Mechanistic Study of Yibi Decoction in Treating Rheumatoid Arthritis via Modulating the JAK2/STAT3 Signaling Pathway

Abstract

Introduction/Objective

To investigate the therapeutic effects of Yibi Decoction (YBD) in rheumatoid arthritis (RA) and to elucidate the underlying mechanisms, with a particular focus on the JAK/STAT signaling pathway.

Methods

Network pharmacology analysis was employed to predict the bioactive compounds and potential therapeutic targets of Yibi Decoction. A collagen-induced arthritis (CIA) rat model was established to validate the predicted effects in vivo. Western blotting, enzyme-linked immunosorbent assay (ELISA), and histopathological analyses were conducted to evaluate alterations in joint tissues and serum inflammatory biomarkers.

Results

Network pharmacology analysis identified 128 bioactive components in YBD, among which quercetin and luteolin were predicted to be key active compounds. In CIA rats, YBD treatment significantly downregulated the expression of IL-6, IL-17, IL-23, JAK2, and STAT3 in ankle joint tissues (P < 0.05), and markedly reduced serum levels of anti–cyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor (P < 0.05). In parallel, the arthritis index was significantly decreased, and histopathological examination revealed substantial attenuation of synovial hyperplasia and cartilage destruction.

Discussion

These findings suggest that YBD exerts anti-rheumatoid arthritis effects through a multi-component, multi-target regulatory mechanism involving inhibition of the JAK/STAT signaling pathway. Quercetin and luteolin may contribute to these effects by suppressing pro-inflammatory cytokine production and ameliorating joint pathological changes.

Conclusion

YBD ameliorates rheumatoid arthritis by suppressing the JAK/STAT signaling pathway and associated inflammatory responses, thereby providing a mechanistic basis for its potential therapeutic application in rheumatoid arthritis.

1. INTRODUCTION

Rheumatoid arthritis (RA) is a complex, chronic systemic autoimmune disease characterized by symmetrical polyarticular synovitis, progressive joint and bone destruction, and persistent systemic inflammation [1-4]. The pathogenesis of rheumatoid arthritis is multifaceted, involving dysregulated activation of both innate and adaptive immune responses, amplification of pro-inflammatory cytokine cascades (including IL-6, TNF-α, and IL-17), and hyperproliferation of synovial fibroblasts, which collectively drive tumor-like invasive behavior of the synovium [5, 6]. Currently, the clinical management of rheumatoid arthritis primarily relies on nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs), and biologic agents, including Janus kinase (JAK) inhibitors such as tofacitinib and TNF-α antagonists [7, 8]. However, long-term use of these medications is associated with significant adverse effects, including gastrointestinal ulceration, osteoporosis, and opportunistic infections, as well as a substantial financial burden. It is estimated that approximately 40% of patients with rheumatoid arthritis discontinue therapy due to adverse drug reactions or insufficient therapeutic efficacy [9]. Therefore, there is an urgent need to develop novel therapeutic strategies that are both highly effective and safe.

Traditional Chinese Medicine (TCM), characterized by principles of holistic regulation and multi-target intervention, offers distinct advantages in the management of rheumatoid arthritis and provides an alternative therapeutic perspective. In recent years, compound Chinese herbal medicines have attracted increasing attention in mechanistic studies of rheumatoid arthritis due to their multi-component and synergistic properties. Previous studies have reported that Guizhi Shaoyao Zhimu Decoction alleviates rheumatoid arthritis by regulating autophagy in pre-osteoclasts via the ROS/NF-κB signaling axis [10], while Wutou Decoction ameliorates immune dysregulation in rheumatoid arthritis by modulating the Th17/Treg balance through inhibition of the JAK2/STAT3 signaling pathway [11]. Modern pharmacological studies have identified Tripterygium wilfordii as a commonly used therapeutic agent in Traditional Chinese Medicine for the management of rheumatoid arthritis. Triptolide and glycosides, the principal active constituents of Tripterygium wilfordii, ameliorate rheumatoid arthritis pathology by inhibiting the NF-κB and MAPK signaling pathways, thereby reducing the secretion of pro-inflammatory cytokines, including IL-6 and TNF-α, and inducing apoptosis in synovial cells [12].

The JAK/STAT signaling pathway serves as a pivotal regulatory hub in the pathological progression of rheumatoid arthritis. Upon binding of pro-inflammatory cytokines, such as IL-6 and IL-23, to their cognate membrane receptors, JAK family kinases (including JAK1 and JAK2) undergo phosphorylation and activation. This activation subsequently leads to phosphorylation of STAT3 and STAT5, which then dimerize and translocate into the nucleus [13]. Within the nucleus, activated STAT proteins regulate the transcription of pro-inflammatory genes, such as matrix metalloproteinase-3 (MMP-3) and receptor activator of nuclear factor-κB ligand (RANKL), thereby promoting synovitis, osteoclast activation, and progressive bone erosion. Notably, STAT3 phosphorylation is markedly elevated in the synovial tissues of patients with rheumatoid arthritis and shows a positive correlation with disease activity, as assessed by the Disease Activity Score in 28 joints (DAS28) [14-16]. Currently, although JAK inhibitors such as baricitinib provide rapid symptomatic relief, their clinical utility is limited by an increased risk of infections and a tendency toward disease relapse following treatment discontinuation [17-19].

Yibi Decoction was developed by Professor Li Guiwen, a distinguished senior traditional Chinese medicine physician in China. Over more than 30 years of clinical application, the formula was continuously refined, ultimately resulting in a formulation with Tripterygium wilfordii as the main component. The specific composition is as follows: Tripterygium wilfordii 30 g, Astragalus membranaceus 30 g, Achyranthes bidentata 20 g, Glycyrrhiza uralensis 20 g. This decoction has achieved good clinical efficacy and can effectively control the progression of rheumatoid arthritis. At present, the synergistic regulatory mechanisms by which compound Chinese herbal medicines modulate the JAK/STAT signaling pathway remain incompletely understood. In particular, the interactions among multiple bioactive constituents and their network-based modes of regulation warrant further in-depth investigation. Accordingly, the present study employed a network pharmacology approach in combination with a collagen-induced arthritis (CIA) rat model, together with Western blotting and enzyme-linked immunosorbent assay (ELISA) analyses, to elucidate the regulatory effects of Yibi Decoction (YBD) on the JAK/STAT signaling pathway and its therapeutic mechanisms in rheumatoid arthritis.

2. MATERIALS AND METHODS

2.1. Research Design

This study was designed as an integrated experimental investigation combining network pharmacology–based prediction with in vivo validation in an animal model to systematically elucidate the therapeutic mechanisms of Yibi Decoction (YBD) in rheumatoid arthritis.

2.2. Network Pharmacology

2.2.1. Data Collection and Preparation

Rheumatoid arthritis–associated datasets were retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/, accessed on 8 March, 2025). Eligible disease-related gene expression datasets were selected according to the following criteria: (1) inclusion of both rheumatoid arthritis patients and normal control samples; (2) a minimum sample size of eight to ensure analytical reliability; and (3) generation based on high-throughput sequencing platforms. A search of the GEO database using the keyword “rheumatoid arthritis” identified three gene expression datasets that met the inclusion criteria for this study: GSE55235, GSE55457, and GSE77298. These datasets were subsequently merged using R software, and batch effects inherent to the high-dimensional data were addressed by applying the “sva” package to estimate and adjust for surrogate variables.

2.2.2. RA Difference Analysis

Following data preprocessing, differential expression analysis was conducted using the Linear Models for Microarray Data (LIMMA) package (version 4.2.2) in R software. The LIMMA package was applied to identify differentially expressed genes (DEGs) between the rheumatoid arthritis (RA) samples and normal control samples. Genes meeting the criteria of an adjusted P value < 0.05 and an absolute log₂ fold change (|log₂FC|) > 0.4 were considered statistically significant DEGs. Subsequently, heatmaps and volcano plots were generated to visualize the differential expression patterns, with blue and red indicating downregulated and upregulated genes, respectively.

2.2.3. Screen for the Active Ingredients and Pharmacodynamic Targets of the Drug

Compound profiles of Glycyrrhiza uralensis, Achyranthes bidentata, Astragalus membranaceus, and Tripterygium wilfordii were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP; https://tcmsp-e.com/index.php/, accessed on 9 March, 2025) and PubMed to identify bioactive constituents relevant to the pathogenesis and treatment of rheumatoid arthritis. Potential active compounds were screened based on oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The corresponding target proteins of the identified compounds were subsequently collected, and target gene names were standardized using the UniProt database.

2.2.4. Venn Diagram Screening for Targets of RA Treatment

Intersecting genes between the differentially expressed targets and the herb-derived compound targets were identified using the Jvenn platform (https://jvenn.toulouse.inrae.fr/app/example.html/, accessed on 10 March, 2025), representing putative therapeutic targets for rheumatoid arthritis. Subsequently, a herb–compound–target interaction network was constructed and visualized using Cytoscape software (version 3.9.1).

2.2.5. Core Gene Preselection and Functional Enrichment Analysis

The overlap between predicted drug targets and differentially expressed genes (DEGs) was analyzed to identify key therapeutic targets. To further elucidate the biological functions and associated signaling pathways of these targets, Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the clusterProfiler package in R. The bar chart illustrates that the functional enrichment results were statistically significant at P < 0.05. Furthermore, the top 10 significantly enriched Gene Ontology (GO) terms—including those from the Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories—as well as the top 30 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, were visualized to highlight the most relevant biological features.

2.3. Animal Experiment

2.3.1. Experimental Animal

Forty 8-week-old specific pathogen-free female Sprague–Dawley rats (180–210 g) were obtained from Beijing Sipeifu Biotechnology Co., Ltd. (SCXK (Jing) 2019-0010). Animals were housed under controlled conditions (20–26°C, 30–70% humidity, 14 h/12 h light–dark cycle) with ad libitum access to food and water in the SPF facility at Guangxi University of Chinese Medicine. All procedures were approved by the Institutional Animal Welfare and Ethics Committee (Approval No. DW20240919-241) and conducted in accordance with NIH guidelines.

2.3.2. Experimental Agents

The Chinese herbal materials were obtained from the pharmacy of Ruikang Hospital, affiliated with Guangxi University of Chinese Medicine. Baricitinib tablets (HJ20215001; Lilly del Caribe, Inc.) and methotrexate tablets (H31020644; Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd.) were purchased from Sinopharm Group Guangxi Co., Ltd.

2.3.3. Reagents and Instruments

Major reagents included bovine type II collagen (Shanghai Yuanye), Complete and Incomplete Freund’s Adjuvant (Servicebio), RIPA lysis buffer, 5× SDS–PAGE loading buffer, 10% Tris-SwePAGE precast gels, PMSF, 10× TBST (Servicebio), 0.45-µm PVDF membranes (Biosharp), BCA protein quantification kit (Biosharp), and ECL substrate (Biosharp). Primary antibodies against IL-6, IL-17A, IL-23A, JAK2, and STAT3 (all from Solarbio) were used, with HRP-conjugated goat anti-rat IgG (Solarbio) as secondary antibody. Serum levels of cyclic citrullinated peptide (CCP) and rheumatoid factor (RF) were measured using commercial ELISA kits (Jingmei Biotechnology). Key instruments included a refrigerated centrifuge (Eppendorf), a multimode microplate reader (SpectraMax), an electrophoresis system (Liuyi), a gel imaging system (Tanon), a high- throughput cryogenic tissue grinder (Servicebio), and a water purification system (Fulham).

2.3.4. Experimental Animal Grouping and CIA Model Preparation

Rats were randomly assigned to five groups (n = 8 per group) using a random number table: Blank, Model, Methotrexate, Baricitinib, and Yibi Decoction. Sample size was determined by a priori power analysis (G*Power 3.1, one-way ANOVA, α = 0.05, power = 0.80), which indicated a minimum of seven rats per group; eight were used to account for an anticipated 15% attrition rate. The study protocol was approved by the Animal Welfare Ethics Committee of Guangxi University of Chinese Medicine (No. DW20240919-241). After a 1-week acclimatization, CIA was induced in all groups except the Blank group by immunization with bovine type II collagen emulsified in Complete Freund’s Adjuvant (primary) and Incomplete Freund’s Adjuvant (booster).

2.3.5. Preparation of Reagents

Emulsions were prepared on ice. Bovine type II collagen (40 mg) was dissolved in 3.6% (v/v) dilute acetic acid to obtain a uniform suspension, then mixed with an equal volume of Complete Freund’s Adjuvant and emulsified to form a stable water-in-oil emulsion. Emulsification was confirmed by a water-droplet test. The final collagen concentration was 1 mg/mL. Incomplete Freund’s Adjuvant emulsions were prepared using the same procedure.

2.3.6. Injection of Immunological Adjuvant

On day 1, the CFA emulsion was administered via subcutaneous injections (0.05 mL/site) at the bilateral hind paw pads and dorsal sites. Booster immunizations with the IFA emulsion (same dosage) were given subcutaneously along the dorsal region on days 7, 14, and 21, with injection sites rotated to mimic the chronic progressive nature of rheumatoid arthritis.

2.3.7. Evaluation of Model Construction Effect

On day 28, arthritis severity in CIA rats was assessed using an Arthritis Index (AI) scoring system (0–4 per paw; total score 0–16). A total score ≥ 8 was considered indicative of successful model establishment. To validate the model, one rat per group was randomly selected for further evaluation: bilateral ankle joints were collected for HE stain, and serum levels of CCP antibodies and RF were measured by ELISA.

2.3.8. Pharmacological Gavage Protocol

Methotrexate (0.8 mg/kg) was administered by oral gavage once weekly. Baricitinib (0.32 mg/kg) was given once daily by oral gavage. Yibi Decoction (YBD) was prepared as a concentrated decoction (5.0 g crude herb/mL) and administered once daily at 3 mL per rat (15 g crude herb/kg). All treatments lasted 4 weeks. The blank control group received an equal volume (3 mL) of purified water daily.

2.3.9. Sample Collection

Sample collection was performed on days 28 and 56. On D28, one rat per group was randomly selected for interim sampling; the remaining rats were sampled on D56. Body weight was recorded before collection. Approximately 4 mL of blood was collected from the abdominal aorta and centrifuged at 3,000 rpm for 15 min. The serum was then stored at −80°C. After blood collection, rats were euthanized by cervical dislocation. One ankle joint was fixed in 4% paraformaldehyde for histopathology, and the contralateral joint was snap-frozen in liquid nitrogen and stored at −80°C for molecular analysis.

2.3.10. Western Blot

Western blotting was used to detect protein expression of JAK2, STAT3, IL-6, IL-17, and IL-23 in rat ankle joint tissues. Briefly, tissue samples were homogenized in lysis buffer (1:10, w/v) using a pre-chilled high-throughput tissue grinder. Homogenates were centrifuged at 12,000 rpm for 7 min at 4°C, and the supernatants were collected. Protein concentrations were determined using a BCA assay. Equal amounts of protein were separated on 10% SDS–PAGE precast gels and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk, incubated with primary antibodies overnight at 4°C, and then with HRP-conjugated secondary antibodies for 2 h at room temperature. Protein bands were visualized using an ECL detection system and captured with a gel imaging system. Band intensities were quantified using ImageJ software.

2.3.11. ELISA

ELISA was performed to measure serum levels of CCP antibodies and RF using commercial kits (Jingmei Biotechnology). Briefly, serum samples and standards were added to designated wells and incubated at 37°C for 30 min. After washing, the enzyme-conjugated reagent was added and incubated for an additional 30 min at 37°C. Chromogenic substrate was then added and incubated for 10 min in the dark. The reaction was terminated, and the optical density was measured immediately using a microplate reader.

2.3.12. HE Stain

Ankle joints were fixed in 4% paraformaldehyde for 48 h, decalcified with 10% EDTA, and then sent to Wuhan Servicebio Technology for paraffin embedding, HE stain, and histopathological evaluation.

3. STATISTICAL METHODS

Statistical analyses were performed using SPSS version 27.0 (IBM, USA), and graphical representations were generated using GraphPad Prism version 10.1 (GraphPad Software, USA). Quantitative data are presented as the mean ± standard deviation (SD). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests for pairwise comparisons. A two-tailed P value < 0.05 was considered statistically significant.

4. RESULT

4.1. Network Pharmacology

4.1.1. Batch Effect Elimination in GEO

Preliminary data preprocessing was performed on the three datasets, including quality control (QC) assessments to identify and remove aberrant samples or probes, thereby ensuring data reliability. Background correction was subsequently applied to each dataset to normalize signal intensities and minimize technical variation. Subsequently, appropriate data transformation methods were applied based on dataset characteristics to satisfy the statistical assumptions required for downstream analyses. Consistent probe or gene nomenclature was ensured across all datasets to facilitate accurate matching and integration. Each sample was annotated with a batch label corresponding to its source dataset to facilitate batch effect correction [20]. The ComBat algorithm from the sva package, which is based on an empirical Bayes framework, was applied to adjust for systematic inter-batch variability. The integrated dataset was subsequently corrected using ComBat to minimize non-biological variation attributable to batch effects. Comparisons before and after batch correction were performed to visualize and evaluate the effectiveness of batch effect removal (Figs. 1 and 2).

Fig. (1).

GEO dataset before batch effect elimination.

Fig. (2).

GEO dataset after batch effect elimination.

4.1.2. DEGs and targets of RA

Using the limma package, a total of 2,495 differentially expressed genes (DEGs) associated with rheumatoid arthritis were identified, including 1,195 downregulated and 1,300 upregulated genes. These DEGs were visualized using a volcano plot (Fig. 3) and a heatmap displaying the top 40 most statistically significant DEGs (Fig. 4), both generated with R software (version 4.2.2). Subsequently, all identified DEGs were annotated and standardized against the UniProt database using Perl scripts, yielding 2,495 refined rheumatoid arthritis–related disease targets.

Fig. (3).

DEGs volcano plot.

Fig. (4).

DEGs heatmap.

Blue and red indicate downregulation and upregulation, respectively, in both graphical representations.

4.1.3. Key Targets and Regulatory Networks for RA Treatment

Based on the predefined screening criteria, a total of 128 bioactive compounds were identified from the prescribed herbs, including 12 from Achyranthes bidentata, 30 from Tripterygium wilfordii, 1 from Astragalus membranaceus, and 85 from Glycyrrhiza uralensis. In addition, 251 putative therapeutic targets associated with these compounds were collected. Intersection analysis between these compound-related targets and the 2,495 rheumatoid arthritis–related disease targets, performed using a Venn diagram, identified 76 overlapping targets (Fig. 5). Subsequently, a herb–compound–target regulatory network was constructed and visualized, incorporating the four herbs, 128 bioactive compounds, and 76 overlapping targets (Fig. 6). The resulting network comprised a total of 383 nodes. Network topology analysis revealed that PPARG, CCNA2, CHEK1, CA2, and IGHG1 exhibited the highest degree values among target nodes, while quercetin, luteolin, and β-sitosterol were identified as the most highly connected bioactive compounds.

Fig. (5).

Venn diagram of drug target and RA.

Fig. (6).

Herb-Compound-Target regulatory network.

4.1.4. Functions and Pathways Involved in the Drug Therapy of RA Targets

GO functional enrichment analysis identified 1,621 significantly enriched terms, of which 1,454 were classified under the Biological Process (BP) category. These BP terms were predominantly associated with responses to reactive oxygen species, lipopolysaccharide, ultraviolet A (UV-A) radiation, oxidative stress, abiotic stimuli, and chemical stress, as well as regulation of neuroinflammatory responses. These enriched functions were closely associated with key pathological processes involved in rheumatoid arthritis, including inflammation, oxidative stress, tissue damage and repair, and immune regulation. In addition, 27 significantly enriched (CC) terms were identified, primarily involving membrane rafts, the external side of the plasma membrane, membrane microdomains, low-density lipoprotein particles, neuronal cell bodies, and banded collagen fibers. MF enrichment analysis identified 140 significantly enriched terms, including histone kinase activity, nuclear receptor activity, ligand-activated transcription factor activity, viral receptor activity, cytokine receptor binding, exogenous protein binding, insulin receptor substrate binding, and protein kinase regulatory activity, among others (Fig. 7). KEGG pathway enrichment analysis identified 113 significantly enriched pathways, primarily including type II diabetes mellitus, the p53 signaling pathway, lipid metabolism and atherosclerosis, rheumatoid arthritis, the IL-17 signaling pathway, Th17 cell differentiation, the chemokine signaling pathway, the Toll- like receptor signaling pathway, and fluid shear stress and atherosclerosis (Fig. 8).

Fig. (7).

GO functional enrichment analysis.

Fig. (8).

KEGG pathway enrichment analysis.

4.2. Animal Experiment

4.2.1. The ELISA Method was used to Evaluate the Modeling Effect

Serum levels of CCP antibodies and RF on D28 were measured using ELISA. Compared with the blank group, the model, methotrexate, baricitinib, and Yibi Decoction groups exhibited significantly elevated serum levels of CCP and RF (P < 0.001; Fig. 9).

Fig. (9).

CCP and RF levels in the serum of rats at D28. In the modeling-treated group, both CCP and RF were significantly elevated. ns: The difference was not statistically significant.

4.2.2. Evaluation of the Modeling Effect by HE Staining

One ankle joint tissue sample collected on D28 was paraffin-embedded and subjected to HE staining for histomorphological evaluation. Histopathological examination revealed that rats in the model, methotrexate, baricitinib, and Yibi Decoction groups exhibited characteristic rheumatoid arthritis–associated pathological changes, including cartilage surface destruction, pannus formation, synovial hyperplasia, pronounced neovascularization, and inflammatory cell infiltration (Fig. 10).

Fig. (10).

(a) model group, (b) methotrexate group, (c) baricitinib group, (d) Yibi Decoction group; Yellow arrows indicate irregular cartilage surfaces with pannus formation; green arrows denote synovial connective tissue hyperplasia; black arrows highlight infiltration of inflammatory cells, including lymphocytes and neutrophils; blue arrows mark cartilage surfaces. Hematoxylin-eosin staining, original magnification 200×.

4.2.3. Evaluation of the Modeling Effect by the AI Index

On day 28, AI scores were evaluated for both the fore and hind paws of all rats. The model, methotrexate, baricitinib, and Yibi Decoction groups all exhibited AI scores ≥ 8, with no statistically significant differences observed among these groups (P > 0.05). In contrast, AI scores in each of these groups were significantly higher than those in the blank control group (P < 0.05) (Fig. 11; Table 1).

Fig. (11).

(a-d) Morphological presentation of rat forepaws and hindpaws of pre-modeling phase; (e-h) Morphological presentation of rat forepaws and hindpaws of post-modeling phase, showing evident redness and swelling in both fore and hind paws, with more pronounced manifestations in the hind paws.

Table 1.

Comparison of AI index scores in each group of rats.

Groups	Index	F	P
Blank group	0±0.00	130.22	<0.001
Model group	10.00±0.93*
Methotrexate group	9.50±1.41*
Baricitinib group	10.00±1.41*
Yibi Decoction group	9.75±1.04*

Note: *Compared with the blank group, P<0.05.

4.2.4. Western Blot

Following pharmacological intervention, the methotrexate, baricitinib, and Yibi Decoction groups exhibited statistically significant reductions in the expression levels of IL-6, IL-17, IL-23, JAK2, and STAT3 to varying extents compared with the model group (P < 0.05) (Fig. 12).

Fig. (12).

Western blot results after drug intervention. In the methotrexate, baricitinib, and Yibi decoction groups, IL-6, IL-17, IL-23, JAK2, and STAT3 all showed a downward trend compared with the non-intervened model group. ns: The difference was not statistically significant. *:P < 0.05; **:P < 0.01; ***:P < 0.001; ****:P < 0.0001.

4.2.5. ELISA

Following pharmacological intervention, serum levels of cyclic citrullinated peptide (CCP) antibodies and rheumatoid factor (RF) were significantly reduced to varying extents in the methotrexate, baricitinib, and Yibi Decoction groups compared with the model group (P < 0.05) (Fig. 13).

Fig. (13).

ELISA results after drug intervention. In the methotrexate, baricitinib, and Yibi decoction groups, CCP and RF were significantly lower than in the non-intervened model group. ns: The difference was not statistically significant. *:P < 0.05; **:P < 0.01; ***:P < 0.001; ****:P < 0.0001.

4.2.6. Morphological Presentation of Rat Paw After Yibi Decoction Intervention

Following intervention, rats in the Yibi Decoction group exhibited a marked reduction in paw swelling, with skin coloration approaching normal; however, residual deformities of the interphalangeal joints remained evident (Fig. 14).

Fig. (14).

Morphological presentation of rat paw after Yibi Decoction intervention, (a) Pre-modeling phase, (b) Post-modeling phase, (c) Post-intervention with Yibi Decoction.

HE staining revealed that, before treatment, ankle joint tissues displayed extensive cartilage destruction, pannus formation, pronounced synovial connective tissue hyperplasia, and dense infiltration of inflammatory cells, including lymphocytes and neutrophils. After treatment with Yibi Decoction, the cartilage surface was largely preserved, although disrupted tidemark integrity and focal nuclear dissolution were observed. In addition, only mild hyperplasia of the synovial lining layer and limited focal lymphocytic infiltration were present (Fig. 15).

Fig. (15).

HE of rat joint tissues in the Yibi Decoction group before and after intervention. (a-b): Post-modeling phase; (c-d): Post-intervention with Yibi Decoction. Yellow arrows indicate irregular cartilage surfaces with pannus formation; green arrows denote synovial connective tissue hyperplasia; black arrows highlight infiltration of inflammatory cells, including lymphocytes and neutrophils; purple arrows mark disrupted tide lines with visible nuclear dissolution; brown arrows indicate hyperplasia of lining cells. Hematoxylin-eosin staining, original magnification 200×.

5. DISCUSSION

The primary pathological features of rheumatoid arthritis include synovial hyperplasia, infiltration of inflammatory cells, and pannus formation—all of which collectively lead to progressive erosion of cartilage and bone [21-23]. A hallmark pathological feature of rheumatoid arthritis is the aberrant activation of fibroblast-like synoviocytes (FLS), which acquire aggressive, tumor-like proliferative and invasive properties [23]. Persistent synovitis leads to progressive destruction of articular cartilage and loss of joint structural integrity, ultimately resulting in clinical manifestations such as joint swelling, pain, and deformity [24]. Rheumatoid arthritis is primarily driven by CD4⁺ T lymphocytes, while B lymphocytes play a crucial role in mediating and sustaining the pathological process [25-27]. In addition, multiple immune and stromal cell types collectively contribute to synovial inflammation and progressive cartilage and bone erosion [28-31]. Notably, IL-6 serves as a critical mediator of bone destruction in rheumatoid arthritis by promoting T-lymphocyte differentiation and activation and amplifying downstream inflammatory responses [32-34]. Rheumatoid arthritis primarily manifests through two major pathological mechanisms. First, aberrant proliferation of synovial tissue is driven by heightened activation of macrophage-like synoviocytes (MLS) and FLS, which secrete large amounts of pro-inflammatory cytokines and matrix-degrading proteases, including IL-1, IL-6, and TNF-α [35]. In addition to expressing IL-6, FLS produce substantial levels of matrix metalloproteinases (MMPs) and lipid-derived inflammatory mediators, such as prostaglandins and leukotrienes. Moreover, FLS actively participate in immune activation by interacting with infiltrating immune cells and promoting the formation of ectopic lymphoid-like structures (ELS) within synovial tissue. The second pathological mechanism involves infiltration of adaptive immune cells into the synovial sublining, leading to the formation of characteristic pannus tissue at the cartilage–bone interface [36-38]. This pannus is composed primarily of macrophages, FLS, dendritic cells, plasma cells, and mast cells, which collectively mediate progressive tissue damage and bone erosion during the later stages of disease [39]. During the early phase of RA, IL-23 promotes the differentiation and stabilization of Th17 cells, which subsequently produce IL-17 and IL-6. IL-17 further stimulates macrophages to generate pro-inflammatory mediators, including TNF-α. In fibroblast-like synoviocytes derived from RA patients, IL-17 upregulates the expression of IL-23p19 at both the mRNA and protein levels, thereby reinforcing the IL-23/Th17 inflammatory axis. This sustained inflammatory loop contributes to B-cell activation and autoantibody production, including RF and anti-citrullinated protein antibodies (ACPAs) [40, 41]. The JAK–STAT signaling pathway is a central mechanism of intracellular signal transduction that conveys extracellular cues from cell-surface receptors to the nucleus, thereby regulating key physiological processes such as cell proliferation, differentiation, and apoptosis. A distinctive feature of the JAK–STAT signaling pathway is its reliance on two core molecular components- Janus kinases (JAKs) and signal transducers and activators of transcription (STATs)—to mediate signal transmission, in contrast to many immune signaling pathways that involve more complex, multi-step cascades [14]. This pathway plays a critical role in a wide range of physiological and pathological processes, including immune regulation, inflammation, neurodegenerative disorders, and oncogenesis. Dysregulation of the JAK–STAT signaling pathway can result in severe immunodeficiency as well as uncontrolled cell proliferation and malignant transformation [42]. During the pathogenesis of rheumatoid arthritis, the JAK/STAT signaling pathway remains chronically activated as a result of impaired negative regulatory mechanisms. This persistent activation leads to markedly elevated production of multiple pro-inflammatory cytokines, including TNF-α, IL-1, IL-6, IL-15, IL-17, IL-18, IL-22, IL-23, IL-32, and granulocyte–macrophage colony-stimulating factor (GM-CSF) [43]. These cytokines, in turn, further enhance JAK/STAT signaling, thereby establishing a self-amplifying positive feedback loop that sustains chronic inflammation. Consequently, this synergistic inflammatory interplay drives the progression of synovitis, wherein hyperplastic and hyperactivated synovial tissue not only facilitates extensive inflammatory cell infiltration but also promotes excessive secretion of matrix-degrading enzymes, increased production of vascular endothelial growth factor (VEGF), and pronounced pannus formation. Together, these pathological alterations establish a self-perpetuating vicious cycle that sustains and exacerbates the progression of rheumatoid arthritis [42]. Sustained activation of the JAK–STAT signaling pathway within the synovium promotes the release of multiple pro-inflammatory cytokines, which in turn stimulate the production of MMPs [44]. These enzymes facilitate osteoclast differentiation and activation, ultimately leading to progressive destruction of cartilage and bone tissues and thereby exacerbating joint pain and swelling [45]. Furthermore, autophagy in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis is constitutively activated, resulting in increased production of receptor activator of nuclear factor kappa-B ligand (RANKL). RANKL binds to its cognate receptor, receptor activator of nuclear factor kappa-B (RANK), on the surface of osteoclast precursors, thereby promoting osteoclast differentiation and bone resorption. Collectively, these pathological processes drive progressive joint bone erosion and destruction and represent a critical mechanism underlying the exacerbation of chronic inflammation and joint damage in rheumatoid arthritis [46-48].

Using the limma package, we identified a total of 2,495 DEGs associated with rheumatoid arthritis, including 1,195 downregulated and 1,300 upregulated genes. These DEGs reflect substantial transcriptomic alterations during RA pathogenesis and provide valuable insights into the underlying molecular mechanisms. Functional characterization indicated that upregulated genes were predominantly associated with inflammatory responses, immune activation, and cell proliferation, whereas downregulated genes were mainly involved in apoptosis, tissue repair, and immune regulatory processes. Among these DEGs, several genes were closely linked to key pathological features of RA. Notably, PPARG was identified as a high-degree target in the network analysis. PPARG is a nuclear receptor that plays an important role in regulating adipocyte differentiation, glucose homeostasis, and inflammatory responses, and has been increasingly recognized as a critical modulator of immune-mediated inflammation in rheumatoid arthritis [49]. In patients with rheumatoid arthritis, activation of PPARG may attenuate joint inflammation by suppressing the production of pro-inflammatory cytokines [50]. Checkpoint kinase 1 (CHEK1) was also identified as a key target in this study. CHEK1 plays a critical role in cell-cycle regulation and DNA damage response, and its aberrant upregulation in rheumatoid arthritis may be associated with sustained cellular proliferation and enhanced inflammatory activity within synovial tissue [51]. A total of 128 bioactive compounds were identified from the selected Chinese medicinal herbs, including Glycyrrhiza uralensis, Achyranthes bidentata, Astragalus membranaceus, and Tripterygium wilfordii. These herbs have been widely used in traditional medicine for the treatment of rheumatoid arthritis, and their bioactive constituents are thought to exert therapeutic effects through anti-inflammatory, immunomodulatory, and tissue-protective mechanisms. Among the identified compounds, quercetin and luteolin were highlighted as key flavonoids with high abundance, particularly in Glycyrrhiza uralensis and Achyranthes bidentata. Quercetin has been extensively reported to possess potent anti-inflammatory and antioxidant properties, including the inhibition of pro-inflammatory cytokines such as TNF-α and IL-6 [52-56].

Consistent with the reported pharmacological activities of the identified compounds, Gene Ontology (GO) functional enrichment analysis demonstrated that rheumatoid arthritis–related differentially expressed genes were predominantly involved in biological processes associated with inflammatory responses, oxidative stress, immune regulation, and tissue repair. These enriched functions closely align with the core pathological features of rheumatoid arthritis, supporting the potential relevance of the identified bioactive compounds in modulating disease-associated molecular pathways.

Inflammation represents a central pathological process in RA and is characterized by sustained activation of multiple cytokines and chemokines. Notably, the enrichment of inflammation-related processes, such as the response to lipopolysaccharide, indicates persistent inflammatory stimulation in RA patients. In addition, oxidative stress plays a critical role in RA pathogenesis by exacerbating synovial inflammation and contributing to progressive joint damage [57]. The enrichment of oxidative stress–related biological processes identified in this study, such as the response to ultraviolet A (UV-A) radiation, further supports the critical involvement of oxidative stress in rheumatoid arthritis pathogenesis. In addition, KEGG pathway enrichment analysis demonstrated that RA-related differentially expressed genes were significantly associated with several key signaling pathways, including the p53 signaling pathway, IL-17 signaling pathway, and Toll-like receptor signaling pathway. These pathways are known to play essential roles in regulating inflammatory responses and immune homeostasis in RA. Notably, the p53 signaling pathway, which is central to cell- cycle control and apoptosis, may contribute to pathological features of RA when dysregulated, such as sustained proliferation of inflammatory cells and resistance to apoptosis within synovial tissue [58-60]. The IL-17 signaling pathway plays a central role in the inflammatory pathology of rheumatoid arthritis by inducing the production of multiple pro-inflammatory cytokines that drive joint inflammation and tissue destruction [61-63]. In addition, the Toll-like receptor signaling pathway contributes substantially to immune dysregulation in RA, as its activation promotes inflammatory cell activation and robust cytokine release, thereby amplifying inflammatory responses [64-66]. These bioinformatic findings provide insight into the potential molecular mechanisms by which Chinese herbal medicine may exert therapeutic effects in rheumatoid arthritis [67]. Specifically, the bioactive components of Chinese herbs may confer anti-inflammatory, immunomodulatory, and tissue-repair effects through interactions with RA-related targets, thereby modulating multiple disease-relevant biological processes and signaling pathways. Although the four herbs examined in this study were not directly associated with the JAK/STAT signaling pathway in the prediction analysis, key bioactive compounds such as quercetin, luteolin, and β-sitosterol may modulate this pathway indirectly by interacting with upstream or downstream regulatory targets.

Specifically, quercetin exhibits significant anti-inflammatory and antioxidant activities, capable of inhibiting the production of inflammatory cytokines such as TNF-α and IL-6. In addition, quercetin can alleviate oxidative stress damage by modulating the Nrf2 signaling pathway. Luteolin, on the other hand, inhibits NF-κB activation by preventing IκBα degradation induced by LPS stimulation and blocking the nuclear translocation of the NF-κB p65 subunit, thereby suppressing the NF-κB signaling pathway and inhibiting inflammatory responses [68-70]. Through these interactions, these compounds may inhibit inflammatory cytokine production or attenuate signal transduction, thereby contributing to the alleviation of rheumatoid arthritis symptoms. These findings suggest that the herbal composition of Yibi Decoction may indirectly modulate the JAK/STAT signaling pathway, thereby contributing to its therapeutic effects against rheumatoid arthritis. Beyond indirect regulation of the JAK/STAT axis, Yibi Decoction may also exert its anti-RA effects through additional molecular targets and signaling pathways. For example, bioactive compounds derived from Glycyrrhiza uralensis may activate the PPARG signaling pathway, leading to suppression of inflammatory responses and immune activation [39]. Furthermore, herbal constituents may attenuate oxidative damage in patients with rheumatoid arthritis by modulating oxidative stress–related pathways. The multi-target mechanisms of these botanical compounds may confer unique therapeutic advantages in RA management. Compared with conventional single-target drugs, Chinese herbal ingredients can regulate RA-associated pathological processes through multiple signaling pathways, potentially reducing the risks of drug resistance and adverse effects. Notably, multiple compounds within herbal formulations may act synergistically to enhance anti-inflammatory efficacy. This multi-target, multi-pathway therapeutic strategy offers a promising and complementary approach for the treatment of rheumatoid arthritis. Although network pharmacology predicted multiple potential targets and pathways, the present in vivo study primarily focused on validating the JAK2/STAT3 axis, and additional targets remain to be experimentally verified.

ELISA results (Fig. 13) demonstrated that serum levels of CCP antibodies and RF were significantly reduced in the methotrexate, baricitinib, and Yibi Decoction groups following intervention compared with the model group (P < 0.05). Notably, the magnitude of reduction observed in the Yibi Decoction group was comparable to that achieved with methotrexate, suggesting that Yibi Decoction exerts a therapeutic effect of similar efficacy in attenuating immune activation associated with rheumatoid arthritis. Furthermore, Western blot analysis demonstrated that the expression levels of IL-6, IL-17, IL-23, JAK2, and STAT3 were significantly reduced in the methotrexate, baricitinib, and Yibi Decoction groups compared with the model group (P < 0.05).

In this study, Western blot analysis revealed that YBD intervention significantly reduced the protein expression of JAK2, STAT3, and the downstream inflammatory cytokines IL-6, IL-17, and IL-23 in the ankle joint tissues of CIA rats. However, it should be acknowledged that the current study design does not fully allow definitive discrimination between direct pharmacological inhibition of the JAK2/STAT3 pathway by YBD and adaptive changes secondary to the amelioration of arthritis severity—a complexity that arises from the inherent bidirectional interplay between the JAK/STAT pathway and the inflammatory microenvironment in RA.

Notably, the magnitude of reduction in JAK2 and STAT3 expression was markedly greater than that observed for the upstream cytokines IL-6 and IL-23 (Fig. 12 ). This disparity provides indirect evidence suggesting that YBD may exert regulatory effects on the JAK2/STAT3 pathway that are at least partially independent of its suppression of upstream inflammatory cytokines. From the network pharmacology perspective, although bioactive compounds in YBD, such as quercetin and luteolin, were not predicted to directly target JAK2 or STAT3, they may act synergistically on multiple nodes upstream and downstream of the pathway. Moreover, given the multicomponent nature of YBD, it is plausible that different active constituents simultaneously target upstream inflammatory signals (e.g., IL-6, IL-23) and the core kinases of the JAK/STAT pathway, thereby producing a dual inhibitory effect.

Collectively, the current data support the interpretation that the inhibitory effect of YBD on the JAK2/STAT3 pathway is at least partially independent of its regulation of upstream inflammatory cytokines. Nevertheless, this inference warrants further validation through in vitro cellular assays (e.g., using JAK2-overexpressing models or combined intervention with specific inhibitors) and molecular interaction studies (e.g., surface plasmon resonance, molecular docking). Future studies will focus on evaluating the direct inhibitory effects of YBD-containing serum or its active constituents on JAK2 phosphorylation in fibroblast-like synoviocytes to further elucidate the underlying molecular mechanisms.

As described above, IL-6 functions as a key positive regulator of T-lymphocyte differentiation and inflammatory responses and also contributes directly to osteoclast activation and bone destruction. Accordingly, the observed reduction in IL-6 levels is likely to attenuate rheumatoid arthritis pathogenic processes and mitigate cartilage and bone degradation [71-73]. Concurrently, decreased IL-23 levels may limit the differentiation and maintenance of Th17 cells, resulting in reduced IL-17 production and subsequent dampening of IL-17–mediated inflammatory cascades. This coordinated downregulation effectively suppresses the IL-23/IL-17 inflammatory axis, thereby alleviating downstream inflammatory responses. As key upstream activators of the JAK/STAT signaling pathway, reduced levels of IL-6 and IL-23 concurrently inhibit activation of the JAK2/STAT3 pathway, disrupting the self-amplifying inflammatory cycle and contributing to disease amelioration [74, 75]. Consistent with these molecular findings, comparative histopathological analysis of ankle joint sections from the Yibi Decoction group before and after treatment demonstrated marked preservation of cartilage surface integrity and a significant reduction in synovial inflammatory cell infiltration following intervention. However, residual pathological features, including disrupted cartilage tidemarks and mild hyperplasia of the synovial lining layer, were still observed following treatment (Fig. 7). In addition, compared with the normal control group, residual deformities persisted in the interphalangeal joints of rats treated with Yibi Decoction. These pathological manifestations closely resemble the sequelae observed in human rheumatoid arthritis following synovial inflammation and progressive bone and cartilage destruction, underscoring the critical importance of early therapeutic intervention—particularly before the onset of irreversible structural damage to joint tissues. Notably, comprehensive Western blot analysis revealed that the magnitude of reduction in JAK2 and STAT3 expression was greater than that observed for IL-6, IL-17, and IL-23, indicating a disproportionate decrease. This finding raises the possibility that, in addition to indirectly suppressing JAK2/STAT3 signaling via the IL-23/IL-17 axis, Yibi Decoction might influence the expression of JAK2 and STAT3 through mechanisms that are partially independent of its effects on these upstream cytokines. However, direct evidence for a direct molecular interaction between YBD components and JAK2/STAT3 is lacking and requires further investigation. Such dual regulatory effects further attenuate activation of the JAK2/STAT3 signaling pathway, thereby contributing to the alleviation of joint inflammation in rheumatoid arthritis. Overall, the therapeutic efficacy of Yibi Decoction was numerically similar to that of methotrexate, although no direct statistical comparison was performed between the two treatment groups. (Fig. 14).

CONCLUSION

This integrated study demonstrates that Yibi Decoction (YBD) ameliorates rheumatoid arthritis through multi-component and multi-target mechanisms. Network pharmacology analysis predicted that key bioactive compounds, such as quercetin and luteolin, interact with RA-associated targets and signaling pathways. In vivo experimental validation further provided direct evidence that YBD effectively alleviates joint inflammation and pathological damage in collagen-induced arthritis (CIA) rats. Mechanistically, these therapeutic effects are mediated, at least in part, by suppression of the JAK2/STAT3 signaling pathway and its downstream pro-inflammatory cytokines. Collectively, these findings elucidate the mechanistic basis underlying the anti-rheumatic effects of YBD and provide a scientific rationale for its potential development as a therapeutic strategy for rheumatoid arthritis.

LIMITATIONS OF THE STUDY

Although this study provides supportive evidence for the therapeutic potential of Yibi Decoction (YBD) in rheumatoid arthritis, several limitations should be acknowledged. First, the network pharmacology predictions, while informative, are inherently computational and therefore require further experimental validation to confirm the specific interactions between individual bioactive compounds and their predicted targets. In addition, the complex multi-component composition of YBD poses challenges in attributing the observed therapeutic effects to any single constituent, and the potential synergistic interactions among its components remain to be fully elucidated.

Second, although the collagen-induced arthritis (CIA) rat model is widely used and well established for studying rheumatoid arthritis, it does not fully recapitulate the chronicity, heterogeneity, and complexity of human RA. Moreover, this study was conducted over a defined experimental period, and therefore, the long-term efficacy and potential toxicity of Yibi Decoction (YBD) were not assessed. In addition, the experimental design evaluated only a single dosage of YBD; consequently, future studies are warranted to establish dose–response relationships and to optimize therapeutic dosing regimens.

Third, the mechanistic investigation in this study primarily focused on the JAK2/STAT3 signaling pathway and a selected set of inflammatory cytokines. Although this focus was supported by both bioinformatic predictions and experimental validation, the potential regulatory effects of Yibi Decoction (YBD) on other key pathways implicated in rheumatoid arthritis—such as NF-κB and MAPK signaling—cannot be excluded and warrant further investigation. In addition, the assessment of protein and mRNA expression at a single time point limits insight into the dynamic temporal regulation of these pathways during disease progression and treatment.

LIST OF ABBREVIATIONS

ACPAs

Anti-Citrullinated Protein Antibodies

AI

Arthritis Index

CC

Cellular Component

CCP

Cyclic Citrullinated Peptide

CIA

Collagen-Induced Arthritis

DEGs

Differentially Expressed Genes

DMARDs

Disease-Modifying Anti-Rheumatic Drugs

ELISA

Enzyme-Linked Immunosorbent Assay

FLS

Fibroblast-Like Synoviocytes

GEO

Gene Expression Omnibus

GO

Gene Ontology

HE

Hematoxylin and Eosin

IL

Interleukin

JAK

Janus Kinase

KEGG

Kyoto Encyclopedia of Genes and Genomes

MAPK

Mitogen-Activated Protein Kinase

MMPs

Matrix Metalloproteinases

NF-κB

Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells

NSAIDs

Non-Steroidal Anti-Inflammatory Drugs

RA

Rheumatoid Arthritis

RANKL

Receptor Activator of Nuclear Factor Kappa-B Ligand

RF

Rheumatoid Factor

STAT

Signal Transducer and Activator of Transcription

TCM

Traditional Chinese Medicine

TCMSP

Traditional Chinese Medicine Systems Pharmacology Database

TNF-α

Tumor Necrosis Factor-Alpha

VEGF

Vascular Endothelial Growth Factor

WB

Western Blot

YBD

Yibi Decoction

AUTHORS’ CONTRIBUTIONS

All authors contributed to the study conception and design. Writing - original draft preparation: C.H.; Writing - review and editing: Y.C.; Conceptualization: C.H.; Methodology: S.C.; Formal analysis and investigation: T.X.; Funding acquisition: Y.C.; Resources: Y.C.; Supervision: Y.C.; Visualization: S.C. All authors read and approved the final manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was conducted in compliance with laboratory animal ethics guidelines and was approved by the Animal Welfare Ethics Committee of Guangxi University of Chinese Medicine, Nanning, Guangxi, China (Approval No: DW20240919-241).

HUMAN AND ANIMAL RIGHTS

This study adheres to internationally accepted standards for animal research, following the 3Rs principle. The ARRIVE guidelines were employed for reporting experiments involving live animals, promoting ethical research practices. The study was conducted in accordance with NIH guidelines.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

FUNDING

This study was funded by Guangxi Natural Science Foundation: Basic Research on the Inhibition of Ferroptosis in Rheumatoid Arthritis Synovial Tissue by Triptolide through the Xc-/GPX4 Axis via the JAK/STAT Signaling Pathway (Project No.: 2024GXNSFAA010321).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.