Five-flavor Sophora flavescens Enteric-coated Capsules Alleviate Experimental Colitis and Ferroptosis-related Changes

Summary

In a mouse model of ulcerative colitis, five-flavor Sophora flavescens enteric-coated capsules alleviated colonic injury and improved oxidative stress, inflammatory cytokine imbalance, JAK2/STAT3 signaling, and changes in ferroptosis-related markers.

Abstract

Ulcerative colitis remains difficult to manage because effective and targeted therapeutic options are limited, and oxidative stress, inflammatory signaling, and regulated cell-death pathways may contribute to mucosal injury. This study evaluated whether five-flavor Sophora flavescens enteric-coated capsules (FSEC) alleviate experimental colitis and explored associated changes in oxidative stress, inflammation, and markers of ferroptosis. A mouse model of ulcerative colitis was established using cyclic exposure to 2.5% dextran sulfate sodium, combined with tumor necrosis factor alpha challenge. Mice were treated with low-, medium-, or high-dose FSEC or with the TLR4 antagonist CRX-526 as a positive-control intervention. Colon histopathology was assessed by hematoxylin and eosin staining. Serum superoxide dismutase, catalase, glutathione, myeloperoxidase, and Fe²⁺ levels were measured using biochemical assays. Colon tissue cytokines were quantified by enzyme-linked immunosorbent assay, JAK2 and STAT3 expression were evaluated by immunohistochemistry, and GPX4, FTH1, and ACSL4 expression were analyzed by western blotting. FSEC treatment reduced colonic mucosal injury and inflammatory-cell infiltration, increased antioxidant indices, and decreased myeloperoxidase levels. FSEC also reduced TNF-α and IL-1α levels, partially restored IL-13 levels, and was associated with weaker JAK2 and STAT3 immunostaining. In parallel, FSEC improved ferroptosis-related marker changes, including reduced Fe²⁺ and ACSL4 levels and increased GPX4 and FTH1 expression. These findings suggest that FSEC alleviates experimental ulcerative colitis in mice, at least in part by improving oxidative-stress status, moderating inflammatory signaling, and restoring ferroptosis-related marker profiles.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory disorder that primarily affects the colonic and rectal mucosa and is characterized by recurrent episodes of mucosal inflammation, ulceration, and impaired barrier function^1,2,3. Although genetic susceptibility, environmental factors, epithelial barrier dysfunction, and immune dysregulation have been implicated in UC pathogenesis, the mechanisms underlying persistent mucosal injury remain incompletely understood. Current pharmacological treatments, including aminosalicylates, corticosteroids, immunosuppressants, and biologic agents, can reduce disease activity in many patients; however, their clinical use may be limited by relapse after treatment discontinuation, adverse effects, high cost, and incomplete therapeutic responses³.

Ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation and altered iron metabolism, has increasingly been implicated in intestinal inflammation and UC-associated epithelial injury^4,5,6,7,8. In parallel, traditional Chinese medicine formulations have attracted increasing attention as complementary approaches for UC treatment because of their multi-target therapeutic potential⁹.

Five-flavor Sophora flavescens enteric-coated capsules (FSEC) are used clinically for the treatment of mild to moderate active UC in traditional Chinese medicine practice. Previous studies suggest that FSEC may improve UC symptoms through multi-component and multi-target mechanisms¹⁰. However, the effects of FSEC on oxidative stress, inflammatory signaling, JAK2/STAT3 activity, and ferroptosis-related changes in experimental UC remain insufficiently characterized.

Oxidative stress and inflammatory signaling are important contributors to intestinal mucosal injury in UC. Excessive production of reactive oxygen species can disrupt epithelial integrity, promote inflammatory-cell infiltration, and impair antioxidant defense systems. Cytokine-mediated pathways such as JAK2/STAT3 signaling contribute to inflammatory amplification and tissue injury^{11,12,13,14,15}. In addition, oxidative stress and ferroptosis are closely linked through alterations in antioxidant defenses, iron metabolism, and lipid peroxidation^{16,17,18,19,20}. Therefore, simultaneous evaluation of oxidative-stress indices, inflammatory cytokines, JAK2/STAT3 signaling, and ferroptosis-related markers may provide a more comprehensive understanding of mucosal injury and therapeutic responses in experimental UC.

In the present study, a cyclic dextran sulfate sodium and tumor necrosis factor alpha–challenged mouse model was used to evaluate the effects of low-, medium-, and high-dose FSEC. A key methodological strength of this design is the integration of histopathological assessment with measurements of serum oxidative-stress indices, colon tissue cytokines, JAK2/STAT3 immunohistochemistry, and western blot analysis of ferroptosis-related proteins within a single experimental framework. This approach was used to determine whether FSEC alleviates experimental UC and to characterize associated changes in oxidative stress, inflammatory signaling, and ferroptosis-related marker profiles. The overall experimental design and analytical workflow are summarized in Figure 1.

Figure 1: Experimental workflow of the mouse model and downstream analyses. Chronic experimental ulcerative colitis was induced by cyclic exposure to 2.5% dextran sulfate sodium (DSS) combined with tumor necrosis factor alpha (TNF-α) challenge during the final week of the experimental protocol. Mice were assigned to normal control, model, low-dose FSEC, medium-dose FSEC, high-dose FSEC, and CRX-526 positive-control groups. FSEC was administered by intragastric gavage at doses of 108, 216, or 432 mg/kg body weight. CRX-526 was used as the positive-control intervention. Following the final treatment, samples were collected for histopathological evaluation, oxidative-stress-related biochemical assays, cytokine analysis by ELISA, immunohistochemistry, and western blot analysis. Please click here to view a larger version of this figure.

Protocol

All animal procedures were reviewed and approved by the Animal Welfare and Ethics Committee of Nanchang Medical College (approval no. NY2SC 20250407). All experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. The chemicals, reagents, and equipment are listed in the Table of Materials.

1. Animals and treatment

Male C57BL/6J mice (wild-type, 8 weeks old) were used in this study. Animals were housed under standard laboratory conditions with controlled temperature and humidity and were provided free access to food and water. Following acclimatization, chronic colitis was induced by administering three cycles of 2.5% dextran sulfate sodium (DSS) in the drinking water. Each cycle lasted 1 week and was separated by a 2-week recovery period with regular drinking water. During the final week of the experimental protocol, mice in the model and treatment groups received tumor necrosis factor alpha (TNF-α; 10 µg/100 g body weight, twice daily) by intragastric gavage to further aggravate colonic injury. Normal control mice received vehicle only and were not exposed to DSS or TNF-α.

Five-flavor Sophora flavescens enteric-coated capsules (FSEC) were used as a commercially available, approved Chinese patent medicine. The formulation contained Sophora flavescens Aiton, Sanguisorba officinalis L., Indigo naturalis, Bletilla striata (Thunb.) Rchb.f., and Glycyrrhiza uralensis Fisch. ex DC. Capsule contents from the same production batch were weighed and suspended in sterile distilled water to prepare fresh dosing suspensions before administration. Suspensions were mixed thoroughly before each gavage to ensure uniform dispersion. Product source, approval number, pharmaceutical specifications, and quality-control information are provided in the Table of Materials. Product quality was described in accordance with the approved pharmaceutical specification and manufacturer-provided information, including capsule specifications, appearance, enteric-release and disintegration requirements, microbial limit requirements, and quality-control standards.

FSEC-treated mice received low-dose FSEC (108 mg/kg body weight), medium-dose FSEC (216 mg/kg body weight), or high-dose FSEC (432 mg/kg body weight) by intragastric gavage twice daily during the final week of modeling. Dose levels were selected based on the approved clinical dose, body-surface-area conversion, and preliminary dose-ranging considerations. Vehicle-treated mice received the same volume of sterile distilled water by intragastric gavage.

The positive-control group received the TLR4 antagonist CRX-526 at 1 mg/kg via tail vein injection during the final week of modeling. CRX-526 was used as a reference anti-inflammatory intervention. The study included six groups (n = 6 per group): normal control, model, low-dose FSEC, medium-dose FSEC, high-dose FSEC, and CRX-526 positive-control groups. The mice were sacrificed 24 h after the last study treatment. Euthanasia was performed by cervical dislocation under isoflurane anesthesia, in accordance with the institutional guidelines for the care and use of laboratory animals.

2. Histological evaluation by hematoxylin and eosin staining

Colon tissues were collected from the macroscopically most affected region between the anus and the ileocecal area. For each mouse, one representative colon segment from this region was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. For histological evaluation, at least three sections were prepared from each animal, and five non-overlapping high-power fields were examined per section. Fields were selected systematically at random by scanning the section from one edge to the other, avoiding torn, folded, or poorly stained areas. Histopathological changes, including mucosal ulceration, glandular destruction, congestion, and inflammatory-cell infiltration, were evaluated under light microscopy.

3. Measurement of oxidative-stress-related biochemical indicators

Blood samples (0.5 mL) were collected into anticoagulant-containing tubes and centrifuged at approximately 1160 × g for 10 min at 4 °C. The supernatant was collected and used to measure serum iron, superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), and myeloperoxidase (MPO) levels using the corresponding assay kits listed in the Table of Materials according to the manufacturers' instructions.

4. Immunohistochemical analysis of JAK2 and STAT3

Paraffin-embedded colon tissue sections (5 µm) were deparaffinized in xylene, rehydrated through a graded ethanol series, and rinsed with phosphate-buffered saline. Antigen retrieval was performed in citrate buffer (pH 6.0) using microwave heating. After cooling to room temperature, endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min. Sections were blocked with 5% normal goat serum for 30 min at room temperature and incubated overnight at 4 °C with primary antibodies against JAK2 and STAT3 at dilutions of 1:50 and 1:80, respectively. Following phosphate-buffered saline washes, sections were incubated with a horseradish peroxidase-conjugated secondary antibody according to the manufacturer's instructions. Immunoreactive signals were visualized using 3,3′-diaminobenzidine, and nuclei were counterstained with hematoxylin. Sections were dehydrated, mounted, and examined under a light microscope.

5. Cytokine quantification by enzyme-linked immunosorbent assay

Colon tissues were cut into 2–3 mm³ pieces, homogenized on ice in phosphate-buffered saline, and centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was collected, and concentrations of TNF-α, IL-1α, and IL-13 were measured using the corresponding ELISA kits according to the manufacturers' instructions. Absorbance values were measured using a microplate reader, and cytokine concentrations were calculated from standard curves generated for each assay.

6. Western blot analysis of ferroptosis-related proteins

Total proteins were extracted from colon tissues, and protein concentrations were determined using a bicinchoninic acid assay. Equal amounts of protein from each sample were separated by SDS-PAGE and transferred onto membranes. After blocking with 5% non-fat milk or an equivalent blocking buffer for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against GPX4, FTH1, ACSL4, and the loading-control protein β-actin, as listed in the Table of Materials. On the following day, membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized using a chemiluminescent substrate and imaged using a chemiluminescence imaging system. Band intensities were quantified using densitometry software. Expression levels of GPX4, FTH1, and ACSL4 were normalized to β-actin, and relative protein expression was calculated for statistical analysis.

7. Statistical analysis

Data are presented as mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance, followed by Bonferroni's post hoc test when the assumptions of normality and homogeneity of variance were met. A value of P < 0.05 was considered statistically significant.

Results

FSEC alleviates histopathological injury and improves oxidative-stress-related indices in mice with ulcerative colitis
As shown in Figure 2, the normal control group exhibited intact colonic mucosal architecture, with well-organized glands and minimal inflammatory cell infiltration. In contrast, the model group displayed severe mucosal injury characterized by epithelial disruption, mucosal ulceration, glandular destruction, congestion, and marked inflammatory-cell infilt...

Discussion

This study investigated the effects of five-flavor Sophora flavescens enteric-coated capsules (FSEC) in a mouse model of experimental ulcerative colitis (UC) and evaluated associated changes in oxidative-stress-related indices, inflammatory cytokines, JAK2/STAT3 signaling, and ferroptosis-related markers. The principal findings were that FSEC treatment alleviated colonic histopathological injury, improved antioxidant-related biochemical indices, reduced TNF-α and IL-1α levels, partially restored IL-13 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3,3′-Diaminobenzidine (DAB) substrate kit	ZSGB-BIO	ZLI-9018	Chromogen substrate for HRP-based immunohistochemistry.
ACSL4 antibody (F-4)	Santa Cruz Biotechnology	sc-365230	Mouse monoclonal primary antibody for western blot; used at 1:500; RRID: AB_10842002.
Antigen retrieval solution, citrate buffer, pH 6.0	Beyotime Biotechnology	P0081	Heat-induced epitope retrieval buffer for paraffin-section immunohistochemistry.
BCA protein assay kit	Beyotime Biotechnology	P0012	Protein quantification before western blotting.
C57BL/6J mice	Fujian Anburui Biotechnology Co., Ltd.	N/A	Male SPF mice, 8 weeks old; animal source consistent with the submitted ethics approval materials.
Catalase (CAT) assay kit	Nanjing Jiancheng Bioengineering Institute	A007-1-1	Colorimetric kit for serum CAT activity measurement.
ChemiDoc XRS+ imaging system	Bio-Rad Laboratories	1708265	Chemiluminescence imaging system for western blot detection.
CRX-526	TargetMol	T27090	TLR4 antagonist used as the positive-control intervention; CAS no. 245515-64-4.
Dextran sulfate sodium salt (DSS), colitis grade	MP Biomedicals	160110	DSS, colitis grade, molecular weight 36,000–50,000 Da; used at 2.5% in drinking water.
ECL chemiluminescent substrate	Bio-Rad Laboratories	1705061	Clarity Western ECL substrate for HRP-based western blot signal detection.
ELISA kit, mouse IL-13	Abcam	ab219634	Colorimetric sandwich ELISA kit for mouse IL-13 quantification; 450 nm readout.
ELISA kit, mouse IL-1α	Abcam	ab199076	Colorimetric sandwich ELISA kit for mouse IL-1α quantification; 450 nm readout.
ELISA kit, mouse TNF-α	Abcam	ab208348	Colorimetric sandwich ELISA kit for mouse TNF-α quantification; 450 nm readout.
Ethanol, absolute	Sinopharm Chemical Reagent Co., Ltd.	10009218	Used for tissue processing, dehydration, and reagent preparation.
Ferrous iron (Fe2+) assay kit	Nanjing Jiancheng Bioengineering Institute	A039-2-1	Colorimetric kit for Fe2+ measurement in serum or tissue samples.
Five-flavor Sophora flavescens enteric-coated capsules	Beijing Zhonghui Pharmaceutical Co., Ltd.	NMPA approval no. Z20150002	Chinese patent medicine; 0.4 g/capsule; formulation includes Sophora flavescens Aiton, Sanguisorba officinalis L., Indigo naturalis, Bletilla striata (Thunb.) Rchb.f., and Glycyrrhiza uralensis Fisch. ex DC.; pharmaceutical specification YBZ00152015
Glutathione (GSH) assay kit	Nanjing Jiancheng Bioengineering Institute	A006-2-1	Reduced glutathione assay kit for serum GSH measurement.
GPX4 antibody (E-12)	Santa Cruz Biotechnology	sc-166570	Mouse monoclonal primary antibody for western blot; used at 1:500; RRID: not listed in the supplier datasheet.
Hematoxylin and eosin staining kit	Solarbio	G1120	H&E staining of paraffin-embedded colon tissue sections.
Horseradish peroxidase-conjugated goat anti-mouse IgG	Santa Cruz Biotechnology	sc-2005	Secondary antibody for western blot; used at 1:50,000; RRID: AB_631736.
ImageJ software	National Institutes of Health	Version 1.54	Image-analysis and densitometry software used for western blot band quantification.
Immunohistochemistry detection kit	ZSGB-BIO	PV-9000	Polymer-based HRP detection kit for paraffin-section immunohistochemistry.
JAK2 antibody (D2E12)	Cell Signaling Technology	3230	Rabbit monoclonal primary antibody for JAK2 immunohistochemistry on paraffin sections; RRID: AB_2128522.
Microplate reader	BioTek Instruments	Synergy H1	Plate reader for ELISA absorbance measurement at 450 nm.
Myeloperoxidase (MPO) activity assay kit	Nanjing Jiancheng Bioengineering Institute	A044-1-1	Colorimetric kit for serum MPO activity measurement.
Neutral buffered formalin, 10%	Sigma-Aldrich	HT501128	Fixation of colon tissue before paraffin embedding.
Normal goat serum	Solarbio	SL038	Blocking reagent for immunohistochemistry.
Paraffin	Leica Biosystems	39601006	Embedding medium for colon tissue processing.
Phosphate-buffered saline (PBS)	Solarbio	P1020	Buffer for tissue homogenization, washing, and immunostaining procedures.
PVDF membrane	MilliporeSigma	IPVH00010	Protein-transfer membrane for western blotting.
Recombinant mouse TNF-α protein	R&D Systems	410-MT	Recombinant mouse TNF-α used for the final-week inflammatory challenge.
SDS-PAGE and transfer system	Bio-Rad Laboratories	1658004	Mini-PROTEAN Tetra system for SDS-PAGE and western blot transfer workflow.
SPSS Statistics software	IBM	Version 24.0	Statistical analysis software for one-way ANOVA and Bonferroni post-hoc testing.
STAT3 antibody (124H6)	Cell Signaling Technology	9139	Mouse monoclonal primary antibody for STAT3 immunohistochemistry; RRID: AB_331757.
Superoxide dismutase (SOD) assay kit	Nanjing Jiancheng Bioengineering Institute	A001-3-2	WST-1 method kit for serum SOD activity measurement.
Xylene	Sinopharm Chemical Reagent Co., Ltd.	10023418	Dewaxing reagent for paraffin-section processing.
β-actin antibody (C4)	Santa Cruz Biotechnology	sc-47778	Mouse monoclonal loading-control antibody for western blot; used at 1:1,000; RRID: AB_626632.