Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), and trypsin were purchased from Life Technology (Grand Island, NY, United States). Scutellarein (Scu, Cat# B21479), with a purity above 98%, was obtained from Earay Bio-tech (Baoji, Shanxi China). Dextran sulfate sodium (DSS, with the highest sulfur content of 19% and a molecular weight ranging from 36 to 50 kDa, Cat# 160110) was obtained from MP Biomedicals (Santa Ana, CA, United States). BAY11-7085 (Cat# HY-10257), 5-aminosalicylic acid (5-ASA, Cat# HY-15027), and IL-1β (Cat# HY-P78459A) were obtained from MedChemExpress (Monmouth Junction, NJ, United States). The Myeloperoxidase (MPO) Activity Assay Kit (Cat# A114-1–1) was supplied by the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Anti-ZO-1 antibody (Cat# 21773-1-AP) was acquired from Proteintech (Wuhan, Hubei, China). The following antibodies were sourced from Cell Signaling Technology (Danvers, MA, United States): antibodies against phosphor (p)-NF-κB p65 (Cat# 3033), NF-κB (Cat# 8242), occludin (Cat# 91131), E-cadherin (Cat# 3195), and anti-rabbit Alexa Fluor^® 488 (Cat# 4412). The primary antibody against GAPDH (Cat# MB001) was purchased from Bioworld Biotechnology Co., Ltd. (Nanjing, Jiangsu, China). Tribromoethanol, Hoechst 33342, and all the inorganic salts were supplied by Sigma-Aldrich (St. Louis, MO, United States) unless otherwise mentioned. HT-29 cells (Cat# HTB-38) were procured from the Cell Bank of Shanghai (Shanghai, China).

Male C57BL/6 mice, aged 7–8 weeks and weighing between 22 and 24 g, were sourced from Yangzhou University (Yangzhou, Jiangsu, China). The animal care and usage were conducted following the guidelines established by the National Institutes of Health, as detailed in NIH Publications No. 8023 (revised in 1978). All experimental procedures involving animals adhered to ethical standards for animal research and received approval from the Ethics Committee of the China Pharmaceutical University (#SYXK, 2023–0019). The mice were housed in a vivarium with a controlled temperature of 23°C ± 2°C, under a 12-hour light and dark cycle, and had unrestricted access to food and water.

A UC mouse model was established following the methodology described in previous research (Xue et al., 2023). In brief, a total of 42 mice were acclimatized for 1 week and then randomly assigned to 7 different experimental groups. To induce colonic inflammation, mice were challenged with a 3% concentration of DSS in their drinking water for seven consecutive days. Mice in the vehicle (Veh) group and 20 mg/kg Scu group were provided with regular drinking water.

Scu and 5-ASA were dissolved in a solution comprising 2% dimethyl sulfoxide (DMSO), 2% Tween-80, and 0.5% sodium carboxymethyl cellulose (CMC-Na). A measure of 200 mg/kg 5-ASA and Scu at varying levels of 5 mg/kg, 10 mg/kg, and 20 mg/kg were administered to the mice once daily by intragastric gavage for seven consecutive days from the first day of DSS administration until the day of sacrifice by tribromoethanol administration. The distal colon tissues were carefully dissected, harvested, and immediately frozen for further analysis.

The disease activity index (DAI) was scored according to Sann’s method by summing the respective scores for body weight loss, hemorrhage, and faecal consistency (Sann et al., 2013; Wang et al., 2021).

The enzymatic activity of MPO in mouse colon samples was evaluated using an MPO activity assay kit (Alkushi et al., 2022). According to the manufacturer’s instructions, the colonic homogenate supernatant reacts with H₂O₂ and o-dianisidine to produce oxidized o-dianisidine, the absorbance of which at 460 nm is linearly correlated with MPO activity. The MPO results were expressed as units per gram (U/g) of tissue wet weight.

Colon samples were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 μm, followed by staining with hematoxylin and eosin. The sections were photographed using the NanoZoomer 2.0 RS Slide scanner (Hamamatsu Photonics, Japan). According to Wang’s method (Wang et al., 2018), histological scores were assessed by summing the respective scores for inflammation, the extent of crypt damage, and the range of lesions.

The HT-29 cells, human intestinal epithelial cells, were maintained in DMEM supplemented with 10% FBS and 100 U/mL of P/S at 37°C with 5% CO₂.

Western blotting was performed, as previously described (Li S. H. et al., 2022). In brief, 30 µg of protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation, followed by transfer to a nitrocellulose membrane. The membranes were blocked by 5% skimmed milk and then incubated with primary antibodies: anti-ZO-1, anti-p-NF-κB p65, anti-NF-κB, anti-occludin, anti-E-cadherin, anti-β-actin, and anti-GAPDH (at the recommended dilution). After the incubation with IRDye (680RD or 800CW)-labeled goat anti-mouse or goat anti-rabbit secondary antibodies (at 1:10,000 dilution), the blots were visualized using the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, United States), and the densitometry was quantified using its application software (version 2.1). The expression levels of proteins were normalized to those of β-actin or GAPDH.

Real-time quantitative polymerase chain reaction (RT-qPCR) was performed, as previously described (Wang et al., 2022). Total messenger RNA (mRNA) of colon tissues or HT-29 cells was extracted using TRIzol reagent (Cat# R401-01, Vazyme) and subsequently reverse-transcripted to cDNA using the HiScript II Q RT SuperMix Kit (Cat# R223-01, Vazyme). RT-qPCR was conducted on a QuantStudio 3 System (Thermo Fisher Scientific, Massachusetts, United States). Relative mRNA expression levels normalized to GAPDH were quantified using the 2^−ΔΔCt method. All primers listed in Table 1 were synthesized by Beijing Tsingke Biotechnology Co., Ltd. (Beijing, China).

As previously described (Srinivasan et al., 2015), HT-29 cells were seeded in the upper chamber, and the transepithelial electrical resistance (TEER) was recorded using Millcell®ERS (Electrical Resistance System) with two electrodes placed in the upper and lower chambers, respectively. The TEER values were reported in units of ohm-centimeter squared (Ω·cm²).

Colon slides were subject to deparaffinization and subsequent antigen retrieval. HT-29 cells were seeded in a confocal petri dish and fixed with 4% paraformaldehyde. After permeabilization using 0.3% Triton X-100, colon slides and HT-29 cells were blocked with 5% BSA at room temperature for 1 h, followed by incubation overnight with primary antibodies against ZO-1 (1:1,200), occludin (1:400), E-cadherin (1:1,000), and NF-κB (1:400), respectively. The goat anti-mouse or anti-rabbit secondary antibodies conjugated with Alexa Fluor^® 488 were used for visualization. Nuclei were stained with Hoechst. Slides and cells were photographed using a Zeiss LSM 800 Confocal Fluorescence Microscope (Le Pecq, France) with a 20 × (slides) or 63 × (cells) objective.

As previously described (Xue et al., 2023), HT-29 cells seeded in 96-well plates were treated with Scu at various concentrations for 24 h. After the replacement with the fresh medium containing 500 μg/mL of MTT, a further incubation was carried out for 1 h. Then, the medium was replaced by DMSO to dissolve the formazan crystals. Absorbance at 570 nm was measured using an Infinite M200 Pro-NanoQuant Reader from Tecan Austria (GmbH, Grodig, Austria).

Statistical analysis was performed using GraphPad Prism version 8.01 (San Diego, CA, United States). Data are presented as the mean ± SEM. Statistical significance was assessed using one-way ANOVA or two-way ANOVA, followed by Bonferroni’s multiple comparison test. A p-value less than 0.05 was deemed statistically significant.

The structure of Scu is illustrated in Figure 1A inset. Consecutive drinking of 3% DSS led to a gradual reduction in the body weight of the mice (Figure 1A). The administration of Scu alone did not affect the mouse body weight and dose-dependently alleviated the body weight loss induced by DSS (Figure 1A). In addition to body weight loss, DSS also induced diarrhea and bloody stools. These symptoms can be quantified using the disease activity index (DAI) score. The DAI scores were significantly increased on day 3 and gradually increased upon drinking of DSS. The administration of Scu also dose-dependently suppressed the DSS-induced severity of UC, as reflected by the decreased DAI scores. At 20 mg/kg, Scu displayed greater protection against DSS-induced UC than the positive control, 5-ASA (Figure 1B). Compared to the Veh group, mice with DSS administration exhibited shortened colon length (7.45 ± 0.14 cm vs. 4.02 ± 0.20 cm, p < 0.01) (Figures 1C, D). While 5 mg/kg Scu showed marginal improvement (4.02 ± 0.20 cm vs. 4.17 ± 0.19 cm, p > 0.01) in DSS-induced colon atrophy, doses of 10 mg/kg, 20 mg/kg Scu, and 200 mg/kg 5-ASA were able to diminish DSS-induced colon atrophy by 29.1% (p < 0.01), 67.0% (p < 0.01), and 27.1% (p < 0.01), respectively (Figures 1C, D). Pathological examination indicated that colon tissues from DSS mice experienced destruction with the disappearance of crypts (black arrows) and edematous lamina propria containing inflammatory cell infiltrates (red boxes). The administration of Scu and 5-ASA preserved the epithelial barrier, reduced edema, and alleviated inflammatory infiltration in the lamina propria of DSS mice (Figure 1E). The pathology score of DSS mice was 10.0 ± 1.1 (p < 0.01 vs. control). While oral administration of 20 mg/kg Scu alone did not impact colon pathology, doses of 10 mg/kg and 20 mg/kg Scu significantly decreased the pathological scores in DSS mice to 6.0 ± 0.5 (p < 0.01) and 2.0 ± 0.3 (p < 0.01), respectively. As the positive drug, 5-ASA also reduced the pathological score to 6.0 ± 1.0 (p < 0.01) (Figure 1F). These findings indicated that Scu yielded superior protection on UC when compared to the clinically used drug, 5-ASA.

Given the superior protection of Scu on UC in DSS-treated mice, we next evaluated the impact of Scu on the inflammatory response in colonic tissues. DSS-administered mice displayed increased MPO activity in colonic tissues and upregulated mRNA expression levels of inflammatory cytokines such as Tnf-α, Il-1β, Il-1α, Cxcl1, and Il-6 (Figure 2). Although oral administration of 20 mg/kg Scu alone did not affect the MPO activity, Scu dose-dependently reduced the MPO activity and the mRNA expression levels of these inflammatory mediators (Figure 2). At the dose of 20 mg/kg, Scu significantly decreased the MPO activity by 66.6% ± 1.6% (p < 0.01), while 5-ASA only achieved 29.8% ± 8.8% reduction (p < 0.01). Scu at 20 mg/kg also remarkably decreased mRNA expression levels of Tnf-α, Il-1β, Il-1α, Cxcl1, and Il-6 by 72.8% ± 8.5% (p < 0.01), 99.4% ± 2.9% (p < 0.01), 96.9% ± 4.6% (p < 0.01), 90.0% ± 1.8% (p < 0.01), and 99.0% ± 0.6% (p < 0.01), respectively, in the colons of DSS-treated mice (Figure 2). As a positive drug, 5-ASA also significantly reduced mRNA expression levels of Tnf-α, Il-1β, Il-1α, Cxcl1, and Il-6 by 60.5% ± 11.9% (p < 0.01), 70.0% ± 13.5% (p < 0.01), 51.8% ± 26.2% (p < 0.01), 58.1% ± 8.7% (p < 0.01), and 85.1% ± 4.5% (p < 0.01), respectively, in the colons of DSS-treated mice (Figure 2). Again, Scu displayed better suppression on colonic inflammation in the DSS-treated mice than 5-ASA.

The disruption of the gut barrier and loss of mucosal homeostasis were observed in active UC patients and shown to trigger intestinal inflammation (Van der Post et al., 2019). To maintain the integrity of the intestinal barrier, the expression, distribution, and fine balance between isoforms of tight junction proteins, including occludins, claudins, and ZO family, must be tightly regulated (Chelakkot et al., 2018). We, therefore, investigated the impact of Scu on the expression of tight junction proteins in DSS-treated mice. Immunofluorescence staining demonstrated that E-cadherin, occludin, and ZO-1 were highly expressed in the crypts of colons in vehicle-treated mice. The administration of Scu (20 mg/kg) alone did not change the expression levels and patterns of E-cadherin, occludin, and ZO-1. However, in the colons of DSS-treated mice, the expressions of E-cadherin, occludin, and ZO-1 were all diminished. The administration of Scu dose-dependently alleviated the downregulated protein expression of E-cadherin, occludin, and ZO-1, a phenomenon also observed by 5-ASA treatment (Figure 3A). Consistent with the immunofluorescence measurement, Western blots also demonstrated that DSS-treatment decreased the protein levels of E-cadherin, occludin, and ZO-1 by 89.8% ± 2.9% (p < 0.01), 94.8% ± 1.4% (p < 0.01), and 69.5% ± 8.6% (p < 0.01), respectively. The administration of Scu dose-dependently reversed the protein expression of E-cadherin, occludin, and ZO-1 in DSS mice. At 20 mg/kg, Scu increased the expression levels of E-cadherin, occludin, and ZO-1 in DSS-treated mice from 10.2% ± 2.9% to 109.2% ± 15.7% (p < 0.01), 5.2% ± 1.4% to 82.8% ± 10.6% (p < 0.01), and 30.5% ± 8.6% to 120.2% ± 25.3% (p < 0.01) of vehicle-treated mice, respectively (Figures 3B, C).

We next investigated whether Scu can affect the epithelial inflammation and epithelial barrier integrity in a cell model. We first examined the cytotoxicity of Scu in cultured HT-29 cells. A 24 h exposure of Scu up to 30 μM had no cytotoxic effect on the HT-29 cell although 100 μM of Scu decreased the HT-29 cell viability by 9.9% ± 1.4% (p < 0.01) (Figure 4A). IL-1β is a critical inflammatory mediator in the development of UC (Al-Sadi et al., 2008; Mannino et al., 2019). IL-1β exposure for 12 h dramatically increased the mRNA levels of IL-6 and IL-8 to 4.59 ± 0.54 (p < 0.01) and 34.69 ± 1.97-fold (p < 0.01) of their respective controls (Figures 4B, C). Although the incubation of Scu did not affect the basal expression levels of IL-6 and IL-8, it abolished IL-1β-induced mRNA expression of IL-6 and decreased that of IL-8 by 45.8% ± 3.4% (p < 0.01) (Figures 4B, C).

IL-1β exposure for 24 h significantly decreased TEER values from 18.76 ± 0.36 Ω⋅cm² to 13.26 ± 0.78 Ω⋅cm² (P < 0.01) in cultured HT-29 cell monolayer (Figure 4D). Although Scu (10 μM) treatment alone did not alter the TEER value, it significantly increased TEER values to 16.45 ± 1.15 Ω⋅cm² (p < 0.05) in IL-1β-treated HT-29 cell monolayer (Figure 4D). HT-29 monolayer cells showed considerable staining of E-cadherin, occludin, and ZO-1 proteins in the cell membrane between cells, consistent with their function to maintain barrier integrity (Figure 4E). IL-1β exposure decreased the immunofluorescence signals of E-cadherin, occludin, and ZO-1 (Figure 4E). While Scu (10 μM) treatment for 24 h did not alter the expression levels and distribution patterns of these tight junction proteins in vehicle (0.1% DMSO)-treated HT-29 monolayer, it increased the expression levels of E-cadherin, occludin, and ZO-1 in IL-1β-treated HT-29 cell monolayer from 0.66 ± 0.06 to 1.19 ± 0.1 (p < 0.01), 0.74 ± 0.04 to 0.99 ± 0.06 (p < 0.01), and 0.66 ± 0.05 to 0.95 ± 0.08-fold (p < 0.01) of their respective controls (Figures 4E–G).

We next explored how Scu exerted its anti-inflammatory on epithelial HT-29 cells. The activation of the nuclear transcription factor NF-κB mediates signaling pathways for a variety of inflammatory mediators, including IL-1β (Lee et al., 2024). Western blot analysis demonstrated that IL-1β exposure rapidly increased NF-κB phosphorylation (Figure 5A) and nuclear translocation (Figure 5C). Treatment with Scu (10 μM) for 30 min significantly suppressed IL-1β-induced NF-κB phosphorylation by 63.4% ± 10.8% (p < 0.01) (Figure 5B) and abrogated the IL-1β-induced NF-κB nuclear translocation (Figure 5C). To validate the promotion of NF-κB activation on epithelial inflammation and barrier integrity, we examined the effects of NF-κB inhibition on IL-1β-induced IL-6 and IL-8 expression and barrier disruption. An NF-κB inhibitor, BAY-11–7082 (3 μM) suppressed IL-1β upregulated mRNA expression of IL-6 and IL-8 by 84.1% ± 3.2% (p < 0.01) and 24.1% ± 3.3% (p < 0.01), respectively (Figures 5D,E). BAY-11–7082 also reversed the value of TEER in IL-1β-treated HT-29 cells from 6.60 ± 1.35 Ω·cm² to 20.24 ± 1.07 Ω·cm² (p < 0.01) (Figure 5F) and IL-1β downregulated protein expression of E-cadherin, occludin, and ZO-1 (Figure 5G).