Naringin ameliorates intestinal injury in ulcerative colitis model mice by modulating the JAK2/STAT3 signaling pathway

Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) primarily affecting the colon which is characterized by mucosal inflammation. Its incidence is gradually increasing worldwide, with rates in China reaching 11.6 per 100,000 individuals (1,2). If left untreated UC can progress to colorectal cancer or result in systemic complications (3). The pathogenesis of UC involves immune dysregulation, excessive production of pro-inflammatory cytokines and gut microbiota imbalance (4). Current first-line therapies include aminosalicylates, such as mesalazine, corticosteroids and immunomodulators. However, up to 50% of patients experience treatment-related adverse events, such as glucose intolerance and hepatotoxicity (5), highlighting the need for safer and more effective therapeutic alternatives. Natural products have therefore attracted growing attention as potential candidates (6,7).

The intestinal barrier serves an important role in the pathogenesis of UC. Disruption of its structural and functional integrity contributes to increased intestinal permeability, microbial translocation and sustained immune activation. Growing evidence suggests that enhancing intestinal barrier function represents a promising therapeutic strategy for UC (8–14). Among the signaling mechanisms implicated, the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway has been extensively studied. Hyperactivation of this pathway is a hallmark of UC, contributing to both inflammation and barrier dysfunction (15,16). The preliminary data of the present study (data not shown), together with prior reports (17,18), have suggested that naringin, a natural flavonoid, may exert anti-inflammatory effects through modulation of kinase signaling pathways. The present study therefore focused on the JAK2/STAT3 pathway to elucidate the mechanism underlying its protective activity.

Citrus aurantium L., a member of the Rutaceae family also known as bitter orange, is widely consumed in China as both a food and traditional medicine, owing to its anti-inflammatory properties (18). Previous studies have demonstrated that Citrus aurantium L. alleviates trintrobenzene sulfonic acid (TNBS)-induced IBD in rats, while its major flavonoid naringin reduces the expression of inducible nitric oxide synthase (iNOS), cytochrome c oxidase subunit 2 (COX-2) and TNF-α (17,18). Naringin has also been shown to ameliorate clinical symptoms in murine models of IBD (17,18). Given that the JAK2/STAT3 pathway is hyperactivated in UC and promotes inflammation (15,16), its pharmacological inhibition has emerged as a promising therapeutic approach (19–21). However, the role of naringin in modulating UC through this pathway remains undefined. The present study aimed to investigate the effects of naringin on sodium sulfate dextran (DSS)-induced colitis and its influence on the JAK2/STAT3 signaling pathway.

Materials and methods

Materials

Naringin (cat. no. B21594-1g; Shanghai Yuanye Bio-Technology Co., Ltd.) and fluorescein isothiocyanate dextrose 4000 (FD-4; molecular weight, 3,000-5,000 kDa; cat. no. FD4-250MG) were purchased from Sigma Chemical Co. Mesalazine (cat. no. 20230605) was obtained from Heilongjiang Tianhong Pharmaceutical Co., Ltd. DSS (molecular weight, 36,000-50,000 kDa; cat. no. 02160110-CF) was purchased from MP Biomedicals, LLC. Caco-2 cells were obtained from Cell Resource Center, Peking Union Medical College. Cell culture reagents (HyClone™; Cytiva) included modified Eagle's medium (MEM; cat. no. SH30024.01), fetal bovine serum (FBS; cat. no. SH30071.03) and penicillin-streptomycin (cat. no. SV30010).

Animals

Healthy male BALB-c mice (aged 6–8 weeks; 18–22 g) were obtained from Sbeifu (Beijing) Biotechnology Co., Ltd. (certificate of conformity no. 110324210106095731). Mice were housed in cages with controlled temperature (22±2°C), relative humidity (40–60%) and a 12-h light/dark cycle throughout the study. All mice were fed a standard diet (crude protein 16%, crude fat 4%, crude fiber 12% and ash 8%) and had free access to water. Animal experiments were approved by the Animal Welfare Committee of Hebei North University (Zhangjiakou, China; approval no. HBNV20240223003) and performed according to institutional guidelines.

DSS-induced colitis mice

A total of 32 mice were randomly divided into four groups, each consisting of eight mice: The normal, control, mesalazine control and naringin groups. Intragastric administration volumes were 0.3 ml per 10 g body weight. Mesalazine and naringin were suspended in 5% acacia gum (cat. no. V900768-500G; MilliporeSigma; Merck KgaA) and the final doses were 0.2 g/kg and 40 mg/kg, respectively. The dose of 40 mg/kg naringin was selected based on previous studies demonstrating efficacy in rodent models of colitis (17,18). Similarly, the mesalazine dose of 0.2 g/kg is a standard dose used in DSS murine models (22,23). Except for the normal group, all the other groups were freely administered 3% DSS (dissolved in drinking water) to establish the UC mouse model, which was administered alongside other treatments (22,23). The naringin and mesalazine groups received their respective drug suspensions (in 5% acacia gum) by oral gavage once daily for 10 days. The normal and control groups received an equivalent volume of the vehicle (5% acacia gum solution) on the same schedule. Diet and hydration, body movement, body weight, diarrhea incidence and bloody stool were recorded daily throughout the present study.

Disease activity index (DAI) and analysis of colon injury

Body weight, stool character and incidence of bloody stools were recorded daily after models were established. The DAI was determined using a previously established scoring system (Table I) (24). On day 11, mice were anesthetized via inhaled isoflurane (induction, 4%; maintenance, 1.5–2% in oxygen) delivered by a precision vaporizer for final blood collection from the retro-orbital plexus. A terminal blood sample not exceeding 10% of total blood volume (0.6–0.8 ml) was collected. Following this, pentobarbital sodium (40 mg/kg) was administered by intraperitoneal injection to ensure a surgical plane of anesthesia, confirmed by the loss of pedal reflex, before euthanasia was performed via cervical dislocation. This approach guaranteed the absence of pain or distress throughout the procedure. Death was verified by the cessation of cardiac and respiratory activity. All animal procedures were strictly performed in accordance with the approved animal protocol. The colon segment was resected to evaluate the macroscopic colon injury, which was scored according to the method reported in the literature (Table II) (18). Routine hematoxylin and eosin-stained colon sections, according to previously described morphological criteria and observed colon tissue damage, were assessed blindly by two investigators according to a modified histological grading scale, which considered both inflammatory cell infiltration and tissue damage (Table III) (18). Any discrepant scores were resolved through a joint re-evaluation of the slide under a multi-head microscope to reach a consensus. If a consensus could not be reached, a third experienced pathologist was consulted for a final decision.

Table I. Evaluation of DAI scores.

Table I.

Evaluation of DAI scores.

DAI score	Weight loss, %	Stool consistency	Occult or gross bleeding
0	None	None	None
1	1-5	Loose	Hemoccult positive
2	5-10	Loose	Hemoccult positive
3	10-15	Diarrhea	Gross bleeding
4	>15	Diarrhea	Gross bleeding

[i] DAI, disease activity index.

Table II. Evaluation of macroscopic scores.

Table II.

Evaluation of macroscopic scores.

Colon damage	Score
No damage	0
Hyperemia with ulcers	1
Hyperemia and wall thickening without ulcers	2
One ulceration site without wall thickening	3
Two or more ulceration sites	4
0.5 cm extension of inflammation or major damage	5
1 cm extension of inflammation or severe damage	6-10

Table III. Evaluation of histological scores.

Table III.

Evaluation of histological scores.

A, Inflammatory cell infiltration
Pathological characteristic	Score
No infiltration	0
Increased number of inflammatory cells in the lamina propria	1
Inflammatory cells extending into the submucosa	2
Transmural inflammatory cell infiltration	3
B, Tissue damage
Pathological characteristic	Score
No mucosal damage	0
Discrete epithelial lesions	1
Erosion or focal ulcerations	2
Severe mucosal damage with extensive ulceration extending into the bowel wall	3

Western blot analysis for JAK2/STAT3 signaling pathway-related proteins from colon tissues

To determine the protective effect of naringin, western blot analysis was performed. Colon tissues were homogenized in RIPA buffer (cat. no. P0013B; Beyotime Biotechnology) supplemented with 1% (v/v) Protease and Phosphatase Inhibitor Cocktail (cat. no. P1050; Beyotime Biotechnology). Protein concentrations were measured using Cytation 5 (BioTek; Agilent Technologies, Inc.) using a bicinchoninic acid (BCA) protein assay kit (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.). A total of 60 µg protein were mixed with 4X loading dye, as well as Laemmli buffer and 2-mercapto ethanol, and were heated at 95°C for 5 min. The proteins were resolved by 8–12% SDS-PAGE and transferred to immunoblot PVDF membranes (cat. no. IPVH00010; Merck KGaA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature, then incubated at 4°C overnight with primary antibodies against IL-6 (1:1,000; cat. no. Bs-0782R; BIOSS), phosphorylated (p-)JAK2 (1:1,000; cat. no. bs-2485R; BIOSS), JAK2 (1:1,000; cat. no. bs-0908R; BIOSS), p-STAT3 (1:1,000; cat. no. bs-1658R; BIOSS), STAT3 (1:1,000; cat. no. bs-55208R; BIOSS), occludin (1:1,000; cat. no. A2601; ABclonal Biotech Co., Ltd.), zona occludens-1 (ZO-1; 1:1,000; cat. no. A0659; ABclonal Biotech Co., Ltd.) and β-actin (1:1,000; cat. no. ab8227; Abcam). The membranes were washed with TBST three times for 10 min each and incubated with a horseradish peroxidase (HRP)-labeled secondary goat anti-rabbit (1:5,000; cat. no. ab6721; Abcam) antibodies for 1 h at room temperature. After washing three times with TBST, bands were detected using ECL Plus Ultra-Sensitive Substrate (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.) and visualized using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.) after washing three times. The band intensities were semi-quantified using ImageJ v1.53 gel analysis software (National Institutes of Health).

IL-6-induced Caco-2 barrier injury model

Caco-2 cells were cultured in MEM supplemented with 20% FBS, penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (4.5 mg/ml) and glucose (4.5 mg/ml), and incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium was refreshed every other day. A total of 2×106 cells/ml Caco-2 cells were then seeded into a 24-well plate and incubated overnight at 37°C. The following day, the medium was changed to a new medium containing 20 ng/ml IL-6 (cat. no. P05231; Boster Biological Technology) and cells were incubated for 45 min at 37°C. This resulted in the formation of mucosal barrier damage in the Caco-2 cells.

Measurement of transepithelial electrical resistance (TEER) and FD-4 permeability

As described in previously published protocols (18) with slight modification, 1.5×105 cells/ml Caco-2 cells were seeded in Transwell cell culture chambers (6.5 mm diameter inserts; 3.0 mm pore size; Costar; Corning, Inc.), and the growth medium was changed every 2 days. A monolayer of Caco-2 cells was formed after cells were cultured for 10 days. Subsequently, cells were incubated with or without IL-6 (20 ng/ml) for 45 min at 37°C and incubated with naringin (40 µmol/l for 2 h at 37°C. The cell culture was replaced with serum-free medium and incubated for another 30 min at 37°C before the sample TEER (Ω/cm2) was assessed.

After the detection of TEER, 100 ml of 1 mg/ml FD-4 was added into the Transwell upper chambers before the cells were incubated in 37°C for 30 min. After that, 100 ml medium from the lower chamber of the Transwell was added into a black well to detect the fluorescence content at an excitation wavelength of 480 nm and emission wavelength of 520 nm using SpectraMax® M5 (Molecular Devices, LLC) as reported in a previous study (25).

Immunofluorescence assay

Cells were fixed with 4% paraformaldehyde (cat. no. BL539A; Beijing Lanjieke Technology Co., Ltd.) at room temperature for 30 min. After discarding the fixative, the cells were washed with Dulbecco's phosphate-buffered saline and blocked with 5% bovine serum albumin sealing solution at room temperature for 2 h. For immunofluorescence experiments, cells were incubated with the primary antibodies ZO-1 (cat. no. A0659; ABclonal Biotech Co., Ltd.) and occludin (cat. no. A2601; ABclonal Biotech Co., Ltd.) at 1:200 dilution overnight at 4°C after blocking. Cells were then washed with PBS containing 0.1% Tween-20 and subsequently incubated with goat anti-rabbit-Alexa Fluor 488 (1:500; cat. no. 4412S; CST Biological Reagents Co., Ltd.) and goat anti-rabbit-Alexa Fluor 647 (1:500; cat. no. 4414S, CST Biological Reagents Co., Ltd.) fluorescent secondary antibodies at room temperature for 2 h in the absence of light. Finally, cells were counter-stained with DAPI (cat. no. D1306; Thermo Fisher Scientific, Inc.) for 10 min, and the images were collected and analyzed using a laser confocal microscope (FV3000; Olympus Corporation).

Small interfering RNA (siRNA) transfection and western blot analysis in Caco-2 cells

siRNA targeting STAT3 (siSTAT3) and negative control siRNA (siNC) were purchased from Suzhou GenePharma Co., Ltd. The sequences were as follows: siSTAT3, sense 5′-CCCGGAAAUUUAACAUUCUTT-3′, antisense 5′-AGAAUGUUAAAUUUCCGGGTT-3′; and siNC, sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′. Caco-2 cells were transfected with siRNA using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, Caco-2 cells were seeded at a density of 2×106 cells/well in a 6-well plate. In each well, 100 pmol siRNA was complexed with Lipofectamine 3000 reagent. The transfection mixture was incubated with the cells for 6 h at 37°C, after which it was replaced with fresh complete medium. A total of 24 h after transfection, the transfected cells were treated with IL-6 (20 ng/ml) for 45 min at 37°C, followed by naringin (40 µmol/l) treatment for 2 h at 37°C, prior to protein extraction. For protein detection, Caco-2 cells were lysed using ice-cold RIPA buffer (Thermo Fisher Scientific, Inc.) and centrifuged 12,000 × g for 5 min at 4°C. Protein concentrations were determined using a BCA assay (Beyotime Biotechnology). Total proteins (20 mg/lane) were subjected to electrophoresis in a 12% polyacrylamide gel and transferred onto a PVDF membrane. Subsequently, the membrane was blocked at 37°C for 1 h with 5% non-fat milk in TBS, with 0.05% Tween-20. Membranes were subsequently incubated separately with primary antibodies for p-JAK2 (1:1,000; cat. no. bs-2485R; BIOSS), JAK2 (1:1,000; cat. no. bs-0908R; BIOSS), p-STAT3 (1:1,000; cat. no. bs-1658R; BIOSS), STAT3 (1:1,000; cat. no. bs-55208R; BIOSS), occludin (1:1,000; cat. no. A2601; ABclonal Biotech Co., Ltd.), ZO-1 (1:1,000; cat. no. A0659; ABclonal Biotech Co., Ltd.) and β-actin (1:1,000; cat. no. ab8227; Abcam), and then with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; cat. no. ab6721; Abcam). Signals were detected by enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). Western blot images were semi-quantified using ImageJ v1.53 software, Inc.). Relative band intensities were normalized to β-actin.

Statistical analysis

Experiments were performed with multiple independent replicates as indicated in the figure legends (typically, n=8 for in vivo studies and n=3 for in vitro assays). Data are expressed as mean ± SEM. Statistical analyses were performed using SPSS v11.0 (SPSS, Inc.). One-way ANOVA followed by Tukey's post-hoc test was applied. P<0.05 was considered to indicate a statistically significant difference.

Results

Naringin ameliorates clinical symptoms of UC in DSS-induced mice

To evaluate whether oral administration of naringin could ameliorate intestinal damage in UC mice, the present study established a DSS-induced colitis model and assessed clinical and pathological indicators, including body weight, diarrhea, bloody-stool incidence colon length and macroscopic injury. Compared with the normal group, DSS-treated mice in the control group exhibited a notable reduction in body weight (Fig. 1A) and a marked increase in DAI (Fig. 1B). In addition, DSS administration caused significant colon shortening (P<0.01; Fig. 1C and D) and a significant increase in macroscopic injury score (P<0.01; Fig. 1E). Notably, naringin treatment effectively alleviated these pathological changes (P<0.01), indicating its protective effect against DSS-induced UC. The positive control drug, mesalazine, showed a comparable protective efficacy in ameliorating these clinical symptoms (Fig. 1A-E).

Figure 1. Naringin ameliorates clinical symptoms in DSS-induced ulcerative colitis mice. (A) Body weight changes during 10-day DSS exposure. (B) Disease activity index changes during 10-day DSS exposure. (C) Representative colon images; scale bar, 1 cm. (D) Colon length quantification. (E) Macroscopic injury scores. Data are presented as mean ± SEM. n=8. **P<0.01 vs. control group; ##P<0.01 vs. normal group. One-way ANOVA followed by Tukey's post-hoc test was applied. N, normal group; C, control group; Nar, naringin group; Mes, mesalazine group; DSS, dextran sulfate sodium.

Naringin suppresses histological injury in DSS-induced colitis in mice

The present study subsequently evaluated the effects of naringin on colorectal histopathology in mice with DSS-induced UC (Fig. 2A). Compared with in the normal group, DSS-treated mice exhibited severe mucosal damage, including epithelial disruption, erosion, crypt abscesses and extensive inflammatory cell infiltration (primarily neutrophils and lymphocytes). Both naringin and mesalazine treatments markedly ameliorated these pathological features, preserving mucosal and crypt architecture.

Figure 2. Naringin attenuates histological damage in dextran sulfate sodium-induced colitis mice. (A) Representative hematoxylin and eosin-stained colon sections from each group. Scale bar, 100 (overview) and 200 µm (zoomed-in view). (B) Histopathological scores of colon sections from each group. n=8. Data are presented as mean ± SEM. **P<0.01 vs. control group; ##P<0.01 vs. normal group. One-way ANOVA followed by Tukey's post-hoc test was applied. N, normal group; C, control group; Nar, naringin group; Mes, mesalazine group.

Pathological scoring further supported these observations (P<0.01; Fig. 2B). Compared with the normal group, DSS treatment significantly increased histological scores. Treatment with either naringin or mesalazine significantly reduced these scores compared with the DSS group.

Naringin repairs intestinal mucosa and decreases JAK2/STAT3 signaling pathway-related protein expression in DSS-induced colitis in mice

The JAK2/STAT3 signaling pathway is abnormally activated in patients with UC and is known to exacerbate colonic inflammation and compromise mucosal barrier function (26,27). Consistent with this, western blot analysis revealed a significant upregulation of p-JAK2 and p-STAT3, as well as significantly elevated IL-6 expression, in DSS-induced mice compared with the normal group. Notably, treatment with naringin or mesalazine significantly suppressed the activation of JAK2/STAT3, as evidenced by reduced p-JAK2 and p-STAT3 levels, and significantly reduced IL-6 expression, showing a clear difference compared with the model group (P<0.05; Fig. 3B-H).

Figure 3. Naringin inhibits JAK2/STAT3 signaling and restores tight junction proteins in dextran sulfate sodium-induced colitis. (A) Representative western blotting images of p-JAK2, JAK2, p-STAT3, STAT3, IL-6, occludin, ZO-1 and β-actin expression in colon tissues. Densitometric semi-quantification of the relative protein expression levels of (B) p-JAK2/β-actin, (C) JAK2/β-actin, (D) p-JAK2/JAK2, (E) p-STAT3/β-actin, (F) STAT3/β-actin, (G) p-STAT3/STAT3, (H) IL-6/β-actin, (I) occludin/β-actin and (J) ZO-1/β-actin. Data are presented as mean ± SEM. n=3. *P<0.05 and **P<0.01 vs. control group; #P<0.05 and ##P<0.01 vs. normal group. One-way ANOVA followed by Tukey's post-hoc test was applied. N, normal group; C, control group; Nar, naringin group; Mes, mesalazine group; p-, phosphorylated; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; IL-6, ZO-1, zona occludens-1.

Tight junction proteins are important structural components of the intestinal mucosal barrier and are closely linked to the pathogenesis of UC. Among these, ZO-1 and occludin serve important roles in maintaining epithelial integrity (28,29). To further investigate the protective effects of naringin, the present study assessed their expression in colonic tissues. Naringin treatment significantly increased the expression of both ZO-1 and occludin in UC mice, with levels significantly higher than those in the DSS model group (P<0.01; Fig. 3I and J). Similarly, mesalazine treatment significantly upregulated the expression of these tight junction proteins compared with the DSS group. As expected, the expression levels of ZO-1 and occludin in the DSS control group were significantly lower than those in the normal group.

Naringin repairs IL-6-induced barrier injury in Caco-2 cells

The intestinal mucosal barrier is an important component of host defense, and its disruption in patients with UC leads to impaired barrier function and pathological immune activation. A decrease in TEER is widely recognized as an indicator of barrier dysfunction (30,31). To evaluate the protective effect of naringin, the present study established an IL-6-induced barrier injury model in Caco-2 monolayers. After 10 days of culture, confluent Caco-2 cells formed a resistant monolayer with a baseline TEER of 927.9 Ω/cm2. IL-6 stimulation caused a significant decrease in TEER, providing evidence of impaired barrier integrity. Treatment of IL-6 stimulated cells with naringin significantly restored TEER values compared with the IL-6 group, whereas naringin alone did not alter TEER in unstimulated cells compared with the normal group (P<0.01; Fig. 4A).

Figure 4. Naringin restores barrier function in IL-6-induced Caco-2 cells. (A) Transepithelial electrical resistance values over time. (B) Fluorescein isothiocyanate-dextran 4 kDa permeability assay. n=6. Data are presented as mean ± SEM. **P<0.01 vs. IL-6 group; ##P<0.01 vs. normal group. One-way ANOVA followed by Tukey's post-hoc test was applied. Nar, naringin.

To further assess barrier permeability, FD-4 was added to the upper chamber of Transwell inserts. In IL-6-treated cells, FD-4 transport into the lower chamber increased 2.3-fold compared with the normal group, indicating elevated paracellular permeability. Naringin co-treatment significantly reduced FD-4 flux compared with the IL-6 group (P<0.01; Fig. 4B). Together, these results demonstrate that naringin effectively repairs IL-6-induced barrier damage in Caco-2 cells.

Naringin enhances fluorescent expression of tight junction proteins in Caco-2 cells

A previous study has shown that naringin can enhance the mRNA expression of tight junction proteins ZO-1 and occludin in Caco-2/RAW264.7 co-culture systems (18). To further clarify whether the mucosal protective effect of naringin was related to the JAK2/STAT3 signaling pathway, the present study used IL-6 to induce mucosal damage in cells and used STAT3 gene silencing to examine the mechanism of action of naringin. Tight junction protein expression was assessed using immunofluorescence (Fig. 5), and JAK2/STAT3 pathway activation was evaluated using western blot analysis (Fig. 6).

Figure 5. Naringin reverses IL-6-induced ZO-1 and occludin impairment in a STAT3-dependent manner. (A) Immunofluorescence staining for ZO-1 (blue) and occludin (green); nuclei were stained with DAPI (cyan). Merged shows the merged images of DAPI and respective protein staining. Quantitative analysis of relative (B) ZO-1 and (C) occludin protein expression. (n=3). Data are presented as mean ± SEM. *P<0.05 and **P<0.01 vs. IL-6 group; ##P<0.01 vs. normal group. One-way ANOVA followed by Tukey's post-hoc test was applied. ZO-1, zona occludens-1; N, normal group; Nar, naringin; STAT3, signal transducer and activator of transcription 3; si, small interfering RNA; NC, negative control.

Figure 6. Naringin suppresses JAK2/STAT3 activation in IL-6-stimulated Caco-2 cells with STAT3 silencing. (A) Western blot analysis of p-JAK2, JAK2, p-STAT3, STAT3, occludin and ZO-1 in Caco-2 cells under indicated treatments. (B) Validation of STAT3 knockdown efficiency: Relative STAT3 expression in cells transfected with siSTAT3 vs. siNC. ##P<0.01 vs. siNC. Densitometric semi-quantification of relative protein expression levels of (C) p-JAK2/β-actin, (D) JAK2/β-actin, (E) p-JAK2/JAK2, (F) p-STAT3/β-actin, (G) STAT3/β-actin, (H) p-STAT3/ STAT3, (I) occludin/β-actin, (J) ZO-1/β-actin. Data are presented as mean ± SEM. n=3. *P<0.05 and **P<0.01 vs. IL-6 group; ##P<0.01 vs. normal group; ΔP<0.05 and ΔΔP<0.01 IL-6 + Nar group vs. IL-6 + Nar + siSTAT3 group. One-way ANOVA followed by Tukey's post-hoc test was applied. N, normal group; Nar, naringin group; Mes, mesalazine group; p-, phosphorylated; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; ZO-1, zona occludens-1; si, small interfering RNA; NC, negative control.

Immunofluorescence analysis revealed that in the normal group, ZO-1 (blue) and occludin (green) displayed strong, continuous fluorescence signals along cell-cell junctions, reflecting intact tight junctions. IL-6 stimulation significantly reduced the fluorescence intensity of both proteins compared with the normal group (P<0.01), indicating barrier disruption. Naringin treatment significantly alleviated this reduction, restoring ZO-1 (P<0.01) and occludin (P<0.05) expression at cell borders. Notably, the combination of STAT3 silencing and naringin treatment (IL-6 + Nar + siSTAT3 group) also significantly restored the fluorescence intensities of ZO-1 and occludin compared with those in the IL-6 group (P<0.01), to a level comparable with that achieved by naringin alone (Fig. 5). This supports the conclusion that naringin-induced protection of tight junctions is mediated, at least in part, through the inhibition of JAK2/STAT3 signaling (Fig. 5).

Western blot analysis showed that IL-6 stimulation significantly increased the expression of p-JAK2 (P<0.01; Fig. 6C and E) and p-STAT3 (P<0.01; Fig. 6F and H) in Caco-2 cells compared with the normal group, while JAK2 and STAT3 levels remained largely unchanged (P>0.05; Fig. 6D and G). Transfection with siSTAT3 alone significantly reduced total STAT3 protein levels compared with siNC transfection (P<0.01; Fig. 6B), confirming knockdown efficiency. Naringin treatment significantly reduced the expression of p-JAK2 and p-STAT3 relative to the IL-6 group (P<0.05; Fig. 6C, E, F, H). The present study subsequently examined the effect of STAT3 silencing in the context of IL-6 and naringin treatment. Compared with in the IL-6 group, the combination treatment of naringin and STAT3 siRNA (IL-6 + Nar + siSTAT3) led to a significant reduction in both p-JAK2 (P<0.01; Fig. 6C and E) and p-STAT3 levels (P<0.05; Fig. 6F and H).

However, when compared specifically to the IL-6 + Nar group, the combination treatment (IL-6 + Nar + siSTAT3) significantly decreased p-STAT3 levels (P<0.05; Fig. 6H). By contrast, p-JAK2 levels were significantly increased in the IL-6 + Nar + siSTAT3 group relative to the IL-6 + Nar group (P<0.01; Fig. 6E). An increase in total STAT3 protein was observed in both siRNA co-treatment groups (IL-6 + Nar + siSTAT3 and IL-6 + Nar + siNC) compared with that in the normal, IL-6 and IL-6 + Nar groups (P>0.05; Fig. 6G), which may represent a compensatory cellular response. Despite this increase in total STAT3, JAK2/STAT3 pathway activity (i.e., the phosphorylation levels of JAK2 and STAT3, semi-quantified as p-JAK2/JAK2 and p-STAT3/STAT3 ratios) in the IL-6 + Nar + siSTAT3 group remained significantly suppressed compared with in the IL-6 group (P<0.01; Fig. 6E and H). Furthermore, analysis of tight junction proteins showed that naringin treatment (IL-6 + Nar) increased the expression of both occludin and ZO-1 compared with in the IL-6 group (P<0.01; Fig. 6I and J). Notably, the combination treatment with STAT3 silencing (IL-6 + Nar + siSTAT3) differentially affected the two proteins: It further enhanced occludin expression but reduced ZO-1 expression relative to the IL-6 + Nar group. This suggests a complex regulatory role of STAT3 in modulating distinct tight junction components.

Discussion

UC is a chronic, non-specific inflammatory disease of the gastrointestinal tract that primarily affects the colon and rectum, and is commonly characterized by abdominal pain, diarrhea and bloody stool (32,33). Current pharmacological interventions, such as sulfonamides, corticosteroids and immunosuppressive agents, can alleviate UC symptoms; however, high recurrence rates and limited long-term efficacy remain notable clinical challenges (34,35). Consequently, the development of effective strategies for treating and preventing UC has attracted considerable attention globally (34,36). Citrus aurantium L., dried immature orange, possesses both nutritional and medicinal value, with flavonoids representing its principal bioactive constituents. Naringin, the dominant flavonoid, exhibits multiple pharmacological activities (17,37). Previous studies have shown that naringin suppresses jejunal contractions, alleviates intestinal spasms (19), enhances the repair of TNBS-induced intestinal mucosal barrier damage in experimental mice, downregulates inflammatory factors such as iNOS, COX-2 and TNF-α, and preserves cellular permeability and integrity (18) in IBD models. These findings suggest that naringin may exert protective effects in UC, thereby supporting its clinical potential as a food-derived therapeutic agent.

In experimental UC models, the DAI provides a comprehensive measure of disease severity by integrating weight loss, stool consistency and presence of blood (38,39). Colon shortening is another hallmark of DSS-induced UC and serves as a reliable morphological indicator of disease severity (33,40). In the present study, DSS administration markedly increased DAI scores and significantly reduced colon length compared with the normal group. Naringin treatment ameliorated these parameters, indicating a protective effect. Histopathological analysis further revealed that naringin alleviated epithelial necrosis, goblet cell loss, crypt destruction and inflammatory infiltration in DSS-treated mice.

Mechanistically, the present study demonstrated that naringin protected against UC by inhibiting the JAK2/STAT3 signaling pathway and enhancing intestinal barrier integrity. Tight junction proteins such as ZO-1 and occludin are important for barrier function, and their disruption leads to increased permeability, microbial translocation and sustained inflammation (8–11). The data of the present study showed that naringin upregulated ZO-1 and occludin expression while suppressing phosphorylation of JAK2 and STAT3 in colon tissue and IL-6-stimulated Caco-2 cells. These findings suggest that naringin restores intestinal barrier integrity through JAK2/STAT3 inhibition.

The JAK2/STAT3 signaling pathway serves a dual role in UC. In inflamed intestinal tissues of patients with UC, this pathway is excessively activated and serves as a key driver of disease progression. However, in healthy intestinal epithelial cells, the activation of STAT3 can promote the repair of the intestinal mucosal barrier (15,23,41). The present study focused on UC mouse intestinal tissue and Caco-2 cells, which are fundamentally different from intestinal epithelial cells; thus, the JAK2/STAT3 signaling pathway observed in Caco-2 primarily reflected its role in promoting inflammatory reactions. This interpretation was consistent with multiple previous reports (22,42,43). ZO-1 and occludin are important proteins in the tight junction structures of the intestinal barrier, and their expression and distribution are usually used to assess the function of the intestinal barrier in the colon (12–14). The present data demonstrated that naringin upregulated ZO-1 and occludin expression while suppressing phosphorylation of JAK2 and STAT3, highlighting its potential as a dietary intervention for UC. Future studies are warranted to explore the efficacy of the clinical translation of naringin.

JAK2 is an important tyrosine protein kinase that serves an important role in the inflammatory cascade of UC, while STAT3 is a key transcription factor involved in cell proliferation, differentiation, apoptosis and the immune and inflammatory response (44). Accumulating evidence indicates that activation of the JAK2/STAT3 pathway exacerbates UC symptoms (45,46). In the present study, IL-6 was selected to induce mucosal barrier damage and inflammatory responses in Caco-2 cells, as it is a primary activator of the JAK2/STAT3 pathway (15,45). Although lipopolysaccharide is commonly used to model inflammation, it primarily activates Toll-like receptor 4/NF-κB signaling; therefore, IL-6 was more appropriate for studying JAK2/STAT3-specific effects. Furthermore, IL-6 is elevated in patients with UC and contributes to barrier dysfunction. By silencing the STAT3 gene the protective effect of naringin on this pathway was explored. IL-6 binds to its soluble receptor, forms a complex with membrane-bound glycoprotein 130, activates JAK2, induces STAT3 phosphorylation and activates downstream transcription factors, regulating the expression of inflammatory cytokines and thus mediating the occurrence of inflammatory responses (15).

The results of the present study showed that naringin significantly reduced the levels of p-JAK2 and p-STAT3 in the colon tissue of UC mice and inhibited JAK2/STAT3 activation in IL-6-stimulated Caco-2 cells. The enhanced suppression of pathway activation in the IL-6 + Nar + siSTAT3 group compared with the IL-6 group confirmed the involvement of the JAK2/STAT3 pathway in mediating the effects of naringin. Although the combination treatment (IL-6 + Nar + siSTAT3) reduced p-STAT3 compared with that in the IL-6 + Nar group, it did not lead to a further reduction in p-JAK2 levels, indicating a complex interaction. Nevertheless, the significant reduction in both p-JAK2 and p-STAT3 in the IL-6 + Nar + siSTAT3 group compared with the IL-6 group underscores that the therapeutic action of naringin is mediated, at least in part, through the suppression of JAK2/STAT3 signaling. Notably, in the siRNA-treated groups (IL-6 + Nar + siSTAT3 and IL-6 + Nar + siNC) under IL-6 stimulation, an increase in total STAT3 protein was observed, which may represent a compensatory cellular response to STAT3 knockdown. Furthermore, despite this increase in total STAT3 protein, the combination treatment (IL-6 + Nar + siSTAT3) still achieved notable suppression of JAK2/STAT3 phosphorylation compared with the IL-6 group. This effective uncoupling of pathway activation from total STAT3 protein levels underscored that the therapeutic benefit of naringin was achieved through suppressing the activation of the JAK2/STAT3 pathway.

Laser confocal microscopy revealed that IL-6 stimulation significantly decreased ZO-1 and occludin expression in Caco-2 cells, whereas naringin treatment significantly restored their expression. This restorative effect persisted even after STAT3 silencing, reinforcing the conclusion that naringin enhances tight junction protein expression primarily through JAK2/STAT3 inhibition, thereby repairing the intestinal mucosal barrier and exerting therapeutic effects in UC.

Inflammatory responses were evaluated indirectly through histological scoring of inflammatory cell infiltration and directly through activation assessment of the JAK2/STAT3 pathway, which functions upstream of multiple pro-inflammatory cytokines. However, direct measurement of cytokine levels, such as those of TNF-α and IL-1β, would provide additional validation. The effects of naringin on other inflammatory factors require further elucidation in subsequent studies.

Although JAK2 inhibition would provide more comprehensive mechanistic insights, experimental focus was placed on STAT3 due to resource constraints. Previous investigations on Citrus aurantium (17,18) have demonstrated inhibitory effects on JAK2 downstream pathways, including key inflammatory factors such as TNF-α and NF-κB. Given that naringin represents a major active component of Citrus aurantium (47), these findings provide biological plausibility for the approach in the present study. The direct targeting of JAK2 by naringin requires further investigation in future studies. Additionally, while naringin may potentially interact with other inflammatory pathways such as the NF-κB, MAPK or NACHT, LRR and PYD domains-containing protein 3 pathways, the present experimental design specifically focused on the JAK2/STAT3 axis based on prior evidence from Citrus aurantium (17,18). These limitations indicate important directions for future research. Studies employing molecular docking or surface plasmon resonance could strengthen mechanistic claims and are recommended for subsequent investigations. Further studies are warranted to explore additional mechanisms and validate direct molecular targets of naringin with the ultimate goal of comprehensively elucidating its therapeutic potential for UC.

In summary, integrated in vitro and in vivo evidence indicates that naringin, a flavonoid from the edible fruit Citrus aurantium L., significantly ameliorates DSS-induced UC in mice. Its therapeutic effects are mediated through suppression of the JAK2/STAT3 signaling pathway, leading to both anti-inflammatory effects and enhanced tight junction protein expression. These findings support the potential of naringin as a food-based intervention for UC and provide a molecular basis for further mechanistic investigation. Although the JAK2/STAT3 pathway serves a notable role in UC pathogenesis, the precise mechanisms underlying naringin-induced modulation of this pathway require further elucidation.

Acknowledgements

Not applicable.

Funding

The present research was supported by the Hebei Youth Fund for Science and Technology Research in Colleges and Universities (grant no. QN2022014).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

WH was responsible for funding acquisition, experimental design, conceptualization, writing the original draft and supervision. YaW contributed to the experimental design, assisted in the interpretation of data and critically revised the manuscript for important intellectual content. MW was contributed towards writing the original draft, reviewing and editing the manuscript, formal analysis and data curation. YA contributed to writing the original draft, reviewing and editing the manuscript, formal analysis and data curation. YL contributed towards experimental design and formal analysis. YiW was responsible for validation and data curation. CW contributed towards validation and formal analysis. MW and YA confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Animal experiments were approved by the Animal Welfare Committee of Hebei North University (approval no. HBNV20240223003) and performed according to institutional guidelines.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References