair, accompanied by metabolic insufficiency. In vivo, the Huanglian–Baiji herbal formulation was administered to evaluate epithelial repair phenotypes in colonic tissues. In vitro, BSP-Diol was used to dissect the molecular mechanisms underlying epithelial repair competence under inflammatory stress. Dashed arrows indicate proposed but unconfirmed regulatory links.\u003c/p\u003e","description":"","filename":"Figuer1.png","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/159063ace8005afb8c33f5a2.png"},{"id":105035783,"identity":"b1b5f93e-a576-4d76-96b1-136719aa6cd4","added_by":"auto","created_at":"2026-03-20 07:26:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17792827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuanglian–Baiji herbal pair promotes epithelial proliferation and goblet cell restoration in DSS-induced colitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative immunohistochemical staining and quantitative analysis of Ki67 (epithelial proliferation marker) and Muc2 (goblet cell marker) in colonic tissues. Compared with the control group, DSS-induced colitis markedly reduced the proportion of Ki67-positive epithelial cells and the number of Muc2-positive goblet cells. Treatment with high-dose Huanglian–Baiji herbal pair (CB-H) significantly restored Ki67-positive cell proliferation and Muc2-positive goblet cell abundance. Scale bar = 50 μm. Data are presented as mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figuer2.png","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/8398e15ac9d0b1de17572322.png"},{"id":105035804,"identity":"5c3180e7-75ae-453e-b20b-9bf313331d32","added_by":"auto","created_at":"2026-03-20 07:26:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7542075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBSP-Diol promotes β-catenin nuclear translocation in intestinal epithelial cells under inflammatory stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative immunofluorescence images showing β-catenin expression and subcellular localization in different treatment groups. Nuclei were counterstained with DAPI (blue), and β-catenin was visualized in red. Quantitative analysis of β-catenin nuclear-positive area is shown below. Data are presented as mean ± SEM (n = 3). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figuer3.png","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/5b7920e8ed090998f7b7de74.png"},{"id":105004666,"identity":"6b80bf6b-cdc1-40f5-8f58-59a22cf454fe","added_by":"auto","created_at":"2026-03-19 18:04:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":885139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBSP-Diol restores PKM2 protein expression under inflammatory conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis of PKM2 protein expression in different treatment groups, with β-actin used as a loading control. Densitometric quantification of PKM2/β-actin is shown below. Data are presented as mean ± SEM (n = 3). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figuer4.png","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/92832eafca2f4df5971d0a05.png"},{"id":105004663,"identity":"07542585-b676-4007-a72e-604385654eed","added_by":"auto","created_at":"2026-03-19 18:04:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":254687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional changes of MAM-associated metabolic regulators in colonic tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR analysis of Ppargc1a, Sirt1, and Sirt3 mRNA expression levels in colonic tissues. Data are presented as mean ± SEM. ns indicates no statistically significant difference.\u003c/p\u003e","description":"","filename":"Figuer5.png","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/ef17c8937cdf77d21289e923.png"},{"id":105004667,"identity":"930efaf7-42a2-4a86-acc2-4e69b2b47073","added_by":"auto","created_at":"2026-03-19 18:04:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9185077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model of BSP-Diol–mediated epithelial repair through PKM2-dependent metabolic licensing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder inflammatory stress, epithelial cells exhibit impaired metabolic capacity, characterized by reduced PKM2 activity, which limits the execution of repair programs. BSP-Diol restores PKM2-dependent metabolic competence, functioning as a permissive metabolic checkpoint that enables β-catenin nuclear translocation and downstream epithelial repair processes. This model highlights metabolic licensing as a prerequisite for effective mucosal regeneration rather than forced activation of proliferative signaling.\u003c/p\u003e","description":"","filename":"Figuer6.png","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/447a9fdd4fb54143ca18c34e.png"},{"id":105562855,"identity":"be13070c-6b29-48f5-b0e6-ac09929bcb5f","added_by":"auto","created_at":"2026-03-27 12:44:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":41788367,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/2e0c3884-f77d-4a3c-a0b4-e9ff8c5ac656.pdf"},{"id":105004664,"identity":"6df0a1fc-9958-4c54-8188-4ec63ff462bf","added_by":"auto","created_at":"2026-03-19 18:04:28","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1569625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFile1UncroppedWesternBlots.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/1a6ade56d6c903fed38357aa.pptx"},{"id":105004675,"identity":"77143352-f926-45b0-bbd6-1abd777b4ac5","added_by":"auto","created_at":"2026-03-19 18:04:29","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12255360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1. Dose-dependent therapeutic effects of Huanglian–Baiji herb pair in DSS-induced colitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with the DSS model group, treatment with the Huanglian–Baiji herb pair significantly attenuated body weight loss, diarrhea, and rectal bleeding. Dynamic analysis of disease activity index (DAI) revealed that all treatment groups exhibited lower DAI scores throughout the intervention period, with a clear dose-dependent improvement. Among them, the high-dose group (CB-H) showed the most pronounced therapeutic effect (A).\u003c/p\u003e\n\u003cp\u003eMacroscopic examination demonstrated that DSS-induced colonic shortening and edema were markedly alleviated following Huanglian–Baiji treatment. Representative colon images and quantitative analysis of colon length showed significant recovery in treated groups, with the CB-H group displaying the greatest improvement (B).\u003c/p\u003e\n\u003cp\u003eHistological assessment by H\u0026amp;E staining revealed severe epithelial disruption, crypt distortion, and inflammatory cell infiltration in the DSS model group. In contrast, Huanglian–Baiji treatment markedly ameliorated mucosal damage, reduced inflammatory infiltration, and restored crypt architecture, with the CB-H group showing the most prominent histological improvement (C).\u003c/p\u003e\n\u003cp\u003eBased on these dose–response comparisons, the high-dose Huanglian–Baiji formulation (CB-H) was selected for subsequent mechanistic studies.\u003c/p\u003e","description":"","filename":"SupplementaryFiguerS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/63b0d3c6eb59b4a668f25338.tif"},{"id":105035634,"identity":"58031102-4940-4e5d-826f-50f2274a9e6c","added_by":"auto","created_at":"2026-03-20 07:26:22","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5933412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S2. In vitro dose screening and experimental grouping design.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) LPS-induced IL-6 production at different concentrations and stimulation durations (6 h, 12 h, and 24 h).\u003c/p\u003e\n\u003cp\u003e(B) Effects of palmatine at indicated concentrations on cell viability assessed by CCK-8 assay.\u003c/p\u003e\n\u003cp\u003e(C) Effects of BSP-Diol at indicated concentrations on cell viability assessed by CCK-8 assay.\u003c/p\u003e\n\u003cp\u003e(D) Schematic illustration of experimental grouping and treatment design, including Control, LPS model, Palmatine, BSP-Diol, and combination groups.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM. *P \u0026lt; 0.05 compared with the control group. Based on these results, palmatine (5 μM) and BSP-Diol (1 μM) were selected for subsequent in vitro experiments.\u003c/p\u003e","description":"","filename":"SupplementaryFiguerS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/ec9e92c695206b360da7681c.tif"},{"id":105004670,"identity":"3be10305-c1b6-4699-9b69-a96fbfa44499","added_by":"auto","created_at":"2026-03-19 18:04:29","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5799136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S3. Molecular docking analysis supporting compound selection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Docking pose of LIMK1 with palmatine.\u003c/p\u003e\n\u003cp\u003e(B) Docking pose of PKM with BSP-Diol.\u003c/p\u003e\n\u003cp\u003eMolecular docking analysis suggested favorable binding energies for PKM–BSP-Diol (−9.8 kcal/mol) and LIMK1–palmatine (−8.4 kcal/mol) (Supplementary Table S2). These results support the structural plausibility of the predicted interactions and provide a computational basis for selecting BSP-Diol and palmatine for subsequent in vitro mechanistic analyses.\u003c/p\u003e","description":"","filename":"SupplementaryFiguerS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/40bd0b5f2c04dd83ed0fe904.tif"},{"id":105004668,"identity":"a0ced29b-6fd5-4fb5-b8c5-a888c8ef8165","added_by":"auto","created_at":"2026-03-19 18:04:29","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4280820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S4. Molecular dynamics simulations of docking complexes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular dynamics simulations were performed to evaluate the dynamic stability of the docking complexes. Two independent 200-ns simulations were conducted for each system. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), total energy profiles, hydrogen bond numbers, and ligand–protein distance analyses indicated that both complexes maintained stable interaction patterns throughout the simulation period. Detailed trajectory analyses are provided in the Supplementary Materials.\u003c/p\u003e","description":"","filename":"SupplementaryFiguerS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/9aee773a1158a387cf25dbc0.tif"},{"id":105004676,"identity":"f6408eab-a17b-43bb-b62b-bdccbb105941","added_by":"auto","created_at":"2026-03-19 18:04:29","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3810216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S5. PKM2 is required for combination-enabled β-catenin nuclear localization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Screening and validation of PKM2 siRNA efficiency by Western blot analysis. Cells were transfected with three independent siRNA sequences targeting PKM2 (si-1, si-2, si-3) or negative control siRNA (si-NC). Total PKM2 protein levels were assessed, with β-actin used as a loading control. Quantitative analysis confirmed a significant reduction of PKM2 protein levels in all PKM2 siRNA groups compared with si-NC (***P \u0026lt; 0.001), while no significant difference was observed between the control and si-NC groups (ns).\u003c/p\u003e\n\u003cp\u003e(B) Immunofluorescence analysis of β-catenin expression and subcellular localization under PKM2 knockdown conditions following combination treatment. Compared with the si-NC + Combination group, PKM2 silencing markedly reduced β-catenin fluorescence intensity and impaired its nuclear localization in the si-PKM2 + Combination group. Quantification of β-catenin nuclear-positive cells is shown on the right (***P \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM. **P \u0026lt; 0.01, ***P \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"SupplementaryFiguerS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/92650fe0221c1c674c5c1ed6.tif"},{"id":105004671,"identity":"8e72ffcc-1c66-41d3-a540-1722dce81d2c","added_by":"auto","created_at":"2026-03-19 18:04:29","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12320,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/1d81ab9dcc09d3104e47e10f.docx"},{"id":105004672,"identity":"fd1cd479-d01c-437f-88da-40b14f341a12","added_by":"auto","created_at":"2026-03-19 18:04:29","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":13491,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9086575/v1/5402d6bab6ab8dcdc53deff7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"BSP-Diol restores PKM2 and facilitates β-catenin–dependent epithelial repair in experimental colitis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eInflammatory bowel disease (IBD), encompassing Crohn\u0026rsquo;s disease and ulcerative colitis, is a chronic relapsing inflammatory disorder of the gastrointestinal tract. The global incidence and prevalence of IBD continue to rise, particularly in newly industrialized regions, imposing a substantial healthcare burden\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Persistent disruption of the intestinal epithelial barrier and defective mucosal repair are central pathophysiological features of IBD, contributing to sustained inflammation and progressive tissue injury\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Although current therapies primarily target immune-mediated inflammation\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, durable remission is closely associated with effective mucosal healing, which depends on the ability of intestinal epithelial cells to re-enter and sustain proliferative programs.\u003c/p\u003e \u003cp\u003eWnt/β-catenin signaling plays a central role in intestinal epithelial regeneration. Nuclear translocation of β-catenin activates transcriptional programs necessary for epithelial proliferation and barrier restoration. However, under inflammatory stress, epithelial repair frequently remains insufficient despite the presence of repair-associated signaling activity. This raises an important question: does repair failure reflect absence of regenerative signals, or does inflammatory stress impose metabolic constraints that limit the effective execution of these signaling programs?\u003c/p\u003e \u003cp\u003eEmerging evidence indicates that chronic inflammation induces mitochondrial dysfunction, altered metabolic reprogramming, and impaired biosynthetic capacity in intestinal epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Such metabolic disturbances may restrict cellular proliferation, migration, and barrier reconstruction even when regenerative signaling pathways are intactt\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that metabolic state may critically modulate signaling-dependent epithelial repair.\u003c/p\u003e \u003cp\u003ePyruvate kinase M2 (PKM2) is a key glycolytic enzyme that also participates in metabolic regulation of proliferative responses. In intestinal epithelium, PKM2 has been implicated in maintaining epithelial integrity and facilitating β-catenin\u0026ndash;dependent repair processes\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, Epithelial-specific deletion of PKM2 exacerbates experimental colitis and impairs regenerative responses, highlighting a potential role for PKM2 in supporting signaling-dependent mucosal healing. These observations position PKM2 as a candidate metabolic regulator of epithelial repair under inflammatory stress.\u003c/p\u003e \u003cp\u003eThe Huanglian\u0026ndash;Baiji herb pair is traditionally used to promote tissue regeneration. Bletilla striata (Baiji) contains bioactive constituents including polysaccharides and small molecules such as BSP-Diol, which exhibit anti-inflammatory and wound-healing properties\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, whether Baiji-derived components modulate epithelial repair through metabolic regulation of signaling pathways remains unclear.\u003c/p\u003e \u003cp\u003eIn the present study, we employed an integrated strategy combining in vivo validation, network pharmacology\u0026ndash;guided component prioritization, and mechanistic analyses to investigate whether BSP-Diol promotes epithelial repair through modulation of PKM2-associated metabolic regulation. Using a DSS-induced colitis model, we examined epithelial proliferation, goblet cell restoration, β-catenin nuclear localization, and PKM2 expression under inflammatory stress. Our findings reveal a PKM2\u0026ndash;β-catenin regulatory axis that links metabolic state to signaling-dependent epithelial regeneration in IBD.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals and experimental colitis model\u003c/h2\u003e \u003cp\u003eSpecific pathogen\u0026ndash;free (SPF) male C57BL/6J mice (8 weeks old, 20\u0026ndash;23 g) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China; production license No. SCXK (Beijing) 2024-0001). Animals were housed under standard SPF conditions with a 12 h light/dark cycle, controlled temperature (22\u0026ndash;25\u0026deg;C), and relative humidity (50\u0026ndash;65%), with free access to food and water. All experimental procedures were approved by the Animal Ethics Committee of Jiangxi Hengqing Technical Testing Co., Ltd. (approval No. JXHQ2025002).\u003c/p\u003e \u003cp\u003eExperimental colitis was induced by administering 3% (w/v) dextran sulfate sodium (DSS; MP Biomedicals, molecular weight 36\u0026ndash;50 kDa, Cat. No. YD05012) in drinking water for 7 consecutive days. After a 5-day acclimatization period, a total of 48 mice were randomly assigned to six groups (n\u0026thinsp;=\u0026thinsp;8 per group): (1) normal control group; (2) DSS model group; (3) mesalazine-treated positive control group (200 mg/kg/day); and (4\u0026ndash;6) Huanglian\u0026ndash;Baiji herb pair low-, medium-, and high-dose groups (100, 200, and 400 mg/kg/day, respectively).\u003c/p\u003e \u003cp\u003eAll pharmacological interventions were administered by oral gavage once daily concomitantly with DSS exposure throughout the 7-day induction period, corresponding to a preventive intervention protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Drugs, reagents, and administration\u003c/h2\u003e \u003cp\u003eHuanglian granules (China National Pharmaceutical Group Zhonglian Pharmaceutical Co., Ltd., batch No. 240041; 1 g granules equivalent to 4.5 g crude drug) and Baiji granules (Beijing Kangrentang Pharmaceutical Co., Ltd., batch No. 24012071; 1 g granules equivalent to 4.3 g crude drug) were dissolved in normal saline prior to use. The high-dose Huanglian\u0026ndash;Baiji group (CB-H) received a total granule dose of 400 mg/kg/day.\u003c/p\u003e \u003cp\u003eMesalazine enteric-coated tablets (Sunflower Pharmaceutical Group, batch No. 240807) were suspended in normal saline at a concentration of 20 mg/mL and administered at 200 mg/kg/day. All gavage volumes were fixed at 0.25 mL per mouse per day.\u003c/p\u003e \u003cp\u003eBSP-Diol (YuanYe Bio, Cat. No. B22600) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution and diluted with culture medium to the desired working concentrations for in vitro experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Tissue collection and histopathological evaluation\u003c/h2\u003e \u003cp\u003eOn day 7, mice were anesthetized and euthanized. The entire colon was excised, measured for length, and photographed. Proximal colon segments were fixed in 4% paraformaldehyde for paraffin embedding, sectioning (5 \u0026micro;m), and histological analysis. Hematoxylin and eosin (H\u0026amp;E)\u0026ndash;stained sections were evaluated using blinded pathological scoring. Remaining tissues were snap-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent molecular analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin-embedded sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% H₂O₂, followed by blocking with 5% bovine serum albumin (BSA) at room temperature for 1 h. Sections were incubated overnight at 4\u0026deg;C with the following primary antibodies: rabbit anti-Ki67 (Proteintech, Cat. No. 27309-1-AP, 1:4000) and rabbit anti-Muc2 (Proteintech, Cat. No. 27675-1-AP, 1:2000).\u003c/p\u003e \u003cp\u003eAfter washing, sections were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (Servicebio, Cat. No. GB23204, 1:500) for 1 h at room temperature. Signal detection was performed using a DAB substrate kit (Wuhan Boster Bioengineering, Cat. No. B0053), followed by hematoxylin counterstaining. Slides were scanned using a digital slide scanner (3DHISTECH Pannoramic MIDI/DESK). Quantitative analysis was conducted using Image-Pro Plus 6.0 software on at least five randomly selected non-overlapping fields per section (200\u0026times;). The percentage of Ki67-positive cells at the crypt base and the area fraction of Muc2-positive staining within the epithelial region were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from colon tissues using TRIzol reagent (TAKARA) according to the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed using a NanoDrop\u0026trade; One spectrophotometer (Thermo Fisher Scientific), with A260/A280 ratios between 1.8 and 2.0. RNA integrity was confirmed by agarose gel electrophoresis.\u003c/p\u003e \u003cp\u003eReverse transcription was performed using the PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (Perfect Real Time, TAKARA) with 1 \u0026micro;g total RNA per sample. Quantitative PCR was conducted on an FQD-96A real-time PCR system (Hangzhou Bioer Technology, China) using SYBR\u0026reg; Green Premix Pro Taq HS (Xiamen Lifeint, Cat. No. A4004M). Actb (β-actin) was used as the internal reference gene, and relative gene expression levels were calculated using the 2^\u0026minus;ΔΔCt method. Primer sequences are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell culture and treatments\u003c/h2\u003e \u003cp\u003eHuman colorectal adenocarcinoma Caco-2 cells (iCell, Cat. No. iCell-h032) were cultured in MEM supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% non-essential amino acids (NEAA) at 37\u0026deg;C in a humidified incubator with 5% CO₂. To establish an in vitro inflammatory model, cells were stimulated with lipopolysaccharide (LPS; 1 \u0026micro;g/mL; Solarbio, Cat. No. L8880) for 24 h. BSP-Diol was administered as a pretreatment prior to LPS stimulation where indicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell viability assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8; GLPBIO, Cat. No. GK10001). Caco-2 cells were seeded in 96-well plates at a density of 1 \u0026times; 10⁴ cells per well and treated with various concentrations of BSP-Diol (0\u0026ndash;100 \u0026micro;M) for 24 h. After incubation with CCK-8 reagent for 2 h, absorbance at 450 nm was measured using a microplate reader (BK-EL10A, Shandong Biogain). Based on preliminary dose-screening experiments (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), BSP-Diol at 1 \u0026micro;M, which did not affect cell viability, was selected for subsequent in vitro experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 siRNA transfection\u003c/h2\u003e \u003cp\u003eSmall interfering RNAs targeting human PKM2 and a negative control siRNA (si-NC) were synthesized by General Biosystems (China). Caco-2 cells were transfected at 70\u0026ndash;80% confluence using Lipo6000\u0026trade; transfection reagent (Beyotime, Cat. No. C0526) according to the manufacturer\u0026rsquo;s protocol. After 4\u0026ndash;6 h, the transfection medium was replaced with complete culture medium, and cells were incubated for an additional 48 h before further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eCells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% goat serum. Cells were incubated overnight at 4\u0026deg;C with rabbit anti-β-catenin antibody (Proteintech, Cat. No. 51067-2-AP, 1:2000), followed by incubation with Cy3-conjugated goat anti-rabbit IgG secondary antibody (Solarbio, Cat. No. SF134, 1:200) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI.\u003c/p\u003e \u003cp\u003eImages were captured using a confocal microscope (ZEISS LSM880) or fluorescence microscope (Nikon Eclipse CI). Nuclear regions were defined based on DAPI staining, and cytoplasmic regions were defined as DAPI-negative areas within the cell boundary. β-catenin nuclear translocation was quantified as the ratio of nuclear to cytoplasmic fluorescence intensity using Image-Pro Plus 6.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blot analysis\u003c/h2\u003e \u003cp\u003eProtein extracts from colon tissues or cultured cells were prepared using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were incubated overnight at 4\u0026deg;C with primary antibodies against PKM2 (Proteintech, Cat. No. 15822-1-AP, 1:10,000) and β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:50,000). After incubation with HRP-conjugated secondary antibodies, signals were detected using enhanced chemiluminescence reagents and imaged with a Tanon-5200 system. Band intensities were quantified using ImageJ software and normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 9.0. Data distribution was assessed for normality prior to parametric testing. Comparisons between two groups were performed using Student\u0026rsquo;s t-test, and multiple-group comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eTo investigate how epithelial repair is regulated under inflammatory stress, we designed an integrated in vivo and in vitro study, as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Huanglian\u0026ndash;Baiji treatment restores epithelial proliferative capacity and goblet cell integrity in DSS-induced colitis\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of the Huanglian\u0026ndash;Baiji herb pair on mucosal renewal and barrier repair, immunohistochemical staining was performed to assess the expression of the epithelial proliferation marker Ki67 and the goblet cell marker Muc2 in colonic tissues.\u003c/p\u003e \u003cp\u003eBased on dose\u0026ndash;response evaluation of the Huanglian\u0026ndash;Baiji formulation in DSS-induced colitis (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), the high-dose treatment group (CB-H) exhibiting the most robust therapeutic efficacy was selected for subsequent mechanistic analyses.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, compared with the normal control group, DSS-induced colitis markedly reduced the proportion of Ki67-positive cells at the crypt base (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating impaired epithelial proliferative capacity under inflammatory conditions. In parallel, the number of Muc2-positive goblet cells was significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting concomitant disruption of mucus secretion and mucosal barrier integrity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing high-dose Huanglian\u0026ndash;Baiji treatment (CB-H), the proportion of Ki67-positive epithelial cells was significantly increased compared with the DSS model group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating restoration of epithelial proliferative activity. Consistently, Muc2 immunoreactivity was markedly enhanced after CB-H intervention (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with goblet cells exhibiting a more continuous and organized distribution along the crypt\u0026ndash;surface axis.\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that Huanglian\u0026ndash;Baiji treatment simultaneously promotes epithelial proliferation and goblet cell recovery in DSS-induced colitis, supporting mucosal regeneration at both structural and functional levels.\u003c/p\u003e \u003cp\u003eWhile these in vivo findings establish the reparative efficacy of the Huanglian\u0026ndash;Baiji herb pair at the tissue level, the molecular determinants underlying this effect remain unclear. To enable mechanistic dissection under controlled conditions, we therefore next focused on BSP-Diol, a representative bioactive constituent identified through network-based screening of the Huanglian\u0026ndash;Baiji formulation(Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 BSP-Diol restores β-catenin\u0026ndash;dependent proliferative signaling under inflammatory stress\u003c/h2\u003e \u003cp\u003eTo investigate the downstream mechanisms by which BSP-Diol regulates epithelial repair, immunofluorescence staining was performed to assess β-catenin expression and subcellular localization under inflammatory stress.\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, inflammatory stimulation markedly attenuated β-catenin signaling, as evidenced by reduced fluorescence intensity and predominant cytoplasmic localization in the model group compared with control cells. This distribution pattern suggests impaired β-catenin nuclear translocation under inflammatory stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon BSP-Diol treatment, β-catenin fluorescence intensity was substantially enhanced, with a significant accumulation in the nucleus. Quantitative analysis of β-catenin nuclear-positive areas further confirmed a significant increase in nuclear localization in the BSP-Diol\u0026ndash;treated group compared with the model group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFurther validation was performed using a loss-of-function approach. siRNA-mediated knockdown of PKM2 significantly diminished BSP-Diol\u0026ndash;induced β-catenin nuclear translocation (Supplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), reinforcing the role of PKM2 as a metabolic checkpoint required for the effective execution of repair programs.\u003c/p\u003e \u003cp\u003eTaken together, these findings demonstrate that BSP-Diol effectively restores β-catenin nuclear translocation under inflammatory conditions, laying the foundation for the activation of transcriptional programs associated with epithelial repair.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 BSP-Diol restores PKM2 expression to support proliferation-associated metabolic competence\u003c/h2\u003e \u003cp\u003eGiven the requirement for adequate metabolic support during epithelial regeneration, we next examined the expression of pyruvate kinase M2 (PKM2), a key regulator of glycolytic flux and metabolic plasticity, by Western blot analysis.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, inflammatory stress markedly reduced PKM2 protein expression compared with control conditions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating compromised metabolic capacity in injured epithelial cells. Upon BSP-Diol treatment, PKM2 protein levels were significantly restored relative to the model group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Densitometric quantification normalized to β-actin confirmed this recovery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings indicate that BSP-Diol reverses inflammation-induced suppression of PKM2 expression, thereby re-establishing a level of metabolic competence required for the effective execution of epithelial repair programs under inflammatory stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Limited transcriptional modulation of MAM-associated metabolic regulators\u003c/h2\u003e \u003cp\u003eGiven the close functional interplay between mitochondria-associated endoplasmic reticulum membranes (MAMs), mitochondrial activity, and cellular energy metabolism, we further assessed the transcriptional expression of selected metabolic regulators, including Ppargc1a, Sirt1, and Sirt3, using RT-qPCR.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, DSS-induced inflammatory stress significantly downregulated Ppargc1a expression compared with the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with suppression of mitochondrial biogenesis\u0026ndash;associated transcriptional programs under inflammatory conditions. Following high-dose Huanglian\u0026ndash;Baiji treatment, Ppargc1a expression exhibited a trend toward recovery, although this change did not reach statistical significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, Sirt1 and Sirt3 displayed modest alterations in the DSS model group, suggesting disturbance of mitochondrial deacetylation\u0026ndash;associated regulatory networks during inflammation. Huanglian\u0026ndash;Baiji intervention tended to normalize the expression of these genes; however, no statistically significant differences were observed between groups.\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that Huanglian\u0026ndash;Baiji treatment does not induce broad transcriptional reprogramming of MAM-associated metabolic regulators. Instead, the reparative effects are more likely mediated through post-transcriptional mechanisms, protein-level regulation, or changes in subcellular organization, consistent with a metabolic licensing rather than a metabolic reprogramming model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Proposed model of BSP-Diol\u0026ndash;mediated epithelial proliferation and repair through PKM2-dependent metabolic licensing\u003c/h2\u003e \u003cp\u003eBased on the findings described above, we propose a working model illustrating how BSP-Diol facilitates epithelial repair under inflammatory stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder inflammatory conditions, epithelial cells exhibit compromised metabolic capacity, characterized by reduced PKM2 expression, which limits the effective execution of repair-associated programs. BSP-Diol treatment restores PKM2-dependent metabolic competence, thereby establishing a permissive metabolic state that enables β-catenin nuclear translocation and the activation of downstream epithelial repair processes, including epithelial proliferation and mucus barrier restoration.\u003c/p\u003e \u003cp\u003eThis dependency on PKM2 is further supported by loss-of-function analyses, in which PKM2 silencing markedly impaired BSP-Diol\u0026ndash;induced β-catenin nuclear localization (Supplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), underscoring the essential role of PKM2 in establishing the repair-associated molecular state.\u003c/p\u003e \u003cp\u003eCollectively, this model positions PKM2-dependent metabolic licensing as a prerequisite for effective mucosal regeneration under inflammatory stress, rather than a consequence of forced activation of proliferative signaling pathways.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eEffective epithelial repair, driven by coordinated epithelial proliferation, is a critical determinant of mucosal healing in inflammatory bowel disease (IBD). However, the molecular requirements that enable epithelial cells to execute repair programs under inflammatory stress remain incompletely defined. While substantial efforts have focused on proliferative and inflammatory signaling pathways, comparatively less attention has been paid to how metabolic status modulates the effectiveness of repair-associated signaling once such pathways are activated.\u003c/p\u003e \u003cp\u003eIn the present study, we identify PKM2 as a metabolic regulator that constrains β-catenin\u0026ndash;dependent epithelial repair under inflammatory stress. Rather than directly activating proliferative signaling, BSP-Diol restores PKM2 protein abundance suppressed by inflammation, thereby enabling effective β-catenin nuclear localization and downstream regenerative responses. These findings indicate that restoration of PKM2-dependent metabolic regulation is required for productive β-catenin signaling during epithelial repair and provides a mechanistic explanation for the reparative phenotype observed following Huanglian\u0026ndash;Baiji treatment.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.1 PKM2 modulates β-catenin signaling under inflammatory stress\u003c/h2\u003e \u003cp\u003eTo elucidate the regulatory logic underlying epithelial repair, it is necessary to consider interactions between metabolic regulation and canonical signaling pathways. PKM2 has been widely characterized as a rate-limiting enzyme in glycolysis and a key mediator of metabolic reprogramming in proliferative tissues\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, whereas β-catenin functions as a central transcriptional regulator driving epithelial proliferation and regeneration\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Within conventional paradigms, β-catenin activation is typically viewed as upstream of metabolic adaptation. However, our findings suggest that metabolic regulation can instead determine the efficiency of β-catenin signaling under inflammatory stress.\u003c/p\u003e \u003cp\u003eAchieving mucosal healing, defined as restoration of epithelial barrier integrity, is a primary therapeutic goal in ulcerative colitis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Under sustained inflammatory conditions, PKM2 protein levels were markedly reduced, coinciding with impaired β-catenin nuclear localization and restricted epithelial proliferation. Restoration of PKM2 by BSP-Diol did not act as a direct upstream activator of β-catenin; rather, it relieved a metabolic constraint that limited β-catenin\u0026ndash;dependent proliferative responses. Loss-of-function experiments further demonstrated that PKM2 knockdown markedly attenuated BSP-Diol\u0026ndash;induced β-catenin nuclear localization, indicating that intact PKM2 expression is required for efficient repair-associated signaling under inflammatory stress.\u003c/p\u003e \u003cp\u003eThis interpretation is consistent with prior observations that PKM2 structural status influences its subcellular localization and regulatory interactions\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Furthermore, context-dependent coupling between PKM2 expression and β-catenin signaling has been reported in other regenerative and pathological settings\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. supporting the existence of a functional PKM2\u0026ndash;β-catenin axis.\u003c/p\u003e \u003cp\u003eBy preferentially restoring PKM2 protein abundance, BSP-Diol re-establishes the metabolic capacity necessary to sustain β-catenin\u0026ndash;dependent proliferative responses and to meet the bioenergetic and biosynthetic demands of epithelial repair, including cytoskeletal remodeling, macromolecule synthesis, and energy supply. This metabolic regulatory role may facilitate the canonical nuclear functions of PKM2 described in other regenerative contexts, where PKM2 directly interacts with β-catenin\u0026ndash;dependent transcriptional machinery\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLoss-of-function experiments further demonstrated that PKM2 knockdown markedly attenuated BSP-Diol\u0026ndash;induced β-catenin nuclear localization, indicating that intact PKM2 expression is required for efficient repair signaling. Collectively, these data suggest that PKM2-dependent metabolic regulation enables effective translation of β-catenin signaling into regenerative outcomes under inflammatory conditions. In vivo evidence from colitis models further supports this regulatory relationship, as PKM2 overexpression has been shown to sustain regenerative responses in the intestinal epithelium\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImportantly, these findings indicate that metabolic state is not merely a downstream consequence of epithelial repair, but an active regulator of signaling-dependent regeneration. By defining a PKM2\u0026ndash;β-catenin regulatory axis under inflammatory stress, our study adds a mechanistic layer to current paradigms of mucosal healing in IBD, complementing established immune-mediated and stromal regulatory mechanisms\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.2 PKM2 restoration is associated with protein-level regulation rather than extensive transcriptional remodeling\u003c/h2\u003e \u003cp\u003eIn the present study, key transcriptional regulators associated with mitochondrial biogenesis and function, including Ppargc1a, as well as deacetylases such as Sirt1 and Sirt3, did not exhibit statistically significant changes in mRNA expression following BSP-Diol treatment. This observation suggests that BSP-Diol\u0026ndash;mediated metabolic regulation occurs primarily at the protein level rather than through broad transcriptional reprogramming.\u003c/p\u003e \u003cp\u003eUnder sustained inflammatory stress, extensive transcriptional remodeling is energetically demanding and may further exacerbate metabolic strain in compromised epithelial cells. In this context, rapid and reversible regulatory mechanisms at the protein and structural levels may represent a more efficient strategy to support repair-associated signaling. Such regulation enables epithelial cells to adjust metabolic capacity without incurring the high energetic cost associated with large-scale transcriptional changes\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsistent with this notion, restoration of PKM2 protein abundance represents a central protein-level regulatory event that provides immediate metabolic support for β-catenin\u0026ndash;dependent proliferative responses. PKM2 is known to exert localization-dependent functions, including modulation of mitochondrial and cytoplasmic signaling processes\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In addition, mitochondria-associated endoplasmic reticulum membranes (MAMs) have been recognized as critical platforms for metabolic coordination and stress adaptation\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Regulation of MAM integrity predominantly involves post-translational modifications and structural reorganization\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, facilitating rapid metabolic adaptation to environmental challenges\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCollectively, these findings indicate that BSP-Diol\u0026ndash;mediated epithelial repair does not require extensive transcriptional reprogramming. Instead, restoration of PKM2 at the protein level appears sufficient to re-establish metabolic capacity and support effective repair signaling under inflammatory stress. This protein-centered regulatory mode may be particularly advantageous in inflammatory microenvironments, where epithelial cells must initiate regenerative responses under conditions of limited metabolic resources\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.3 From herb pair to mechanistic node: implications and perspectives\u003c/h2\u003e \u003cp\u003eIn this study, we employed an integrated strategy combining in vivo validation of therapeutic efficacy, network pharmacology\u0026ndash;guided identification of bioactive constituents, and in vitro interrogation of specific regulatory nodes\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.Through this approach, BSP-Diol was identified as a principal contributor to the mucosal repair\u0026ndash;promoting effects of the Huanglian\u0026ndash;Baiji herb pair, and a PKM2\u0026ndash;β-catenin regulatory axis under inflammatory stress was delineated. This workflow provides a practical framework for mechanistic dissection of complex herbal formulations and enables translation of empirically defined therapeutic effects into experimentally tractable molecular pathways\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrom a translational perspective, our findings suggest that restoration of metabolic regulation may represent a viable approach to support epithelial repair. Unlike strategies that directly amplify proliferative signaling, modulation of metabolic capacity may enhance the efficiency of endogenous repair pathways without excessive pathway activation. In chronic inflammatory diseases such as IBD, persistent stimulation of proliferative signaling carries the risk of disrupting tissue homeostasis. By contrast, targeting metabolic regulation may provide a more balanced means to support regenerative responses within physiologically constrained environments. Similar principles have been observed in metabolic modulators such as metformin, which exert protective effects through coordinated regulation of metabolic and signaling networks\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e][\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. BSP-Diol represents a single bioactive component and does not fully recapitulate the compositional complexity of the Huanglian\u0026ndash;Baiji formulation. In addition, complete mucosal healing in vivo requires coordinated interactions between epithelial cells, immune regulation\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, and the intestinal microbiota\u003csup\u003e[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. PKM2 may also exert context-dependent functions in epithelial and immune compartments\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, raising the possibility that BSP-Diol may influence repair indirectly through immunometabolic pathways. Future studies incorporating multi-cellular and microbiota-integrated systems will be required to further define these interactions\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, our study demonstrates that BSP-Diol promotes epithelial repair by restoring PKM2 expression and enabling β-catenin\u0026ndash;dependent regenerative signaling under inflammatory stress. These findings identify metabolic regulation as an important modulator of signaling-dependent mucosal healing and provide mechanistic insight into how restoration of metabolic capacity may complement existing anti-inflammatory therapies in IBD.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn conclusion, this study demonstrates that BSP-Diol promotes proliferation-dependent epithelial repair under inflammatory stress by restoring PKM2 expression and enabling efficient β-catenin\u0026ndash;dependent signaling. Rather than directly activating proliferative pathways, BSP-Diol alleviates metabolic constraints that limit repair-associated signaling execution.\u003c/p\u003e \u003cp\u003eBy defining a PKM2\u0026ndash;β-catenin regulatory axis in inflamed epithelium, our findings provide mechanistic insight into how metabolic state modulates signal-dependent epithelial regeneration. This work offers a molecular interpretation of the traditional concept of \u0026ldquo;promoting regeneration\u0026rdquo; and underscores the importance of metabolic regulation in mucosal healing.\u003c/p\u003e \u003cp\u003eCollectively, these results suggest that therapeutic strategies aimed at restoring metabolic capacity\u0026mdash;rather than excessively amplifying proliferative signaling\u0026mdash;may represent a balanced and physiologically aligned approach for enhancing mucosal repair in inflammatory bowel disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following grants:\u003c/p\u003e\n\u003cp\u003e(1) Superior Specialized Department of Spleen and Stomach Diseases, State Administration of Traditional Chinese Medicine (Guozhongyi Yizheng Han [2024] No. 90);\u003c/p\u003e\n\u003cp\u003e(2) The Fifth Batch of National Outstanding Chinese Medicine Clinical Talent Training Program, State Administration of Traditional Chinese Medicine (Guozhongyi Renjiao Han [2022] No. 1).\u003c/p\u003e\n\u003cp\u003eThe funding bodies had no role in the study design; data collection, analysis, or interpretation; or manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Jiangxi University of Traditional Chinese Medicine\u003c/p\u003e\n\u003cp\u003e(Approval No.: JXHQ2025002; Animal Experimentation Facility License No.: SYXK[Jiangxi]2025-0005)\u003c/p\u003e\n\u003cp\u003eand were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011).\u003c/p\u003e\n\u003cp\u003eNo human participants were involved in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are included within the article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003eUncropped Western blot images corresponding to all immunoblotting results are provided in Supplementary File 1.\u003c/p\u003e\n\u003cp\u003eAdditional datasets generated during the current study are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHou-Shu Tu: Conceptualization, Investigation, Data Curation, Writing \u0026ndash; Original Draft.\u003c/p\u003e\n\u003cp\u003eMeng-Lin Chen: Molecular Docking, Data Analysis, Visualization.\u003c/p\u003e\n\u003cp\u003eCui Xu: Animal Experiments, Data Collection.\u003c/p\u003e\n\u003cp\u003eChengming Yang: Cell-based Experiments, Data Analysis.\u003c/p\u003e\n\u003cp\u003eSai Li: Statistical Analysis, Figure Preparation.\u003c/p\u003e\n\u003cp\u003eJing Hong: Supervision, Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003eLing He: Supervision, Project Administration, Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank the State Administration of Traditional Chinese Medicine and the affiliated institutions for their support. We also acknowledge our colleagues for technical assistance and valuable discussions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNg, S. C., Shi, H. Y., Hamidi, N., Underwood, F. E., Tang, W., Benchimol, E. I.,... Kaplan, G. G. (2017). Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. \u003cem\u003eLancet (London, England)\u003c/em\u003e, 390(10114), 2769-2778. doi: 10.1016/S0140-6736(17)32448-0\u003c/li\u003e\n\u003cli\u003eKaplan, G. G., \u0026amp; Windsor, J. W. (2021). The four epidemiological stages in the global evolution of inflammatory bowel disease. \u003cem\u003eNature reviews Gastroenterology \u0026amp; hepatology\u003c/em\u003e, 18(1), 56-66\u003c/li\u003e\n\u003cli\u003eDunleavy, K. A., Raffals, L. E., \u0026amp; Camilleri, M. (2023). 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Although repair-associated signaling pathways such as β-catenin can remain active during inflammation, epithelial regeneration is frequently insufficient. The mechanisms limiting proliferation-dependent repair in the context of inflammatory colitis remain incompletely defined.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe therapeutic effects of the Huanglian\u0026ndash;Baiji herb pair were evaluated in a dextran sulfate sodium (DSS)\u0026ndash;induced colitis model. An integrated strategy combining component prioritization with experimental validation was applied to identify active constituents and regulatory nodes. The effects of BSP-Diol on epithelial repair were examined in vivo and in vitro, focusing on epithelial proliferation, goblet cell recovery, β-catenin nuclear localization, and PKM2 regulation. The requirement of PKM2 was assessed using protein profiling and loss-of-function approaches.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHuanglian\u0026ndash;Baiji treatment significantly promoted epithelial proliferation and goblet cell restoration in DSS colitis. BSP-Diol was identified as a key component that restored β-catenin nuclear localization in inflamed epithelium. Inflammatory conditions markedly reduced PKM2 protein abundance, coinciding with impaired proliferative responses. Restoration of PKM2 by BSP-Diol facilitated β-catenin\u0026ndash;dependent epithelial proliferation. In contrast, transcriptional regulators of mitochondrial biogenesis exhibited only modest changes, indicating predominant regulation at the protein and subcellular levels. PKM2 knockdown significantly attenuated BSP-Diol\u0026ndash;induced β-catenin nuclear localization, demonstrating that PKM2 is required for effective repair signaling.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings identify PKM2 as a critical metabolic regulator of β-catenin\u0026ndash;dependent epithelial repair in experimental colitis. Restoration of PKM2 relieves inflammation-associated metabolic constraints and promotes mucosal regeneration. Targeting metabolic regulation may represent a complementary strategy to enhance epithelial repair in IBD.\u003c/p\u003e","manuscriptTitle":"BSP-Diol restores PKM2 and facilitates β-catenin–dependent epithelial repair in experimental colitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 18:04:23","doi":"10.21203/rs.3.rs-9086575/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-11T04:51:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T13:56:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169058009631981391567867535859983439625","date":"2026-04-23T11:25:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T03:57:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92934107659736823216231112737794008967","date":"2026-04-13T23:31:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T07:16:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T03:42:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T03:41:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation and Regeneration","date":"2026-03-10T16:58:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-and-regeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ireg","sideBox":"Learn more about [Inflammation and Regeneration](http://inflammregen.biomedcentral.com/)","snPcode":"41232","submissionUrl":"https://www.editorialmanager.com/ireg/default2.aspx","title":"Inflammation and Regeneration","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27dd5258-9ed4-47c0-8eb5-d8fe1895a9ef","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-11T04:51:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T13:56:23+00:00","index":67,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T04:55:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 18:04:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9086575","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9086575","identity":"rs-9086575","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}