Abstract
Objective:
Rheumatoid arthritis (RA) is a chronic autoimmune disease with limited treatment options and associated side effects or resistance. This study aims to investigate the therapeutic potential of the natural compound cucurbitacin B (CuB) in RA treatment.
Methods:
We utilized a collagen-induced arthritis (CIA) mouse model to evaluate the effects of CuB. Arthritis scores, histological damage, and pro-inflammatory cytokine expression (TNF-α, IL-17A) were assessed. In addition, network pharmacology analysis was performed to explore CuB’s molecular mechanisms, focusing on Th17 cell differentiation, IL-17 signaling, and the JAK-STAT pathway.
Results:
CuB significantly reduced arthritis severity, decreased histological damage, and lowered the expression of pro-inflammatory cytokines in CIA mice. CuB was found to inhibit STAT3 phosphorylation and reduce the proportion of Th17 cells in the spleen, indicating its potential anti-inflammatory effects.
Conclusion:
These findings suggest that cucurbitacin B may serve as a promising novel therapeutic agent for rheumatoid arthritis by targeting key inflammatory pathways.
Keywords: anti-inflammatory therapy, cucurbitacin B, JAK/STAT pathway, rheumatoid arthritis (RA), Th17 cell differentiation
Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily affects the joints, causing pain, swelling, and functional impairment. 1 The pathogenesis of rheumatoid arthritis (RA) involves multiple T cell subsets, among which T helper 17 (Th17) cells are key contributors to chronic inflammation and joint damage. Th17 cells secrete interleukin-17 (IL-17), which further stimulates the production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and matrix metalloproteinases (MMPs), exacerbating synovial inflammation and accelerating cartilage destruction.1–3 Compared to T helper 1 (Th1) cells, Th17 cells play a dominant role in the early stages of RA development, while Th1 cells have a more limited impact.4,5
Given the pivotal role of Th17 cells and IL-17 in the pathogenesis of rheumatoid arthritis (RA), the Th17/IL-17 axis has emerged as a significant therapeutic target in autoimmune and inflammatory diseases. Although IL-17A inhibitors, such as secukinumab, have been approved for the treatment of psoriatic arthritis and non-radiographic axial spondyloarthritis, clinical trials in RA have not shown superior efficacy compared to IL-6 inhibitors or rituximab following anti-TNF therapy failure. 6 As a result, IL-17 blockade has not been approved for RA treatment. Nevertheless, due to its central involvement in RA pathophysiology, targeting the Th17/IL-17 axis remains a promising avenue for future therapeutic strategies.
Despite the availability of various treatments, the therapeutic outcomes remain unsatisfactory for some patients, and some treatments may come with adverse effects or the development of drug resistance. 7 Therefore, finding new treatment methods and developing drugs with high efficacy and low toxicity have become important directions in current medical research. 8 In this context, natural products have become a widely focused research direction. 9 Traditional Chinese medicine (TCM) is a potential source of various active natural components with anti-inflammatory, anti-tumor, and immune-regulating properties. 10 TCM may play significant roles in treating different diseases and may also be a potential source of drugs for treating RA. 11
Cucurbitacin B (CuB), a natural compound, has demonstrated significant anti-inflammatory12,13 and immunomodulatory effects. 14 It has been shown to suppress nuclear factor-κB (NF-κB) and NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis, inhibiting interleukin-1β (IL-1β) in various inflammatory diseases.12,15 CuB has been tested as an NLRP3 inflammasome inhibitor for treating gouty arthritis and osteoarthritis.15,16 Moreover, CuB also influences autoimmune responses in multiple sclerosis (MS) by reducing STAT3 activation, inhibiting microgliosis, and modulating the IL-23/IL-17 axis. 16 Given its role in immune regulation, inflammation suppression, and therapeutic potential for other inflammatory and autoimmune diseases, we are particularly interested in exploring CuB’s potential as a treatment for rheumatoid arthritis (RA). However, there is a lack of experimental evidence regarding its effects on RA, making this study of both theoretical and clinical significance.
This study aims to explore the anti-arthritis efficacy of CuB using an experimental mouse model of arthritis for the first time. Additionally, we further investigate the potential mechanisms of action through network pharmacology and cellular experimental platforms on different cell lines, providing new insights and methods for RA treatment.
Materials and methods
Mice
Six to eight-week-old female DBA/1 mice (18–20 g), obtained from the Jackson Laboratory (Jackson Laboratory, Bar Harbor, ME, USA), were utilized for conducting collagen-induced arthritis (CIA) experiments. All mice were housed following the ethical standards of the Institutional Animal Care and Use Committee (IACUC) of the hosting institution. Each cage contained a maximum of six mice, living under standard light and temperature conditions, with free access to standard laboratory chow.
Collagen II-induced arthritis
Chicken collagen type II (CII) (Sigma-Aldrich, St. Louis, MI, USA) is dissolved at a concentration of 2 mg/mL in 0.1 M acetic acid, then mixed with an equal volume of complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MI, USA) to form an emulsion. Intradermal injections are performed at the base of the mouse’s tail, administering 100 µL of the emulsion per mouse to induce arthritis. After 21 days, a booster immunization is conducted by injecting an emulsion containing 100 µg of CII in incomplete Freund’s adjuvant. The entire experiment is completed within 6 to 8 weeks.
CuB treatment
The CIA mice were randomly divided into four groups, with 10 mice in total per group divided into two independent trials (n = 10 per group). Cucurbitacin B (CuB; MCE, Monmouth Junction, NJ, USA) treatment was initiated on day 21 after CIA induction, coinciding with the booster immunization. Treatment was administered simultaneously to all animals at this fixed time rather than initiated based on individual clinical arthritis scores. From day 21 to day 42, doses of 0.5 mg/kg and 1 mg/kg CuB dissolved in a 95% (v/v) olive oil and dimethyl sulfoxide (DMSO) mixture were administered daily via oral gavage. The control group received only the 95% olive oil and DMSO mixture without CuB. Additionally, methotrexate (MTX; 10 mg/kg, intraperitoneal injection; MCE, Monmouth Junction, NJ, USA) was administered as the positive control
Clinical evaluation of arthritis
After boosting immunity, all DBA/1 mice are checked every 2–3 days to assess the presence of arthritis. To evaluate the severity of arthritis, a clinical scoring system from 0 to 3, which is adapted from previous literature, is used for each mouse’s paws.17–19 A score of 0 indicates no signs of inflammation, a score of 1 indicates mild swelling or erythema or both, a score of 2 indicates moderate swelling and erythema, and a score of 3 indicates severe swelling and erythema. The arthritis index for each mouse comprised the total score from all four paws. To minimize bias, all scoring was performed by trained researchers under blind conditions, following previously validated methodologies.
Histological examination
On the 42-day post-initiation of the experiment, the hind limbs of the euthanized mice were collected and preserved in 10% formalin solution. After fixation, tissue sections were prepared and subjected to hematoxylin and eosin (H&E) staining (Merck, Melbourne, VIC, Australia). These sections were then examined using a light microscope (Olympus, Tokyo, Japan) at 40× magnification for an overview and 100× magnification for detailed analysis. The evaluation of histological alterations included cellular infiltration, synovial hyperplasia, and cartilage degradation. Each parameter was assessed using a scoring system ranging from 0 to 3, where 0 indicated no observable changes, 1 represented mild changes, 2 denoted moderate changes, and 3 showed severe changes, as referenced in multiple previous studies related to CIA.
Cytokine production measured by enzyme-linked immunosorbent assay (ELISA)
On day 42, the hind paws of the mice were collected. Initially, tissues were homogenized using T-PER Tissue Protein Extraction Reagent (Thermo Fisher, Waltham, MA, USA) combined with UltraCruz® Protease Inhibitor Cocktail (MCE, Monmouth Junction, NJ, USA) at a ratio of 50 mg tissue per mL. Proteins were then isolated. The concentrations of proteins were assessed using the Micro BCA™ Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Subsequently, the protein levels of TNF-α, IL-6, IL-17A, and IL-10 were measured using specific ELISA kits (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. T cells were cultured in the Th17 cell culture medium with or without CuB for 72 hours, and then cytokine levels, including IL-17A, were measured using ELISA (BioLegend, San Diego, CA, USA).
Construction of protein-protein interaction (PPI) network
The targets of CuB were sourced from various databases, including the Comparative Toxicogenomics Database (CTD), the Drug Gene Interaction Database (DGID), the Encyclopaedia of Traditional Chinese Medicine (ETC), SwissTargetPrediction, Supertarget, TargetNet, and the Traditional Chinese Medicine Database and Analysis Platform (TCMSP). As referenced in a previous study, 20 the RA treatment targets were gathered from Genecards, Drugbank, DisGeNET, OMIM, PharmGKB, and the Therapeutic Target Database. The common targets between CuB and RA were identified and analyzed using the STRING database (https://www.string-db.org/) with the species set to “Homo sapiens”. Only targets with a “Medium confidence” score above 0.4 were displayed, and the combined scores were calculated and saved. Additionally, the common target genes of CuB and RA were visualized using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/), and the intersected target genes were considered candidate target genes. The data, in TSV format, was imported into Cytoscape 3.8.2 software (Cytoscape, San Diego, CA, USA) for PPI network analysis. Within Cytoscape, the CytoHubba plugin was used to identify the top 10 most relevant genes based on their connectivity.
The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
The candidate targets underwent KEGG pathway functional enrichment analyses through the DAVID online platform (https://david.ncifcrf.gov/summary.jsp). For visualization, the OmicShare web-based tool (http://www.omicshare.com/tools/Home/Index/index.html) was employed. A diagram was created to display the top 20 immune-related pathways (P < 0.05).
Th17 cell differentiation in vitro
Following the manufacturer’s instructions, CD4+ T cells were isolated from the spleens of female DBA/1 mice using magnetic bead sorting and a mouse primary CD4+ T cell isolation kit (Miltenyi Biotec, CA, USA). These cells were then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (MCE, Monmouth Junction, NJ, USA).
To induce Th17 differentiation, naïve CD4+ T cells were stimulated with 1 μg/mL of plate-bound anti-CD3 (Acro, Newark, NJ, USA) and 1 μg/mL of soluble anti-CD28 (Invitrogen, Waltham, MA, USA), along with 50 ng/mL of IL-6 (Invitrogen, Waltham, MA, USA), and 10 ng/mL of transforming growth factor beta 1 (TGF-β1) (Acro, Newark, NJ, USA). In addition, 10 μg/mL of anti-interferon-γ (IFN-γ) antibody (Invitrogen, Waltham, MA, USA) and 10 μg/mL of anti-IL-4 antibody (Invitrogen, Waltham, MA, USA) were added. CuB was administered at the indicated dose at the start of induction. Following a 72-hour culture period, the cells were analyzed using flow cytometry and quantitative RT-PCR.
RNA isolation and real-time RT-PCR
Total RNA was extracted from stimulated CD4+ T cells using TRIzol (EBL, Taipei, Taiwan) according to the manufacturer’s instructions. cDNA was then synthesized using the Superscript III reverse transcriptase system (Invitrogen, Waltham, MA, USA). Following the addition of SYBR Green Master Mix (ABclonal, Woburn, MA, USA), RT-qPCR was performed using the AB 7500 Fast System (Applied Biosystems, Waltham, MA, USA). The primer sequences used are as follows: mouse IL-17A, forward 5′-TTTTCAGCAAGGAATGTGGA-3′ and reverse 5′-TTCATTGTGGAGGGCAGAC-3′; mouse retinoic acid-related orphan receptor (ROR)γt, forward 5′-CCGCTGAGAGGGCTTCAC-3′ and reverse 5′-TGCAGGAGTAGGCCACATTACA-3′; and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-GGCATCCTGGGCTACACTGA-3′ and reverse 5′-GGAGTGGGTGTCGCTGTTG-3′.
Flow cytometry
Under Th17 polarizing conditions, isolated T cells were placed in 96-well plates and treated with or without CuB for 72 hours. Subsequently, during the final 4 hours, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) (MCE, Monmouth Junction, NJ, USA), ionomycin (500 ng/mL) (Sigma-Aldrich, St. Louis, MI, USA), and GolgiStop (BD Biosciences, Franklin Lakes, NJ, USA). Next, CD4+ T cells were stained at 4°C with PerCP/Cyanine 5.5-conjugated anti-CD4 antibody (Invitrogen, Waltham, MA, USA) for 20 minutes in the dark. Following this, cells were fixed and permeabilized using the Cytofix/Cytoperm Plus kit (BD Biosciences, Franklin Lakes, NJ, USA) for intracellular staining. The cells were then stained with PE-conjugated anti-IL-17A antibody (Invitrogen, Waltham, MA, USA) and incubated for 30 minutes at room temperature in the dark. The Th17 cell subset was identified by recognizing CD4 and IL-17A double-positive cells, and the mean fluorescence intensity of these cells was quantified using an Accuri C5 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Spleen cells were extracted from mice using a spleen dissociation kit (Miltenyi Biotec, Gaithersburg, MD, USA) and suspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 50 μg/mL gentamicin, 2 mM L-glutamine, and 50 μM 2-mercaptoethanol. And then, these cells were stimulated with 50 μg/mL of bovine CII for 72 hours. After stimulation, the cell surface was stained with PerCP/Cyanine 5.5-labeled anti-CD4 antibody. Intracellular staining was performed using PE-labeled anti-IL-17A antibody. Finally, the stained cells were analyzed on an Accuri C5 flow cytometer to determine their phenotypes.
Western blot analysis
Under Th17 polarizing conditions, T cells, either treated with CuB or untreated, were lysed using lysis buffer (Visual protein, Taipei, Taiwan), and total protein was extracted and quantified using the BCA Protein Assay Kit (Visual protein, Taipei, Taiwan). The cell lysates were then transferred onto PVDF membranes, which were incubated overnight at 4°C with the following primary antibodies: anti-phosphorylated STAT3 (Cell Signaling, Danvers, MA, USA), anti-STAT3 (Cell Signaling, Danvers, MA, USA), anti-phosphorylated JAK2 (Abcam, Cambridge, UK), anti-JAK2 (Abcam, Cambridge, UK), and anti-GAPDH (Cell Signaling, Danvers, MA, USA). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA), followed by the addition of LumiFlash Ultima chemiluminescent substrate (Visual protein, Taipei, Taiwan; LF08-500) for detection. The emitted light was detected using the Hansor Luminescence Imaging System (Taichung, Taiwan). Finally, band densities were evaluated using ImageJ 1.47 (Bethesda, MD, USA).
CuB toxicity experiment in mice
To analyze the toxic effects, we determined the safe dosage of CuB in normal DBA/1 mice. The experiment utilized 6- to 8-week-old DBA/1 mice, which were randomly assigned to two administration routes: oral (Oral) and intraperitoneal injection (i.p.). In the oral group, mice were allocated to Vehicle, 0.5, 1, 5, and 10 mg/kg CuB dose groups, consisting of five mice in each group (n = 5). In the i.p. group, mice were assigned to vehicle, 0.5, and 1 mg/kg CuB dose groups, with five mice in each group (n = 5). Each mouse received an administration of 100 μL of CuB or Vehicle daily for 14 consecutive days. The survival of each mouse was monitored and recorded daily.
To evaluate the effects of CuB on hematology and organ function in DBA/1 mice, a complete blood count (CBC) and multiple blood biochemical analyses were performed. On day 14, blood samples were collected via orbital blood sampling and cardiac puncture. Whole blood samples were treated with EDTA as an anticoagulant and analyzed using an automated hematology analyzer to measure key CBC parameters, including white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), and platelets. The serum was separated by centrifugation and subjected to biochemical analysis to assess the liver function including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), kidney function including blood urea nitrogen (BUN) and creatinine, cardiac function including creatine kinase (CK) and lactate dehydrogenase (LDH), inflammatory markers (C-reactive protein, CRP), and pancreatic function (amylase, lipase).
Power analysis
To evaluate the adequacy of our sample size, we performed a post hoc power analysis using G*Power (version 3.1.9.7). The analysis was based on a one-way ANOVA (fixed effects, omnibus) comparing five independent groups. The parameters used were: effect size (f) = 0.4 (moderate-to-large), α = 0.05, total sample size = 50 (n = 10 per group), and number of groups = 5.
Statistical analysis
All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism v8.0 (GraphPad Software, Tampa, FL, USA), and a P-value less than 0.05 was considered statistically significant. Comparisons between two groups were conducted using a two-tailed unpaired Student’s t-test. For mouse weight and arthritis scores, a two-way repeated measures ANOVA was performed to assess the effects of treatment and time. Post hoc multiple comparisons were conducted using Tukey’s test on the main impacts to compare group means across treatment and time. For other animal and cell-based experiments involving more than two groups, one-way ANOVA was performed, followed by Dunnett’s post hoc test to compare treatment groups with the control. Survival rates were calculated as the percentage of mice surviving each day. Survival analysis was performed using Kaplan–Meier curves, and statistical differences between groups were assessed using the log-rank test.
Result
The inhibitory effect of cucurbitacin B (CuB) on the symptoms of collagen-induced arthritis (CIA) in mice
We utilized the CIA mouse model to evaluate the therapeutic effects of CuB (Figure 1a) on RA progression, as depicted. The mice were administered daily doses of CuB at 0.5 and 1 mg/kg in two separate groups, and received the current RA medication, MTX, at a dose of 10 mg/kg. The clinical RA scores were regularly assessed following immunization with type II collagen (CII). While no significant changes in body weight were observed (Figure 1b), the arthritis scores in both the 0.5 mg/kg and 1 mg/kg CuB-treated mice were lower than those in the vehicle-treated group and comparable to those in the 10 mg/kg MTX-treated mice (Figure 1c). It was noted that only in the later stages, approximately from day 39 to day 42, did the 1 mg/kg CuB and MTX groups show a relatively better suppression of arthritis compared to the 0.5 mg/kg CuB group. However, statistically, there were no significant differences among the three groups.
Figure 1.
Effects of cucurbitacin B on CIA symptoms in mice. (a) Chemical structure of cucurbitacin B (CuB). (b) Body weights of normal mice and CIA-induced mice treated with vehicle, methotrexate (MTX, 10 mg/kg), or CuB (0.5 mg/kg and 1 mg/kg) were monitored periodically up to day 42. (c) Arthritis scores were assessed using a visual scoring system, with higher scores indicating more severe symptoms.
Each point on the graph represents the mean ± SD of ten mice. Statistical analyses were performed using two-way ANOVA followed by Tukey’s HSD post hoc test.
*P < 0.05 and ***P < 0.001 versus the vehicle-treated CIA mice group.
Additionally, histological examination of the ankle joints in mice revealed that CIA mice exhibited joint symptoms such as significant infiltration of inflammatory cells into the joint tissues, synovial hyperplasia, and cartilage erosion, which were not seen in normal mice. However, the histological scores of CIA mice treated with 0.5 and 1 mg/kg CuB were significantly reduced (Figure 2a and b).
Figure 2.
Histological analysis of ankle joints in CIA-induced mice treated with cucurbitacin B. (a) Representative histological images of ankle joint sections from normal mice, CIA-induced mice treated with vehicle, and CIA-induced mice treated with methotrexate (MTX, 10 mg/kg) or cucurbitacin B (CuB) at doses of 0.5 mg/kg and 1 mg/kg. Hematoxylin and eosin staining were used to evaluate joint tissue pathology. Low-magnification images were captured at 40×, and higher-magnification images at 100×. (b) Quantitative histological scores for inflammatory cell infiltration, synovial hyperplasia, and cartilage erosion.
Each bar represents the mean ± SD for five mice per group. Statistical analyses were performed using oneway ANOVA, followed by Dunnett’s post hoc test to compare each treatment group with the vehicle-treated CIA mice group.
*P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant compared to the vehicle-treated CIA mice group.
CuB reduced the expression of inflammatory cytokines in paw tissues
Next, we assayed the major pro- and anti-inflammatory cytokines, including TNF-α, IL-6, IL-17A, and IL-10, in the paw tissues of CIA mice treated with CuB, MTX, or vehicle. As shown in Figure 3, the ELISA data consistently demonstrated that the levels of TNF-α and IL-17 cytokines were significantly reduced in CuB-treated mice, bringing these cytokine levels down to those comparable with the normal control and MTX-treated groups. In contrast, IL-10 levels were significantly increased only in the MTX group (P < 0.05). Neither the 0.5 mg/kg nor the 1 mg/kg CuB treatment groups showed a statistically significant change in IL-10 expression compared with the vehicle control. These findings suggest that, although CuB did not enhance IL-10 production, it effectively alleviated joint inflammation primarily by suppressing pro-inflammatory cytokines, supporting its overall therapeutic efficacy in the CIA model.
Figure 3.
Effects of cucurbitacin B on cytokine levels in paw tissues of CIA-induced mice. Levels of cytokines TNF-α, IL-6, IL-17A, and IL-10 in paw tissue homogenates were measured by ELISA on day 42 in each group, with results expressed in pg/100 mg tissue. Data are presented as means ± SD for five mice per group.
Each bar represents the mean ± SD for five mice per group. Statistical analyses were performed using one-way ANOVA, followed by Dunnett’s post hoc test to compare each treatment group with the vehicle-treated CIA mice group.
*P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant compared to the vehicle-treated CIA mice group.
CuB and RA potential target genes and network pharmacology analysis
Using GeneCards and DisGeNET, a total of 2053 RA-related target genes were identified. In comparison, 250 CuB-related target genes were obtained from the Comparative Toxicogenomics Database, DGID (The Drug Gene Interaction Database), ETC (Encyclopedia of Traditional Chinese Medicine), SwissTargetPrediction, Supertarget, TargetNet, and the Traditional Chinese Medicine Database and Analysis Platform (TCMSP). Using VENNY 2.1 for cross-analysis of RA- and CuB-related genes, 134 candidate common target genes were identified (Figure 4a). These 134 candidates were imported into the STRING database to create a PPI network, which depicted the interaction relationships between these target genes, with different colored edges representing various types of interactions. The network was then created and its topological properties were evaluated using the STRING database. Subsequently, the STRING analysis results were imported into Cytoscape 3.8.2 to visualize the interactions between the target genes (Figure 4b). The top ten key target genes were determined based on the “Degree” ranking in CytoHubba (Figure 4c). Subsequently, 20 immune system-related pathways were identified by KEGG analysis (P < 0.05) (Figure 5). Many of these pathways have been reported to be associated with RA symptoms.
Figure 4.
Screening of potential gene targets for cucurbitacin B (CuB) in RA. (a) Venn diagram showing 134 common genes associated with RA and CuB. (b) Protein-protein interaction (PPI) network of the 134 shared target genes, with different colored edges representing various interaction types. (c) The top 10 key genes identified based on the “Degree” ranking, including TNF, IL1B, STAT3, and others, were visualized for further analysis.
Figure 5.
KEGG pathway enrichment analysis of potential target genes associated with RA. The KEGG pathway analysis on the left displays 20 enriched immune-related pathways (P < 0.05), with the enrichment scores represented as −Log10 (Q value). Key pathways include the Toll-like receptor, Chemokine, and T cell receptor signaling pathways associated with RA pathogenesis. Notably, three pathways are highlighted explicitly due to their relevance: Th17 cell differentiation (enrichment score 3.91), JAK-STAT signaling pathway (enrichment score 2.88), and IL-17 signaling pathway (enrichment score 2.09), all of which play critical roles in modulating immune responses and inflammatory processes in RA. On the right, the bubble plot illustrates pathway enrichment scores with bubble size representing the number of genes covered by each pathway out of the 134 overlapping genes, and color intensity indicating statistical significance.
CuB inhibits IL-17 production by CD4 T cells under Th17-polarizing conditions
Previous studies have shown that CuB can modulate the IL-23/IL-17 axis. 16 Furthermore, through network pharmacology and KEGG analysis, we found that CuB influences Th17 cell differentiation, the IL-17 signaling pathway, and the JAK-STAT pathway. Based on these findings, we further investigated the effect of CuB on Th17 cell differentiation in Th17-polarized CD4 T cells from DBA/1 mice. Compared to the untreated control group, the mRNA expression of IL-17 and RORγt, IL-17 secretion, and the proportion of CD4+ IL-17+ T cells were significantly induced under Th17-polarizing conditions. However, the inhibition of these increases is dose-dependent on CuB (Figure 6a–d; Supplemental Figure 1). These results suggest that CuB can prevent the differentiation of naive T cells into Th17 cells.
Figure 6.
CuB Suppresses STAT3 Phosphorylation and Th17 Cell Differentiation. CD4+ T cells from DBA/1 mice were cultured under Th17-polarizing conditions with or without CuB (50 nM or 100 nM) for 72 hours. (a) CuB reduced IL-17 mRNA expression in a dose-dependent manner. (b) IL-17 protein levels in the culture medium decreased with increasing CuB concentration. (c) Flow cytometry showed a dose-dependent reduction in IL-17A+ Th17 cells. (d) CuB inhibited RORγt mRNA expression, a key transcription factor for Th17 differentiation, in a dose-dependent manner. (e) Western blot analysis of phosphorylated STAT3 (p-STAT3, Tyr705) and JAK2 (p-JAK2, Tyr1007/1008) in Th17-polarized CD4+ T cells treated with CuB (100 nM) for 48 and 72 hours. Vinculin was used as a loading control.
Data are presented as the mean ± SD of three wells from one of three experiments. Statistical analysis for (a–d) was performed using one-way ANOVA followed by Dunnett’s post hoc test (compared to Th17-polarizing condition without CuB). Statistical significance in (e) was determined using Student’s t-test.
*P < 0.05, **P < 0.01, and ***P < 0.001 versus Th17-polarizing condition without CuB treatment.
CuB treatment inhibits STAT3 signaling
Since IL-6-induced JAK2/STAT3 signaling plays a key role in Th17 cell differentiation, 21 we further investigated whether CuB regulates Th17 cell differentiation by affecting JAK2/STAT3 phosphorylation (specifically pSTAT3, Tyr705, and pJAK2, Tyr1007). The results showed that in Th17-polarized CD4 T cells, the expression levels of pSTAT3 and pJAK2 were elevated. However, CuB significantly inhibited the phosphorylation of STAT3 without obviously affecting the expression of JAK2 (Figure 6e). This suggests that the mechanism of CuB in Th17 cell differentiation primarily involves inhibiting STAT3 phosphorylation through a pathway independent of JAK2.
CuB decreases the percentage of Th17 cells in the spleens of CIA mice
Finally, we aimed to confirm whether CuB has the same inhibitory effect on Th17 cell differentiation in the CIA mouse model. Therefore, we evaluated the distribution of CD4+ T cell subsets in the spleens of CIA mice. The results showed that the proportion of CD4+IL-17A+ Th17 cells was significantly reduced after CuB treatment (Figure 7a; Supplemental Figure 2). Additionally, the RNA expression level of RORγt in the spleens of CIA mice was significantly reduced after CuB treatment (Figure 7b). Taken together, these results indicate that CuB can reduce the number of Th17 cells in the spleens of CIA mice.
Figure 7.
Cucurbitacin B (CuB) inhibits Th17 cell differentiation in the spleens of CIA mice. (a) Proportion of CD4+ IL-17A+ Th17 cells in the spleen gated from total lymphocytes in CIA mice treated with vehicle, methotrexate (MTX, 10 mg/kg), or CuB at 0.5 mg/kg and 1 mg/kg. (b) Relative mRNA expression levels of RORγt, a transcription factor critical for Th17 cell differentiation, in the spleens of CIA mice.
Each bar represents the mean ± SD for five mice per group. Statistical analyses were performed using one-way ANOVA, followed by Dunnett’s post hoc test to compare each treatment group with the vehicletreated CIA mice group.
*P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant compared to the vehicle-treated CIA mice group.
Discussion
CuB is a highly researched bioactive compound that plays a key role in innate and adaptive immunity. It is known for its broad pharmacological effects, including controlling immune proteins, reducing oxidative stress, and inhibiting proinflammatory cytokines. Due to its strong impact on immune responses, CuB attracts increasing attention in therapeutic development. 22 CuB is notably recognized for its antitumor properties, as it inhibits the JAK/STAT pathway, leading to G2-M-phase arrest and apoptosis of pancreatic cancer cells and reduces the size of pancreatic tumor xenografts in nude mice. 23 Additionally, CuB has been applied in the topical treatment of imiquimod-induced skin inflammation due to its immunoreactive and antiproliferative effects. It helps reduce inflammation, slow keratinocyte proliferation, alleviate hyperplasia, and improve psoriasis symptoms. 24 Building on these findings, we investigated the therapeutic effects and underlying functions of CuB on RA progression using a CIA mouse model in this study.
During RA treatment, we identified the top ten key target genes of CuB, including TNF, IL1B, SRC, STAT3, CASP3, NFKB1, JUN, TLR4, BCL2, and PTGS2 through network pharmacology analysis. These targets were thought to and demonstrated to affect RA’s pathogenesis and were possible targets for CuB to directly or indirectly affect the improvement of RA. Among these targets, STAT3 was confirmed to be directly binding to CuB by molecular docking experiment. 25 STAT3 drives the expression of inflammatory cytokines and receptor activator of nuclear factor κB ligand (RANKL), resulting in concomitant inflammation and osteoclastogenesis and leading to joint destruction. Thus, STAT3 was suggested as a potential therapeutic target for RA.26,27
To gain insight into the influence of CuB and how this would translate to RA, we measured the levels of pro-inflammatory and anti-inflammatory cytokines in the paw tissues of CIA mice treated with CuB, MTX, or a vehicle. The results revealed that CuB has anti-inflammatory properties to reverse the CIA-induced TNF-α and IL-6 cytokines. This reduction was also observed in the CuB treatment on cholestatic liver injury (CLI). 28 According to the downregulation of IL-6, STAT3, and hypoxia-inducible factor (HIF-1α) expression and inhibiting STAT3 phosphorylation, CuB was thought to prevent the CLI inflammatory response by inhibiting the IL-6/STAT3/HIF-1α pathway. 28 According to KEGG analysis, 20 immune-related pathways were identified as associated with CuB therapy and RA disease. Among these, Toll-like receptor, chemokine, and T cell receptor signaling pathways were found to be linked to RA pathogenesis. Notably, three pathways stood out due to their significance: Th17 cell differentiation, JAK-STAT, and IL-17 signaling pathways. That is to say that CuB may modulate RA immune responses and inflammatory processes to control rheumatoid arthritis onset and progression by regulating these critical pathways. Combined KEGG and network pharmacology analyses revealed that STAT3 was a common target of CuB to RA. Considering the role in the pathogenesis of rheumatoid arthritis of the Th17/STAT3 pathway, we focused on this axis, which is consistent with our experimental results. Not to be overlooked, TLR signaling was shown as the most influential pathway in KEGG. The TLRs play a role in RA pathogenesis, identified by many studies from both murine and human. 29 In other disease models, CuB can protect from neuroinflammatory injury 30 and suppress gout arthritis in mice 15 by regulating TLR signaling. Further verification of how CuB regulates the TLR signaling pathway in RA will be an important research direction in the future.
It’s well known that the JAK/STATs pathway is a common signaling pathway for classical immune cytokines to regulate cytokine signaling and pathogenic Th17 differentiation. 31 Blocking the JAK/STAT signaling can ameliorate this major pathogenic process of RA. 32 The JAK2 inhibitor AG490 improved CIA phenotype by inhibiting Th17 cell differentiation and markedly upregulating Th17 cell/regulatory T cell (Treg) development markers. AG490 was shown to precisely modulate the JAK2/STAT3 pathway by reducing STAT3/STAT5 phosphorylation. 33 STAT3 is also a target protein of CuB to RA. CuB can also significantly decrease STAT3 phosphorylation in Th17-polarized CD4 T cells. We further found that this inactivation did not affect JAK2 expression and phosphorylation. It’s indicated that CuB can serve as a JAK2 inhibitor to influence Th17 cell development primarily by suppressing STAT3 phosphorylation. Comparing the effective doses, 5 μM AG490 decreased IL17 mRNA by 50%, but 50 nM CuB decreased IL17 mRNA by more than 60%. Therefore, CuB is more suitable for developing RA drugs than AG490.
In addition, CuB has potent anticancer properties by suppressing JAK/STAT signaling, specifically in colorectal cancers. 34 It binds to STAT3 and decreases M2 macrophage polarization, inhibiting the JAK2/STAT3 signaling pathway, leading to apoptosis in colorectal cancer cells. In vitro studies showed that it inhibited CRC cell proliferation and reduced cell migration, while in vivo therapy produces anti-tumor immunity by modulating M2-like macrophages and increasing CD4 and CD8 expression. 35 Our investigations confirmed that CuB has the same inhibitory effect on Th17 cell differentiation in the CIA mouse model. The CuB treatment significantly reduced the proportion of CD4+IL-17A+ Th17 cells and the RNA expression level of RORγt in the spleens of CIA mice. This suggests that CuB can reduce the number of Th17 cells in the spleens of CIA mice.
According to PubChem and the Cayman Chemical Safety Data Sheet, the oral and intraperitoneal (i.p.) LD₅₀ of CuB in mice is 14 mg/kg and 1 mg/kg, respectively.36,37 It was reported that i.p. administration of CuB at doses ranging from 1 to 5 mg/kg exhibits anti-arthritis, anti-multiple sclerosis, and anti-cancer pharmacological effects without apparent toxicity,12,38 but differed from our findings in DBA/1 mice. In the i.p. group, the survivals of the vehicle, 0.5 mg/kg, and 1 mg/kg CuB groups are 100%, 60%, and 20% by day 2, respectively, and no mice survive by day 3 (Supplemental Figure 3A). In the oral administration group, mice in the vehicle and low-dose (0.5 and 1 mg/kg) groups maintained a 100% survival rate. However, in the 10 mg/kg group, survival dropped to 40% by day 4, with no surviving mice by day 5. Similarly, in the 5 mg/kg group, survival decreased to 60% by day 4 and reached 0% by day 8 (Supplemental Figure 3B). The results indicated that when the intraperitoneal (i.p.) dose exceeded 0.5 mg/kg or the oral dose exceeded 5 mg/kg, significant toxicity was observed, resulting in mortality within 1 to 5 days (Supplemental Figure 3). Additionally, CBC and biochemical analysis results showed no significant differences in hematological or organ function alterations in different groups (Supplemental Table 1). Based on these findings, we selected the relatively safer oral doses of 0.5 mg/kg and 1 mg/kg for further experimentation in this study. However, we speculate that the observed differences in CuB toxicity may be influenced by animal strain, dosage, administration route, and physiological characteristics. Nevertheless, given our findings and previous reports indicating that excessive accumulation of cucurbitacin can lead to food poisoning,39,40 we emphasize the need for a thorough safety evaluation when considering CuB as a potential therapeutic agent.
One limitation of this study is the relatively low statistical power resulting from the sample size used. Although we conducted two independent experiments and have now combined the datasets for analysis (n = 10 per group), a post hoc power analysis indicated that the achieved power was approximately 0.55, below the generally recommended threshold of 0.8. This suggests that the study may not be adequately powered to detect more minor but biologically meaningful differences. We acknowledge this limitation and will apply a priori power calculations in future experimental designs to ensure that the number of animals used is sufficient to achieve statistically robust conclusions. Despite this limitation, the observed differences between the CuB-treated and control groups remained statistically significant after data consolidation, supporting the potential efficacy of the treatment.
Conclusion
Our study demonstrates that cucurbitacin B (CuB) plays a significant role in modulating immune response pathways, specifically the JAK/STAT and IL-17 signaling pathways, both critical in the progression of rheumatoid arthritis (RA). CuB effectively alleviates collagen-induced arthritis (CIA) symptoms by reducing pro-inflammatory cytokines such as TNF-α and IL-17 and inhibiting Th17 cell differentiation. These findings highlight CuB’s potential as a therapeutic agent in RA management, offering novel insights into anti-inflammatory strategies for autoimmune diseases.
Supplemental Material
Supplemental material, sj-docx-1-iji-10.1177_03946320251348715 for Cucurbitacin B inhibits Th17 cell differentiation via the suppression of the JAK/STAT pathway and alleviates collagen-induced arthritis in mice by Shu-Ping Kung, Hira Umbreen, Jou-Hsuan Wang, Chih-Ming Tsia, Tim Chi-Chen Lin and Yu-Ting Chen in International Journal of Immunopathology and Pharmacology
Acknowledgments
The authors would like to thank the team members at the Institute of Biomedical Science, National Chung Hsing University, for their invaluable support and assistance in facilitating this research.
Footnotes
Author contributions: Shu-Ping Kung was responsible for conceptualization, methodology, data analysis, and the preparation of the original draft. Hira Umbreen contributed to data collection, data curation, and participated in manuscript review and editing. Jou-Hsuan Wang assisted with data analysis and data collection, while Chih-Ming Tsai was involved in data collection and contributed to manuscript review and editing. Tim C.C. Lin secured funding, provided supervision, managed project administration, and participated in manuscript review and editing. Yu-Ting Chen provided supervision, managed project administration, and contributed to the manuscript review and editing.
Data availability statement: The data supporting this study’s findings are available from the corresponding author upon reasonable request.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The present study was supported by grants from the Taichung Veterans General Hospital (Grant No. TCVGH-1137306C), National Science and Technology Council, R.O.C (110-2313-B-005-042-MY3, 113-2320-B-005-010-MY3) and in part by the Advanced Plant and Food Crop Biotechnology Center from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (113S0023A).
Ethical considerations: All animal housing and experiments were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals at National Chung Hsing University (NCHU-IACUC No.113-143).
Animal welfare: The present study followed international, national, and/or institutional guidelines for humane animal treatment and complied with relevant legislation.
ORCID iDs: Shu-Ping Kung 
https://orcid.org/0009-0009-1467-4827
Yu-Ting Chen
https://orcid.org/0000-0003-4579-6498
Supplemental material: Supplemental material for this article is available online.
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Associated Data
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Supplementary Materials
Supplemental material, sj-docx-1-iji-10.1177_03946320251348715 for Cucurbitacin B inhibits Th17 cell differentiation via the suppression of the JAK/STAT pathway and alleviates collagen-induced arthritis in mice by Shu-Ping Kung, Hira Umbreen, Jou-Hsuan Wang, Chih-Ming Tsia, Tim Chi-Chen Lin and Yu-Ting Chen in International Journal of Immunopathology and Pharmacology