Dextran sulfate sodium and β-sitosterol (SIT, purity >95% and HPLC ≥98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Carboxymethyl cellulose was purchased from Biotopped Technology Co. Ltd. (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits of TNF-α, IL-1β, and IL-18 were obtained from Cloud-Clone Corp (Wuhan, China). Antibodies against NLRP3, occludin, β-Actin, goat-anti-rabbit IgG H&L (HRP), and goat-anti-mouse IgG H&L (HRP) were purchased from Abcam (Cambridge, United Kingdom). Antibodies against Caspase-1, GSDMD, IKB alpha, and ZO-1 were obtained from Proteintech (Wuhan, China). Antibodies against NF-kappaB p65, phospho-NF-kappaB p65, and phospho-IkB alpha were purchased from Invitrogen (Carlsbad, CA). Antibody against cleaved Caspase-1 was purchased from Cell Signalling Technology (Danvers, MA, United States). Antibody against GSDMD-N terminal was purchased from ABclonal (Wuhan, China). The Cell Counting Kit-8 (CCK-8) was purchased from Biorigin (Beijing, China).

The following reagents were obtained from GenePool Biotechnology (Beijing, China): Total RNA Extraction with DNase Ⅰ Kit, mRNA cDNA Synthesis Kit, mRNA qPCR Kit, RNA Loading Buffer, BSA Blocking Buffer, SDS-PAGE Gel Ki, SDS-PAGE Loading Buffer, Tris-Glycine Running Buffer, WB Transfer Buffer, and TBST. The following reagents were purchased from Beyotime Biotechnology (Shanghai, China): Cell lysis buffer for Western and IP, Protein Extraction Kit, BCA protein assay kit, enhanced chemiluminescence (ECL) kit, Citrate-EDTA Antigen Retrieval Solution, and Hematoxylin and Eosin Staining Kit. The following reagents were purchased from Gibco (Carlsbad, CA, United States): fetal bovine serum (FBS), minimum essential medium (MEM), Penicillin-Streptomycin antibiotics, non-essential amino acids (NEAA), and 0.25% trypsin solution with EDTA. All other chemicals were of reagent grade.

Sixty male Sprague-Dawley rats (6–8 weeks old, weighing 180–200 g) were purchased from Beijing Sibeifu Bioscience, Co., Ltd. (Beijing, China) (License NO. SCXK [Beijing] 2019-0010). All animals were raised under standard specific pathogen-free (SPF) conditions (temperature, 25°C ± 1°C; humidity, 60% ± 5%) with a 12 h light/dark cycle per day and allowed ad libitum eat and drink. Ethical approvals for the animal experiments were obtained from the animal ethics committee of Beijing University of Chinese Medicine (NO. BUCM-4-2022102901-4029).

This experimental design consists of two steps: a DSS-induced colitis stage and a drug treatment stage as described in Figure 2A. 60 rats were randomly assigned to five groups: 1) Control group (CON): received only double-drilled water; 2) DSS group (DSS): received 4% DSS (molecular weight: 40 000 Da; purity >98%) dissolved in double-drilled water in their daily drinking water; 3) Low-dose group (LD): received DSS and then gavaged with 50 mg/kg of SIT (purity >95%) suspended in 0.1% carboxymethyl cellulose; 4) Middle-dose group (MD): received DSS and then gavaged with 100 mg/kg SIT; 5) High-dose group (HD): received DSS and then gavaged with 200 mg/kg SIT. The intervention period of DSS was from day 1 to day 7 and the period of drug treatment was from day 8 to day 21.

Following the 14-day intervention, rats were all deeply anesthetized after 24 h fasting with 1% sodium pentobarbital (40 mg/kg). After recording the colon length and weight, blood samples were collected and centrifuged for serum. Then the colon specimens were immediately removed and then fixed in 4% paraformaldehyde for histopathological studies or stored at −80°C for molecular biology detection.

During this experimental period, body weight, stool status, and rectal bleeding were recorded every day to calculate disease activity index (DAI) scores on a previously established scoring system (Kihara et al., 2003) and listed in Table 1. DAI score is the average of the three categories. All evaluations were performed while unaware of the conditions.

Hematoxylin and eosin (H&E) detection was conducted with a standard protocol. The colon tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Then, the paraffin-blocked samples were cut into 5-µm sections on slides for staining with hematoxylin and eosin. Slides were scanned using a Panoramic MIDI Scan Whole Slide Scanner (3DHISTECH Ltd., Budapest, Hungary) and viewed with Panoramic Viewer 1.15.4 (3DHISTECH); in addition, histopathological scores for histopathological changes were calculated based on a previously developed scoring system (Dieleman et al., 1998) and listed in Table 2. The individual scoring was blinded to the identity of the slides.

Human epithelial colorectal adenocarcinoma (Caco-2) cell lines (originally obtained from ATCC) were purchased from IMMOCELL (Xiamen, China). The Caco-2 cells were maintained in minimum essential medium (MEM) containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin, 100 μg/mL of streptomycin, and 1% non-essential amino acids supplements. Cells were maintained at 37°C with 5% CO₂ atmosphere.

Caco-2 cell viability was measured by CCK-8 assay followed by a standard protocol. After preparing single-cell suspensions, cells (5 × 10⁴ cells/well) were added to 96-well plates and continued to culture till 80% confluency. Then, 10 μL/well of CCK-8 solution was added and incubated protected from light for 2 h at 37°C. The 450 nm absorbance values were detected with a multifunctional microplate reader (Thermo, Manassas, United States) to measure the cell growth inhibition rate.

Cells were cultured overnight with or without SIT (HPLC ≥98%) dissolved in absolute ethanol (concentrations of 8 μg/mL as Low-dose group and 16 μg/mL as High-dose group). Supernatants of cells were discarded and washed with PBS, and 50 mg/mL DSS was added to establish the inflammation model.

The serum of rats and supernatant of cells were collected and then calculated for TNF-α, IL-1β, and IL-18 levels using ELISA kits according to the manufacturer’s scheme.

Relative levels of NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18 mRNAs in colonic tissues were analyzed by a quantitative real-time (qRT)-PCR. Total RNA of colonic tissues was isolated using the Total RNA Extraction Kit and cDNA was synthesized by using the mRNA cDNA Synthesis Kit in accordance with the manufacturer’s protocols. The specific primers for NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18 were designed and synthesized by GenePool Biotechnology (Beijing, China) and listed in Table 3. The PCR reaction parameters were predetermined: 95°C for 5 min, followed by a 35 cycle-denaturation for 30 s at 95°C, 55°C for 30 s, and extension for 1 min at 72°C. Each sample underwent three biological replications for statistical analysis to determine significant differences. GAPDH was an internal control and relative gene expression levels were calculated using the 2^−ΔΔCT method.

The total proteins of tissues and cells were extracted using Western and IP lysate buffer, BCA protein assay kit was applied to determine protein concentration. After homogenization, an equal quantity of proteins was separated with SDS-PAGE and transferred to PVDF membranes (Millipore corp., Massachusetts, United States), then blocked with 5% BSA. Primary antibodies of NLRP3 (dilution 1:1 000), Caspase-1 (dilution 1:2 000), cleaved-Caspase-1 (dilution 1:1 000), GSDMD (dilution 1:2 000), GSDMD-N (dilution 1:1 000), p65 (dilution 1:1 000), p-p65 (dilution 1:1 000), IκBα (dilution 1:1 000), p-IκBα (dilution 1:1 000), ZO-1 (dilution 1:5 000), occludin (dilution 1:1 000), and β-Actin (dilution 1:5 000) were incubated overnight at 4°C. After being washed with TBST three times, membranes were then incubated with secondary antibodies against Goat Anti-Rabbit IgG H&L (HRP) (dilution 1:5 000) or Goat Anti-Mouse IgG H&L (HRP) (dilution 1:5 000) for 1 h at 37°C, and bands were visualized with an ECL kit captured with Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Finally, the relative grey values of the target proteins blots normalized to β-Actin were analyzed by the ImageJ software (National Institutes of Health, United States).

Statistical analysis was performed by SPSS 26.0 system. Quantitative results were expressed as arithmetic mean plus or minus the standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) followed by least significant difference (LSD)’s multiple-comparison test, while Kruskal-Wallis test was performed for difference analysis of non-parametric data. p-value < 0.05 was considered statistically significant.

In order to investigate the therapeutic effects of SIT in vivo, UC model rats were induced by 4% DSS for 7 consecutive days. During the experiment period, rats in the control group remained in a normal state, and body weight was rising steadily, while those in the model group represented a poor mental state with disheveled fur, decreased feeding, persistent fecal bleeding, and obvious body weight loss. Compared to the control group DAI score in the DSS-induced model group was significantly increased, which was consistent with the clinical characteristics. Notably, administration of SIT recovered the body weight and colon length gradually (Figures 2C–E) as well as decreased DAI score (Figure 2B) in DSS-induced rats, especially at the dose of 200 mg/kg.

Furthermore, colonic shortening caused by DSS was evidently mitigated after SIT intake. As can be seen from Figure 2F, DSS in the UC group led to a significant decrease in colon length/weight ratio compared with the control group. High dose of SIT agonist partly offset the decrease of colon length/weight ratio. Under microscopy, results by H&E staining showed that there were pathological lesions and superficial inflammation in the DSS-induced colonic tissues characterized by inflammatory cell infiltration, crypt architectural distortion or absence and mucosa defects or damage. Consistent with the remission of clinical signs, the pathological changes were improved to varying degrees following the administration of SIT (Figures 2F,G). Collectively, these results indicated that SIT is protective against DSS-induced colitis and the effects might be dose-dependent.

Before the formal in vitro experiments, toxicity evaluation of different concentrations of SIT (1, 2, 4, 8, 16, 32, and 64 μg/mL) on Caco-2 was carried out using the CCK-8 assay. After the 24 h-intervention, we observed that there was a significant decline in cell viability of Caco-2 following the higher concentrations of SIT (Figure 3A). Based on this, we chose the relatively safe concentrations for low-dose SIT (LD, 8 μg/mL) and high-dose SIT (HD, 16 μg/mL), respectively. In addition, at the concentration of 46.34 mg/mL DSS, growth of 50% cells was inhibited (Figure 3B). For convenience, we used 50 mg/mL DSS for subsequent experimental Caco-2 modeling, and found that SIT serves protectively against DSS-induced Caco-2 damage, (Figure 3C).

To validate the suppressive effects of SIT on inflammation involved in UC, we detected the levels of inflammatory-related factors. The ELISA results showed that there was a significantly increase of TNF-α, IL-1β, and IL-18 levels after DSS treatment compared with the control group in rats and SIT effectively decreased the expression (Figure 4A). We further noticed the protective effects of SIT on Caco-2 from inflammation after being exposed to DSS (Figure 4B).

NF-κB signaling participates in the regulation of inflammatory response. We wondered whether the anti-inflammatory role of SIT was associated with the signaling. The expression levels of the major proteins p-p65, p65, p-IκBα, and IκBα in the NF-kB signaling pathways were consequently measured by Western blot. The experimental results showed that the levels of p-p65 and p-IκBα in DSS-induced colonic tissues were abnormally higher compared to the control group. In comparison with the DSS group, the protein expression ratio of p-p65/p65 and p-IκBα/IκBα were attenuated after stimulation with SIT (Figures 4C,D) while none were statistically significant. These results indicate that NF-κB signaling might be involved in the reduction of DSS-induced UC inflammation by SIT administration.

Pyroptosis is a type of programmed cell death (PCD) and participates in inflammatory disease processes involved in UC. For this reason, we further measured the expression of critical indicators of NLRP3/Caspase-1/GSDMD-mediated pyroptosis to explicit the anti-inflammatory effects of SIT. RT-qPCR analysis was carried out to assess the gene expression of Caspase-1, NLRP3, GSDMD, IL-1β, and IL-18 in colonic tissues. When compared to the control group, the mRNA expression levels of the above genes were upregulated in the DSS group while significantly downregulated with SIT administration (Figure 5A).

As expected, Western blot analysis showed that protein expression levels of Caspase-1, Cleaved-Caspase-1, NLRP3, GSDMD, and GSDMD-N distinctly increased after DSS treatment in colonic tissues and Caco-2 cells and significantly counteracted following the intervention with SIT (Figures 5B–E). Meanwhile, ELISA indicated that SIT treatment significantly inhibited DSS-induced increased levels of IL-1β and IL-18 in serum and cell supernatant; it has been shown that secretion of cytokines IL-1β and IL-18 were associated with the NLRP3 inflammasome and induced an inflammatory cell death mode termed as pyroptosis (He et al., 2015). Comprehensively, the above results suggest that SIT plays a possible therapeutic role in UC via regulating Caspase-1-mediated pyroptosis.

Tight junction proteins are the essential parts for the maintenance of the gut mucosal barrier integrity, which could against the invasion of luminal detrimental substances and lower the risk of microbe-induced inflammation (Parikh et al., 2019). Therefore, we further verified whether the protective mechanism of SIT on the colon is related to the restoration of intestinal barrier function. ZO-1 and occludin, are important two tight junction-associated proteins (Ji et al., 2016), which were measured by Western blot. The expression levels of ZO-1 and occludin were decreased compared with the control group, which were significantly reversed by SIT as provided in Figure 6, suggesting that SIT could alleviate colitis through enhancing mucosal barrier integrity.