An Herbal Enema Formula Alleviates Ulcerative Colitis by Inhibiting Enteric Glial Activation via the S100β/RAGE/NF-κB Pathway

Summary

An herbal enema formula combining Sanguisorba officinalis, Bletilla striata, and Acacia catechu was evaluated using LC-MS/MS profiling, S100β docking, a DSS colitis model, and LPS/IFN-γ-activated enteric glia. The formula engaged S100β, suppressed glial activation, and reduced p-NF-κB/iNOS expression, indicating amelioration via the S100β-RAGE-NF-κB axis.

Abstract

Activation of enteric glial cells (EGCs) contributes to the pathogenesis of ulcerative colitis (UC). This study examined whether Dibai Enema Liquid (DBE)-an herbal enema combining Sanguisorba officinalis, Bletilla striata, and Acacia catechu-modulates EGC-driven inflammation. LC-MS/MS profiles of DBE constituents and prioritized compounds were docked to S100β. Efficacy was assessed in dextran sulfate sodium (DSS)-induced murine colitis using body weight, colon length, histopathology, and cytokine levels (ELISA). Glial activation markers were measured by immunohistochemistry, immunofluorescence colocalization, quantitative PCR (qPCR), and immunoblotting. An in vitro model used CRL-2690 enteric glia activated with lipopolysaccharide plus interferon-γ, with DBE-containing rat serum (15%) as the intervention. Docking suggested a strong predicted binding of several DBE components to S100β. DSS increased TNF-α and IL-1β, disrupted tight junctions, and elevated S100β/GFAP at both mRNA and protein levels, consistent with glial hyperactivation; DBE significantly reversed these changes. In vitro, DBE reduced S100β, RAGE, iNOS (NOS2), and phosphorylated NF-κB p65. Collectively, DBE appears to alleviate UC by inhibiting EGC activation and downregulating the S100β/RAGE/NF-κB/iNOS axis, thereby preserving mucosal integrity.

Introduction

Ulcerative colitis (UC) is a major subtype of inflammatory bowel disease (IBD) characterized by chronic mucosal inflammation of the colon, with a rising global burden since 1990, and UC accounting for a substantial proportion of incident IBD cases^1,2. Typical manifestations include abdominal pain and bloody diarrhea, leading to considerable physical and psychological distress and substantial treatment costs³. Although treatment options have expanded, long-term exposure to immunomodulators and biologics that suppress systemic immunity is associated with increased risks of lymphoma and opportunistic infections, and a proportion of patients remain refractory or intolerant⁴. UC is widely regarded as an immune-mediated condition in which disruption of mucosal immune homeostasis is central to pathophysiology⁵. However, accumulating evidence indicates that mucosal-immune mechanisms alone are insufficient to explain all features of IBD; the enteric nervous system (ENS) contributes to intestinal inflammation, extending the concept of disease to neuroinflammation within IBD⁶.

Enteric glial cells (EGCs), which outnumber enteric neurons in adult mammals, are now recognized as key regulators of intestinal homeostasis and inflammation^7,8. EGCs modulate epithelial barrier function by releasing mediators such as GDNF, TGF-β1, S100β, and 15d-PGJ2, thereby influencing permeability and mucosal integrity⁹. Among these mediators, S100β can activate downstream cascades-including the RAGE/NF-κB pathway-leading to barrier injury and inflammatory amplification^10,11. EGCs also shape immune responses by regulating macrophage phenotype and visceral sensitivity via connexin-43 and M-CSF, and by exerting suppressive effects on T cells in vitro, highlighting EGC activation as a tractable therapeutic target in UC^12,13.

Traditional Chinese medicine (TCM) has been increasingly investigated in UC and has shown promising benefits in clinical and experimental settings^14,15. The classical pairing of Sanguisorba officinalis (Diyu) and Bletilla striata (Baiji), together with Acacia catechu (Ercha), has long been documented for dysenteric disorders in materia medica sources and is listed in the Chinese Pharmacopoeia for hemostatic, analgesic, and wound-healing indications. Contemporary studies report antibacterial, anti-inflammatory, antioxidative, and anti-ulcer activities for these botanicals, while A. catechu also demonstrates neuroprotective properties with catechin as a prominent constituent^16,17. Relative to conventional systemic immunosuppression, a formulation that engages EGC-centered neuroimmune pathways could offer complementary efficacy with a distinct mechanism and potentially fewer systemic immune-related risks^18,19.

This study evaluates whether the combined preparation of Diyu, Baiji, and Ercha (DBE) mitigates UC by suppressing EGC activation. An integrated approach is used: untargeted LC-MS/MS profiling to prioritize constituents, molecular docking against S100β, validation in a dextran sulfate sodium-induced colitis model, and mechanistic assays in EGCs. The work emphasizes a neuroimmune mechanism-S100β/RAGE/NF-κB/iNOS-less explored in prior UC therapies, and outlines practical implications for dose selection, formulation optimization, and subsequent translational studies.

Protocol

All animal procedures were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine [BUCM-2023-0912] and were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals. No human subjects were involved.
The reagents and the equipment used are listed in the Table of Materials.

1. Reagents and materials

Analytical-grade solvents and molecular-biology-grade reagents were used throughout. All commercial information, including manufacturers, catalog numbers, and software license details, is provided in the Table of Materials (xlsx). Water for all preparations was deionized and filtered. All buffers were prepared fresh or stored under conditions specified below.

2. Equipment and software setup

A UPLC-MS/MS system coupled to a quadrupole time-of-flight mass spectrometer; a refrigerated centrifuge with swing-bucket rotor; a CO₂ incubator maintained at 37 °C, 5% CO₂, and ≥95% relative humidity; a microplate reader (450 nm); a real-time PCR thermocycler; electrophoresis and blotting apparatus; and a fluorescence microscope with fixed excitation/emission filters were used. Instrument control and data analysis were performed in MassLynx v4.2 for chromatographic and mass-spectrometric data and AutoDock 4.2 with AutoDockTools 1.5.7 for docking. Image analysis was performed in ImageJ/Fiji v1.53.

3. Preparation of Dibai Enema Liquid (DBE)

The herbal mixture comprising Diyu (50 g), Baiji (50 g), and Ercha (20 g) was coarsely crushed and soaked in 1,200 mL deionized water at room temperature for 30 min to ensure full hydration. The soaked mixture was decocted under a vigorous boil for 30 min with occasional stirring to prevent adhesion. The hot decoction was filtered through sterile multi-layer medical gauze into sterile glassware to remove insoluble material. The filtrate was vacuum-concentrated in a water bath not exceeding 45 °C until a concentrate corresponding to a final stock concentration of 1.98 g/mL was obtained (high-dose stock). Medium- and low-dose working solutions at 0.99 g/mL and 0.50 g/mL were prepared by dilution with sterile deionized water to final volumes of 100 mL each. Each solution was passed through a 0.22 µm membrane under sterile conditions, dispensed into labeled sterile containers, and stored at 4 °C for up to seven days. Before use, solutions were gently mixed; an acceptable appearance was clear to slightly opalescent without visible particulates.

4. UPLC - MS/MS analysis of DBE

Powder from each medicinal component was weighed accurately at 50 mg and extracted with 1.0 mL of 80% (v/v) methanol by ultrasonic agitation for 30 min at ambient temperature. Extracts were returned to room temperature after sonication, centrifuged at 15,000 × g for 10 min at 4 °C, and supernatants were filtered through 0.22 µm membranes into autosampler vials. Chromatographic separation was carried out on a reversed-phase column (2.1 × 100 mm, 1.8 µm; polar-endcapped C18-type) maintained at 35 °C; column brand/model and catalog number are provided in the Table of Materials. Mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile. Flow rate: 0.30 mL/min; injection volume: 10 µL. Gradient: 5% B for 0-2 min; 5→95% B from 2-20 min; 95% B for 20-23 min; 95→5% B from 23.0-23.1 min; re-equilibrate at 5% B until 26 min (full gradient table is provided in Table 1).

Mass spectrometry used electrospray ionization with positive/negative switching and data-independent acquisition with low- and high-energy (MSE) scans. Mass range: m/z 100-1,500; capillary voltage: 2.0 kV; sampling cone: 20 V; source temperature: 120 °C; desolvation temperature: 360 °C; cone gas: 50 L/h; desolvation gas: 600 L/h. High-energy function employed stepped collision energy of 20-50 eV. Data were processed in MassLynx v4.2 with lock-mass correction and standard peak-detection parameters. A comprehensive list of 105 identified components (molecular formulae, observed m/z, retention times, and confidence scores) is provided in Supplementary Table 1.

5. Molecular docking

Docking was conducted in AutoDock 4.2 under an induced-fit protocol. The three-dimensional structure of S100β (PDB 3HCM) was prepared by removing crystallographic water molecules, adding polar hydrogens, and assigning Kollman charges. Ligand structures representing characteristic DBE constituents were built, protonated at physiological pH, and energy-minimized. The binding site was defined by the native-ligand coordinates; amino-acid residues within 6 Å of the native ligand were treated as flexible during docking. Grid boxes encompassed the entire pocket with a grid spacing of 0.375 Å. Lamarckian genetic-algorithm parameters were: population size 150; up to 2.5 × 10⁶ energy evaluations; 100 independent runs per ligand. Final poses were ranked by predicted binding free energy and clustered by RMSD; interaction patterns (hydrogen bonds, hydrophobic contacts, π-π interactions) were visualized for consistency with known S100β complexes.

6. Animal model of DSS-induced colitis and DBE treatment

Male C57BL/6J mice (8 weeks, 20-22 g) were acclimated for at least 7 days at 22 ± 2 °C, 50%-60% relative humidity, 12 h light-dark cycle, with chow and water ad libitum. Animals were randomly assigned into six groups (n = 10/group): control, dextran sulfate sodium (DSS) model, sulfasalazine (SASP) positive control, and low-, medium-, and high-dose DBE treatments. Personnel responsible for histological scoring and image quantification were blinded to group allocation.

Experimental colitis was induced by 2.0% (w/v) DSS in drinking water for 7 days, followed by 1.0% DSS for 3 days, then regular water for 4 days prior to euthanasia. DBE was administered once daily by enema at 10 mL/kg using a flexible catheter inserted 2.5 cm into the rectum; animals were held vertically for 30 min to minimize leakage. Daily DBE doses were 12.35, 27.4, and 49.4 g/kg/day for the low, medium, and high groups, respectively; SASP was administered at 0.5 g/kg/day as a positive control. Body weight, stool consistency, and fecal occult blood were recorded daily to compute the disease-activity index. At the endpoint, mice were anesthetized with pentobarbital sodium 50 mg/kg i.p.; blood was collected from the retro-orbital sinus using heparinized capillaries; animals were euthanized by cervical dislocation. The colon was excised from the ileocecal junction to the rectum, gently extended, and the length measured under minimal tension. Representative segments were fixed in 4% paraformaldehyde; remaining tissue was snap-frozen in liquid nitrogen and stored at −80 °C.

7. Preparation of drug-containing serum

Male Sprague-Dawley rats were randomized into control and DBE groups (n = 6/group). The DBE group received 12.34 g/kg/day by oral gavage once daily for 7 days; controls received equal-volume purified water. Twenty-four hours after the final dose, animals were anesthetized, and whole blood was collected from the abdominal aorta. Serum was separated after clotting for 30 min at room temperature, followed by centrifugation at 1,500 × g for 10 min at 4 °C. Sera were heat-inactivated at 56 °C for 30 min and filtered through 0.22 µm membranes under sterile conditions before storage. Serum was aliquoted into sterile tubes and stored at −80 °C. Hemolyzed samples, identified by elevated absorbance near 414 nm, were excluded.

8. Histological examination (H&E)

Colon tissue fixed in 4% paraformaldehyde for 24 h was dehydrated through graded ethanols, cleared in xylene, and embedded in paraffin. Sections (5 µm) were cut, deparaffinized, rehydrated, and stained with hematoxylin for 5 min, blued in running water for 5 min, and counterstained with eosin for 2 min. Slides were mounted with resin and examined for crypt architecture, goblet-cell density, epithelial continuity, and inflammatory infiltration. Representative alterations will be indicated with arrows/arrowheads and defined in the figure legends; scale bars (e.g., 200 µm) and predefined visual thresholds will be specified in the legends.

9. Immunohistochemistry (IHC)

Paraffin sections underwent antigen retrieval in citrate buffer (pH 6.0) for 15-20 min at 95-98 °C, quenching of endogenous peroxidase with 3% hydrogen peroxide for 10 min, and blocking with 5% normal serum for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C in a humidified chamber. After washing, sections were processed using a streptavidin-peroxidase detection system, developed with DAB for 2 min (microscopic monitoring), counterstained with hematoxylin, dehydrated, and mounted. Negative controls lacking the primary antibody were included in each batch.

10. Immunofluorescence (IF) analysis

Paraffin-embedded sections were deparaffinized and subjected to antigen retrieval in EDTA buffer (pH 8.0). After cooling, nonspecific binding was blocked with 3% bovine serum albumin for 1 h at room temperature. Sections were incubated overnight at 4 °C with primary antibodies against GFAP (1:500) and S100β (1:200), followed by fluorophore-conjugated secondary antibodies (1:500) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI, and slides were mounted in anti-fade medium. Images were captured using identical objectives, a fixed exposure time of 100 ms, and a detector gain of 1.0 across groups. Mean fluorescence intensity was quantified in ImageJ/Fiji v1.53 after calibration of pixel size, background subtraction (rolling-ball radius 50 pixels), and normalization to DAPI-positive area. At least five biological replicates per group and three non-overlapping fields per replicate were analyzed.

11. Cell culture and treatments

Rat enteric glial cells (designation CRL-2690) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO₂. Cell identity was confirmed by short tandem repeat profiling, and cultures were mycoplasma-free. Experiments were performed at passages 5-15. Cells were seeded into 6-well plates at 2.0 × 10⁵ cells/well in 2.0 mL/well medium and allowed to attach for 24 h. Experimental groups were: untreated control, vehicle control, model, DBE-serum, and inhibitor.

For model induction, cells were exposed to lipopolysaccharide (LPS, 1 µg/mL) plus IFN-γ (20 ng/mL) for 24 h in complete medium; the corresponding U/mL value for IFN-γ is reported in the Table of Materials according to lot-specific potency. For DBE-serum treatment, cells received 15% (v/v) heat-inactivated (56 °C, 30 min), 0.22 µm-filtered DBE-containing rat serum in complete medium for 24 h (dose range 5%-20% is used in viability optimization as specified in the CCK-8 section). For inhibitor treatment, the selective S100β inhibitor SBI-4211 (10-50 µM; 45 µM for key readouts) was applied for 24 h. Vehicle controls matched solvent content (0.1% DMSO (v/v)) with otherwise identical medium. Unless otherwise stated, treatments were prepared in pre-warmed (37 °C) medium, and cells were rinsed 2× with PBS before treatment replacement. After treatment, cells were collected for RNA extraction, protein analysis, and immunofluorescence as detailed in subsequent sections. Biological replication was n ≥ 5 independent cultures per condition.

12. CCK-8 cell viability assay

Cells were seeded into 96-well plates at 5 × 10³ cells/well in 100 µL medium and allowed to adhere overnight. Medium was replaced with medium containing DBE-treated serum at 5%, 10%, 15%, or 20% (v/v) for 24 h. CCK-8 reagent (10 µL/well) was added, and plates were incubated for 1 h at 37 °C. Absorbance was read at 450 nm with blank subtraction (medium + reagent, no cells). Viability was expressed as % of vehicle control. Each condition used n ≥ 5 biological replicates (independent cultures) with three technical replicates per culture.

13. ELISA for tissue cytokines

Colon tissue was homogenized on ice in PBS (1:9, w/v) containing protease inhibitors. Homogenates were clarified by 12,000 × g for 10 min at 4 °C; supernatants were assayed for IL-1β and TNF-α according to the kit protocols. Standards and samples were run in duplicate, and curves were fitted with a four-parameter logistic (4PL) model with R² ≥ 0.99; back-calculated standards were accepted within 85%-115% of nominal. Absorbance was read at 450 nm with a 620-650 nm reference when available. Cytokine concentrations were normalized to tissue weight (pg/mg tissue). Each group included n ≥ 5 biological replicates.

14. Quantitative Real-Time PCR (qPCR)

Total RNA from colon tissue (or cells, as applicable) was isolated with a silica-membrane spin-column kit. RNA integrity was verified by A₂₆₀/A₂₈₀ = 1.8-2.1 and agarose-gel inspection. First-strand cDNA was synthesized from 1 µg RNA with genomic DNA removal. qPCR used SYBR Green master mix on a calibrated thermocycler with the program: 95 °C 30 s; 40 cycles of 95 °C 5 s, 60 °C 30 s; melt curve 65-95 °C in 0.5 °C/5 s steps. GAPDH served as the endogenous control. Relative expression was calculated by 2⁻ΔΔCt after verifying primer efficiencies (90%-110%, from standard-curve slopes). No-RT and no-template controls were included. Primer sequences are listed in Table 2. Each group had n ≥ 5 biological replicates and two technical repeats per sample.

15. Western blot analysis

Proteins from colon tissue (or cells) were extracted in RIPA buffer with protease/phosphatase inhibitors and quantified by BCA assay. Equal protein (30 µg/lane) was denatured, resolved on 4-20% gradient SDS-PAGE, and transferred to PVDF (0.45 µm; 0.2 µm for ≤ 20 kDa targets). Membranes were blocked in 5% skim milk for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies against GFAP (1:1,000), S100β (1:1,000), iNOS (1:1,000), NF-κB p65 (1:1,000), phospho-NF-κB p65 (Ser536, 1:1,000), Occludin (1:1,000), Claudin-1 (1:1,000), and GAPDH (1:5,000). HRP-conjugated secondary antibodies (1:5,000) were applied for 1 h at room temperature. Signals were developed with ECL; exposure times were constrained to the linear dynamic range (10-90 s). Transfers were verified by reversible membrane staining prior to blocking. Band intensities were quantified in ImageJ/Fiji v1.53 after background subtraction and normalized to GAPDH. Expected molecular weights: GFAP ~49 kDa, S100β ~10-12 kDa, NF-κB p65 ~65 kDa, iNOS ~130 kDa, Occludin ~65 kDa, Claudin-1 ~22 kDa, GAPDH ~36-37 kDa. Each analysis included n ≥ 5 biological replicates.

16. Statistical analysis

Data were analyzed in GraphPad Prism 8.0 and are presented as mean ± SEM. For multiple-group comparisons, one-way ANOVA followed by Tukey's post hoc test was used; for pairwise comparisons, unpaired two-tailed Student's t-tests were applied when appropriate. For time-course or two-factor designs (e.g., treatment × dose or treatment × time), two-way ANOVA (repeated measures when applicable) with Tukey's or Dunnett's correction was used as specified in the figure legends. Shapiro-Wilk and Levene's tests were used to examine normality and homoscedasticity prior to parametric testing; when assumptions were violated, data were log-transformed or analyzed with appropriate non-parametric tests (Kruskal-Wallis with Dunn's post hoc; Mann-Whitney U for pairwise). Exact p-values and effect-size measures (η² for ANOVA, Cohen's d for t-tests) are reported in figure legends where applicable. A two-sided p < 0.05 was considered statistically significant. Figure legends use the following notation for significance: *p<0.05, **p<0.01, *** p<0.001 vs Control; # p<0.05, ## p<0.01, ### p<0.001 vs DSS; ns, not significant.

17. Expected results and quality control

DSS is expected to shorten colon length, elevate disease-activity index, and induce histological damage (crypt architectural distortion, goblet-cell depletion, epithelial erosion). DBE treatment should dose-dependently improve these indices and reduce enteric-glial activation markers. Western blots should show bands at the molecular weights listed above, with increased signals in DSS and attenuation with DBE or SBI-4211; exposures should remain within the linear dynamic range. Immunofluorescence for GFAP and S100β should show elevated mean intensities in DSS and a reduction after DBE treatment. LC-MS/MS chromatograms should display stable retention times (drift ≤ 0.2 min) and mass accuracies within ± 5 ppm for reference ions, with characteristic fragment ions for key constituents. Negative controls (e.g., omission of primary antibodies) should yield no specific signal. Imaging acquisition parameters (objective, exposure, gain, gamma) must remain fixed across groups to enable valid quantitative comparisons.

18. Troubleshooting notes

Turbidity or particulates in DBE solutions indicate inadequate filtration or precipitation; repeat 0.22 µm filtration and confirm pH 6.5-7.5. Excessive mortality or severe weight loss during DSS exposure suggests over-induction; reduce DSS concentration, verify batch molecular weight, and replace DSS solutions daily. Inconsistent immunofluorescence quantification typically reflects variable exposure; fix exposure time and gain, and include a reference slide for batch calibration. Weak western-blot bands may reflect under-loading or transfer inefficiency; verify protein quantitation, select appropriate gel percentage or gradient, and confirm transfer by reversible membrane staining. Poor ELISA fit (R² < 0.99 or standard back-calculation outside 85-115%) warrants repeating the standard curve and re-assaying diluted samples. For qPCR runs with primer efficiency outside 90-110%, re-optimize annealing temperature or primer concentration before analysis.

Results

Identification of DBE components and drug-containing serum

LC-MS/MS identified 105 unique chemical constituents in DBE, with mass error < 10 ppm. Identification was confirmed by molecular formula, isotope pattern, and fragment matching. Major classes included triterpenes, flavonoids, biphenanthrenes, and organic acids. Drug-containing serum revealed 59 DBE-derived components after blank subtraction. Represent...

Discussion

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with multifactorial pathogenesis. In addition to dysregulated immune responses, intestinal epithelial barrier dysfunction and gut microbiota dysbiosis are well-recognized contributors, while abnormalities of the enteric nervous system (ENS) are increasingly identified as a key driver in UC progression²³. Specifically, both structural and functional dysregulation of the ENS can exacerbate mucosal inflammation and disease severity

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AutoDock 4.2	Molecular docking software	-	The Scripps Research Institute
Dextran Sulfate Sodium (DSS)	Chemical for inducing colitis in mice	9011-18-1	Sigma-Aldrich
Dibai Enema Liquid (DBE)	Herbal mixture for ulcerative colitis treatment	-	Beijing University of Chinese Medicine
DMEM (Dulbecco's Modified Eagle Medium)	Cell culture medium	11965-092	Thermo Fisher
Fetal Bovine Serum (FBS)	Supplement for cell culture	16000-044	Thermo Fisher
Fluorescence Microscope	Instrument for immunofluorescence imaging	-	Olympus
LC-MS/MS System	Mass spectrometer for LC-MS analysis	-	Waters Corporation
Lipopolysaccharide (LPS)	Inducer of inflammation in cell culture	L2630	Sigma-Aldrich
PCR Thermal Cycler	Equipment for qPCR	T100	Bio-Rad
Primary Antibody (S100)	Antibody for S100 protein detection	ABC1234	Abcam
Protein Assay Kit (BCA)	Protein quantification kit	23225	Thermo Fisher
Recombinant Interferon- (IFN)	Cytokine for EGC activation in cell culture	-	Sigma-Aldrich
Secondary Antibody (HRP-conjugated)	Secondary antibody for immunohistochemistry	XYZ5678	Thermo Fisher
Sulfazalazine (SASP)	Positive control for ulcerative colitis	-	Sigma-Aldrich
Western blot Equipment	Electrophoresis system for protein analysis	-	Bio-Rad