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 op....

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 Materia....

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