Suberosin attenuates rheumatoid arthritis by repolarizing macrophages and inhibiting synovitis via the JAK/STAT signaling pathway

Abstract

Background

Rheumatoid arthritis (RA) is a systemic disease that primarily manifests as chronic synovitis of the symmetric small joints. Despite the availability of various targeted drugs for RA, these treatments are limited by adverse reactions, warranting new treatment approaches. Suberosin (SBR), isolated from Plumbago zeylanica—a medicinal plant traditionally used to treat RA in Asia—possesses notable biological activities. This study aimed to investigate the effects and potential underlying pathways of SBR on RA.

Methods

Tumor necrosis factor-alpha (TNF-α) induced inflammation in RA-derived fibroblast-like synoviocytes (RA-FLS), and the expression of proinflammatory mediators was assessed using q-RT PCR and ELISA after treatment with various SBR concentrations. Bone marrow-derived macrophages (BMDMs) were induced to differentiate into M1 and M2 macrophages, followed by treatment with various SBR concentrations and macrophage polarization assessment. Low-dose (0.5 mg/kg/d) and high-dose (2 mg/kg/d) SBR regimens were administered to a collagen-induced arthritis (CIA) mouse model for 21 days, and the anti-arthritic effects of SBR were evaluated. Network pharmacology and molecular docking analyses were used to predict the anti-arthritic targets of SBR. The effect of SBR on the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was evaluated.

Results

SBR suppressed macrophage polarization toward the M1 phenotype while enhancing their polarization toward the M2 phenotype. SBR reduced the levels of proinflammatory mediators in TNF-α-induced RA-FLS. Mechanistically, SBR inhibited the phosphorylation of the JAK1/STAT3 signaling pathway in RA-FLS and M1 macrophages and promoted the phosphorylation of the JAK1/STAT6 pathway in M2 macrophages, enhancing M2 polarization. In vivo, prophylactic treatment of low-dose SBR reduced M1 macrophage infiltration into synovial tissue, increased the proportion of M2 macrophages, and decreased the expression of inflammatory mediators in the serum and synovial tissue, alleviating synovial inflammation. SBR significantly alleviated arthritis in CIA mice through macrophage repolarization and inhibition of inflammation.

Conclusion

SBR significantly reduced clinical symptoms, joint pathological damage, and expression inflammatory cytokine expression in CIA mice. SBR exhibited anti-arthritic effects via the JAK1/STAT3 and JAK1/STAT6 signaling pathways, inhibiting synovial tissue inflammation and M1 macrophage polarization while promoting M2 macrophage polarization. Therefore, SBR may be an effective candidate for RA treatment.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13075-025-03481-3.

Background

Rheumatoid arthritis (RA) is a systemic disease that primarily manifests as chronic synovitis of the symmetric small joints. The most prevalent clinical manifestations include joint pain and functional impairments. Chronic synovitis affects the cartilage and bone beneath the synovial tissue, leading to joint deformities and stiffness, ultimately compromising the quality of life of affected individuals [1–3]. In recent years, targeted therapeutic drugs have gained increasing popularity for the treatment of arthritis. These drugs are specifically designed to intervene in and regulate immune system function. Specifically, these drugs reduce inflammation, inhibit cell activation, and interfere with abnormal autoimmune responses by targeting specific molecules or pathways of the immune system, thereby retarding the development of autoimmune diseases. Compared with traditional immunosuppressive agents, these drugs are more specific, accurately intervening in key steps of disease occurrence while reducing interference with normal immune functions [1, 4]. Targeted drugs for RA include antitumor necrosis factor inhibitors, recombinant interleukin-1 (IL-1) receptor antagonists, anti-interleukin-6 (IL-6) receptor, anti-CD20 antibodies, anti-CD80/86 antibodies, and Janus kinase (JAK) inhibitors [5]. However, these targeted drugs are associated with adverse reactions, which can vary depending on the drug category and individual differences. Common adverse reactions include risk of infection, allergic reactions, liver damage, gastrointestinal discomfort, cell reduction, cardiovascular effects, pulmonary effects, neurological discomfort, immune system abnormalities, and tumor occurrence [6]. Therefore, it is imperative to develop new treatment approaches.

Fibroblast-like synoviocytes (FLS), the predominant cell type in the synovium, are crucial for the occurrence and development of RA. Under normal conditions, FLS contribute to joint lubrication by producing hyaluronic acid and lubricin. Additionally, FLS contribute to the formation of the extracellular matrix by secreting type IV collagen [7]. However, in RA, FLS transform into an aggressive phenotype, producing various inflammatory mediators, including matrix metalloproteinases (MMPs), interleukins, tumor necrosis factor-alpha (TNF-α), and chemokines [8]. Among these, monocyte chemoattractant protein-1 and fractalkine secreted by RA-derived FLS (RA-FLS) influence monocytes in the circulatory system, causing their migration and accumulation in the RA synovium [9]. Once recruited to synovial tissue, these cells differentiate into macrophages—the most abundant immune cells in the RA synovium. Macrophages exhibit different phenotypes in diverse immune microenvironments, with classical activation (M1) and alternative activation (M2) being the two most typical polarization states [10]. M1 macrophages produce proinflammatory cytokines such as TNF-α and IL-1β, triggering joint erosion and damage, whereas M2 macrophages participate in tissue repair and reconstruction by releasing anti-inflammatory cytokines including IL-10 and transforming growth factor-β (TGF-β). An imbalance in the ratio of M1 to M2 macrophages is implicated in the etiology of RA and associated with disease progression, with patients with RA exhibiting a higher M1/M2 ratio than that in patients with osteoarthritis [11].

In recent years, there has been increasing interest in natural remedies for RA, which may offer unique advantages such as reduced side effects, sustainable long-term use, overall health benefits, and lower risk of drug interactions These natural remedies have opened new avenues for the future development of RA treatments [12]. Plumbago zeylanica (P. zeylanica) is an herb medicinal plant that has been documented in traditional Chinese medicine to treat RA [13, 14]. Up to date, the underlying pharmacological mechanisms of P. zeylanica remain unclear. Suberosin (SBR), a compound isolated from P. zeylanica, possesses notable biological activities, including anti-inflammatory, antitumor, antiviral, and enzyme-inhibitory properties [15–17]. Despite its reported biological activities, the potential role of SBR in RA and underlying anti-inflammatory mechanisms are not well understood, necessitating further investigation.

In the present study, we evaluated the therapeutic efficacy of SBR in a murine model of collagen-induced arthritis (CIA), aiming to explore whether SBR could improve experimental arthritis symptoms by modulating macrophage polarization and FLS inflammation. Additionally, we investigated the downstream signaling pathways targeted by SBR.

Methods

Cells, media, and reagents

SBR (PubChem CID: 68486, purity = 99.98%) was obtained from Shanghai Haoyuan ChemExpress Co. Ltd. (Shanghai, China; Item No. HY-N1196, CAS number: 581-31-7). RA-FLS and bone marrow-derived macrophages (BMDMs) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 U/mL penicillin–streptomycin double antibiotics (HyClone, USA). Roswell Park Memorial Institute (RPMI) 1640 culture medium (Gibco, USA) was used for additional experiments. Type IV collagenase and Escherichia coli bacterial lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (St. Louis, USA). Recombinant mouse IL-4 and interferon-γ (IFN-γ) were procured from PeproTech (Cranbury, USA), while recombinant mouse macrophage colony-stimulating factor (M-CSF) was obtained from BioLegend (San Diego, USA). Recombinant human TNF-α was acquired from Sino Biological (Beijing, China).

Isolation and cultivation of FLS

In a sterile operating room environment, synovial tissue was acquired from individuals diagnosed with RA from West China Hospital, considering the 1987 classification criteria of the American College of Rheumatology (ACR) and the 2010 classification criteria of the ACR/European Alliance against Rheumatism; exclusion of other diseases was also confirmed. The excised synovial tissues underwent meticulous processing, including the elimination of surplus adipose and connective tissue. The synovial tissues were enzymatically digested in an RPMI-1640 medium containing 0.5 mg/mL of type IV collagenase with shaking at 37 °C for 2 h. After filtration and centrifugation, the resultant cell pellet was resuspended in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. The isolated cells were cultured at 37 °C and 5% CO₂. Cells from passages three to five were utilized in the subsequent experiments. This study was approved by the Ethics Committee of West China Hospital, Sichuan University (approval number: 20201151). Informed written consent was secured from all participating individuals.

Polarization of BMDMs

BMDMs were isolated from C57BL/6J wild-type mice, according to a previously established protocol [18]. The retrieved cells were cultured in DMEM supplemented with 20 ng/mL M-CSF, at 37 °C and 5% CO2. The medium was refreshed after 5 d of cultivation. After 7 d, BMDMs were induced to differentiate into M1 macrophages by adding 250 ng/mL LPS and 25 ng/mL IFN-γ. Alternatively, BMDMs differentiated into M2 macrophages after induction with 20 ng/mL IL-4.

Cytotoxicity of SBR

RA-FLS viability was determined by CCK-8 assay as described previously [2]. Approximately 5,000 RA-FLS were cultured in 96-well plates. After 24 h, added in different concentrations SBR. After 24 h, cells were washed and incubated in 90 µL DMEM with 10 µL of CCK8 reagent (GlpBio, USA). After 3 h, the absorbance at 450 nm were measured.

Drug treatment

For cell experiment, SBR was solubilized in dimethyl sulfoxide (DMSO) to achieve a stock concentration of 20 mM and preserved at -80 °C. For RA-FLS, the working concentrations were sequentially 0.1 µM, 0.5 µM, 1µM and 5µM. For macrophage, the working concentrations were 0.1 µM and 1µM. Cells were treated with 20µM Itacitinib (SelleckChem, USA), 100 µM STAT3 inhibitor (S3I-201) (SelleckChem) or 10 µM STAT6 inhibitor (AS1517499) (SelleckChem). For animal experiment, SBR was diluted with PEG300, Tween-80, and normal saline to various concentrations. Starting on the 21st day after the establishment of the CIA model, SBR was administered to mice via daily oral gavage using a syringe. Dosage options for SBR included a low-concentration group (0.5 mg/kg/d) and a high-concentration group (2 mg/kg/d). The model and normal groups received equivalent volumes of solvent.

Inducing and evaluating CIA

For the CIA model, male DBA/1 mice aged eight weeks were procured from Beijing Huafukang Biotechnology Co. Ltd. (Beijing, China). A total of 28 mice were randomly allocated to four groups: control, CIA, 0.5 mg/kg/d SBR, and 2 mg/kg/d SBR, with seven mice in each group. CIA model induction involved anesthetizing the mice via intraperitoneal injection of pentobarbital sodium. Concurrently, chick type II collagen (Chondrex, USA) was emulsified with complete Freund’s adjuvant (Chondrex) at a 1:1 ratio. Subsequently, the emulsion (100 µL) was subcutaneously administered at the base of the tail (1.5–2 cm from the root). Twenty-one days after the first immunization, a newly prepared emulsion (100 µL per mouse) was injected subcutaneously at a site close to the root of the tail. Following enhanced immunization, joint inflammation was assessed every other day, with scores assigned as follows: 0, normal; 1, red or swollen on one finger, 2 red or swollen on two fingers or more, 3 moderate redness and swelling throughout the entire wrist/ankle or all toes; and 4, severe redness and swelling throughout the entire wrist/ankle or all toes. The maximum score for each limb was 4, and the maximum score for each mouse was 16 [13]. On day 42 of modeling, the mice were humanely euthanized and both blood and joint specimens were collected. The animal studies were approved by the Animal Ethics Committee of the West China Hospital, Sichuan University (approval number: 20221103002).

Hematoxylin and eosin (H&E) staining and histological evaluation

The collected joint specimens were fixed with 4% paraformaldehyde (Servicebio, China) at 25 °C for 1 d and then decalcified at 4 °C using 10% EDTA (pH = 7.4). The decalcification solution was replaced every 2 d. After completion of decalcification (one month later), the joint specimens were embedded in paraffin and sectioned into 5-µm-thick slices. Slices were successively deparaffinized in xylene and hydrated in a gradient ethanol solution. Subsequently, H&E staining was performed using an H&E staining kit (Servicebio). The stained sections were sealed with resinene at 25 °C and observed under a light microscope (Leica, Germany). H&E-stained sections of the knee and ankle joints of mice were scanned using a SLIDEVIEW™ VS200 Slide Scanner (Olympus, Japan). Following published scoring criteria [19, 20], H&E-stained sections were evaluated for three aspects: inflammation, cartilage destruction, and bone erosion.

Immunofluorescence

BMDMs were inoculated directly onto round coverslips. After differentiation, cells were treated with 0.1 µM or 1 µM SBR for 1 h. Subsequently, BMDMs were polarized into either M1 or M2 macrophages under different conditions and fixed with 4% paraformaldehyde (Servicebio) at 25 °C for 10 min. For animal tissues, mouse knee tissue sections required antigen retrieval at 95 °C using Tris-EDTA buffer (Servicebio) at pH 9. Following retrieval, sections were blocked with 5% goat serum (Servicebio) at 25 °C for 30 min. M1 macrophages were stained overnight at 4 °C with anti-F4/80 (1:100, ab6640; Abcam, UK) and anti-iNOS (1:100, AF0199; Affinity, China) primary antibodies. For M2 macrophages, overnight staining at 4 °C was performed using anti-F4/80 (1:100, ab6640; Abcam) and anti-CD206 (1:100, DF4149; Affinity) primary antibodies. After three washes with 1× phosphate-buffered saline (PBS) containing Tween^® detergent, the sections were exposed to fluorescent secondary antibodies at 37 °C for 1 h. Subsequently, nuclei were stained with 4’,6-diamidino-2-phenylindole (Abcam, USA). Imaging was performed using an inverted fluorescence microscope (Leica, Germany).

Enzyme-linked immunosorbent assay (ELISA)

Serum samples were collected from normal control, model, and SBR treatment groups. The concentrations of TNF-α, IL-1β, IL-6, and MMP-3 were assessed using ELISA kits (Ruixinbio, China). Cell cultural supernatants were collected and centrifuged with 300 g for 10 min, the IL-1β, IL-6, and MMP-3 levels of RA-FLS were tested by human ELISA kits (Multi Sciences, China). In M1 macrophage, the levels of TNF-α and IL-6 were assessed, in M2 macrophage, the levels of IL-10 was detected by mouse ELISA kits (Multi Sciences).

Western blot analysis

RA-FLS were pretreated with 1 µM SBR for 2 h, then stimulated with 25 ng/mL TNF-α for 15, 30, 60, 120, or 240 min, and the cells were finally harvested. BMDM was polarized to M1 and M2 as described above, then treated with different concentrations of SBR for 24 h, and the cells were finally harvested. total protein of cells was isolated using radioimmunoprecipitation assay (RIPA) buffer (Solabio, China) supplemented with a protease and phosphatase inhibitor cocktail (MCE, China). Synovial tissues were collected from the joints of mice, and placed into 1.5 mL centrifuge tube containing Grinding beads, RIPA, protease and phosphatase inhibitor cocktail, and ground for 20 min at 4 °C by a cryogenic tissue grinder (Servicebio, China). Then, centrifuged and the protein-containing supernatant was collected.

Protein concentration was determined using the bicinchoninic acid protein assay kit (Beyotime, China), and the protein samples were mixed with a loading buffer (Beyotime), then were heated in a metal bath at 95 °C for 10 min. Subsequently, western blot was performed as described previously [2]. Equal amounts of the protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (0.45 μm). The membranes were blocked with a rapid blocking buffer (EpiZyme Biotech, China) at 25 °C for 20 min and then incubated overnight at 4 °C with the respective primary antibodies. The following primary antibodies were used: anti-JAK1 (1:1000, 66466-1-Ig; Proteintech, Wuhan, China), anti-p-JAK1 (1:1000, 3331; Cell Signaling Technology [CST], USA), anti-signal transducer and activator of transcription (STAT) 3 (1:1000, 60199-1-Ig; Proteintech), anti-p-STAT3 (1:1000, 9145T; CST), anti-STAT6 (1:1000, 5397 S; CST), anti-p-STAT6 (1:1000, 9361T; CST), and anti-GAPDH (1:10000, ET1601-4; Huabio, China). Membranes were washed three times with TBST and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies at 25 °C for 90 min. After incubation, the membranes were washed, incubated with an enhanced chemiluminescence substrate (Abbkine, China), and imaged using an Image Lab system (Bio-Rad Laboratories, USA).

Quantitative real-time-PCR (qRT-PCR)

Total RNA was isolated from cells using the RaPure Total RNA kit (Magen, China) and from joint tissues using the TRIzol reagent (Invitrogen, USA). Subsequently, mRNA was reverse transcribed into cDNA using 5× All-In-One RT Master Mix (Applied Biological Materials, Richmond, Canada). qRT-PCR was conducted on the CFX96 System (Bio-Rad Laboratories) using 2× qPCR MasterMix (Applied Biological Materials). The resulting values were standardized to the expression levels of Ga mRNA and analyzed utilizing the 2-^ΔΔCq method. Primer sequences used are listed in Additional File 1: Table S1.

Flow cytometry

Briefly, BMDMs were treated with 0.25% trypsin, and after centrifugation, they were resuspended in PBS supplemented with 1% bovine serum albumin. F4/80-PE (565410; BD Pharmingen, USA) and CD11b-FITC (101206; BioLegend) were used as markers of non-polarized macrophages. CD80-BV711 (104743; BioLegend) was used as a marker of M1 macrophages, whereas CD206-APC (141707; BioLegend) was used as a marker of M2 macrophages. Flow cytometry results were analyzed using FlowJo v10.8.1 software.

Suberosin analysis based on network pharmacology

The SwissTargetPrediction website (http://swisstargetprediction.ch/) was used to predict the prospective targets of SBR. The GeneCards website (https://www.genecards.org/) was used to identify the targets associated with RA. Venny2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was employed to identify the intersection of these target sets, revealing 60 overlapping target genes as candidate targets of SBR in RA treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the DAVID website (https://david.ncifcrf.gov/).

Protein-protein interaction (PPI) analysis was conducted using STRING (http://string-db.org/, ver.10), and the results were visualized using Cytoscape (ver3.8.2; http://cytoscape.org/). The TargetNet website (http://targetnet.scbdd.com) was used to explore the potential combined targets of SBR. Molecular docking studies of drugs and targets were performed using the COACH-D site (https://yanglab.nankai.edu.cn/COACH-D).

Statistical analysis

The experimental results are presented as the mean ± standard deviation (M ± SD). All statistical analyses were performed using Prism 8 software. Statistical significance was assessed using the unpaired two-tailed Student’s t-test for comparisons between two groups, whereas one-way analysis of variance followed by Tukey’s multiple comparison test was used for comparisons among multiple groups. Statistical significance was set at P < 0.05.

Results

Effects of SBR on RA-FLS in vitro

Firstly, the cytotoxicity of SBR was examined in RA-FLS using CCK-8. The rustle showed that SBR is nontoxic when the concentration is 5 µM or lower (Fig. 1A). To assess the effect of SBR on inflammation, we examined the levels of IL-1β, IL-6 and MMP-3 by ELISA (Fig. 1B). Then, we conducted qRT-PCR to examine the mRNA levels of inflammatory mediators in RA-FLS (Fig. 1C-D). As depicted Fig. 1, SBR at concentrations of 0.1, 0.5, 1, and 5 µM markedly reduced the levels of TNF-α, IL-1β, IL-6, IL-8, MMP-1, MMP-3, MMP-9, and MMP-13 in TNF-α-stimulated RA-FLS. Notably, 1 µM SBR exerted the most pronounced inhibitory effect.

Fig. 1.

Effects of SBR on inflammation in RA-FLS in vitro. (A) Cytotoxicity of SBR in RA-FLS. (B) The levels of IL-1β, IL-6 and MMP-3 in TNF-α-stimulated RA-FLS was dected by ELISA. (C) The impact of SBR on the expression levels of TNF-α, IL-1β, IL-6 and IL-8 in TNF-α-stimulated RA-FLS was investigated. (D) The impact of SBR on the expression levels of MMP-1, MMP-3, MMP-9, and MMP-13 in TNF-α-stimulated RA-FLS was investigated. DMSO served as the blank group, whereas TNF-α-stimulated RA-FLS served as the positive control. Data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Effect of SBR on bone marrow-derived macrophage polarization in vitro

Our qRT-PCR results revealed a dose-dependent reduction in the expression of M1-related genes IL-6 and iNOS following SBR treatment (Fig. 2A). Concurrently, SBR significantly increased the expression of M2-related genes Arg1 and CD206 (Fig. 2B). Both levels of TNF-α and IL-6 were decreased in M1 macrophage treated by SBR, whereas IL-10 level was increased in M2 macrophage treated by SBR (Fig. 2C). Flow cytometry analysis indicated that the proportion of CD80-positive BMDMs (M1 macrophages) in the LPS + IFN-γ group was 94%, which further decreased to 84.9% and 77.5% in the 0.1 and 1 µM SBR-treated groups, respectively (Fig. 2D). Conversely, the percentage of CD206-positive BMDMs (M2 macrophages) in the IL-4 group was 82.6%, which further increased to 86.9% and 93.7% in the 0.1 and 1 µM SBR-treated groups, respectively (Fig. 2E). Additionally, SBR suppressed M1 macrophage polarization (Fig. 2F) and facilitated the generation of M2 macrophages (Fig. 2G), as evidenced using immunofluorescence. These findings indicate that SBR induced moderate repolarization of M1 macrophages toward the M2 phenotype.

Fig. 2.

Effect of SBR on BMDM polarization in vitro. (A-B) The expression levels of the M1-related genes iNOS and IL-6, as well as the M2-related genes ARG-1 and CD206, were assessed using qRT-PCR. (C) The levels of TNF-α and IL-6 in M1 macrophage, the level of IL-10 in M2 macrophage were detected by ELISA. (D-E) Macrophage subpopulations were characterized using flow cytometric staining of CD80 and CD206. (F-G) The M1 macrophage marker iNOS and M2 macrophage marker CD206 were visualized by immunofluorescence. Data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 50 μm

Effect of SBR prophylactic treatment on the severity of CIA in mice

To assess the potential inhibitory effects of SBR on arthritis in vivo, we generated a CIA model in DBA/1 mice. We recorded the arthritis scores every other day, starting from day 21 after the initial immunization. The clinical symptoms of CIA mice were significantly ameliorated following prophylactic treatment with 0.5 mg/kg/d SBR (Fig. 3A). We observed reduced serum levels of IL-1β, IL-6, TNF-α, and MMP-3 in CIA mice treated with 0.5 mg/kg/d SBR compared to those in the CIA model group (Fig. 3B). Additionally, qRT-PCR analysis of ankle tissues demonstrated that prophylactic treatment with 0.5 mg/kg/d SBR effectively suppressed the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and MMPs family (MMP-1, MMP-3, MMP-9 and MMP-13) whereas promoted the expression of IL-10 and TGF-β in inflamed joints compared to those in the CIA model group (Fig. 3B-C). But the expression of TGF-β was not statistically difference between normal and CIA model group. H&E staining revealed that the knee and ankle joint structures in CIA mice were destroyed and characterized by inflammatory cell infiltration, cartilage destruction, and bone erosion. Conversely, prophylactic treatment with 0.5 mg/kg/d SBR improved the histopathological damage in the joints (Fig. 3E). Further analysis of histopathological scores of tissues revealed that prophylactic treatment with 0.5 mg/kg/d SBR significantly reduced the knee and ankle pathological scores (Fig. 3F). Consistently, immunofluorescence demonstrated that 0.5 mg/kg/d SBR inhibited the aggregation of M1 macrophages in the knee joint (Fig. 3G) but promoted that of M2 macrophages (Fig. 3H). These results demonstrate the protective effect of low doses of SBR on CIA.

Fig. 3.

SBR administration alleviated arthritis severity in CIA mice. (A) Clinical symptoms of CIA mice improved with the administration of 0.5 mg/kg/d SBR (SBR 0.5) compared to those in the CIA model group. (B) ELISA determination of serum inflammatory factor levels, including TNF-α, IL-1β, IL-6, and MMP-3. (C)qRT-PCR evaluation of the expression of TNF-α, IL-1β, IL-6, IL-10 and TGF-β in ankle joint tissues. (D) qRT-PCR assessment of MMP-1, MMP-3, MMP-9, and MMP-13 expression in ankle joint tissues. (E) H&E staining of the knee and ankle joints in each group. (F) Histopathological scores of the knee and ankle joints in each group. (G-H) Immunofluorescent staining of iNOS/CD206, markers of M1/M2 macrophages in knee joint tissues (F4/80 in red, iNOS in green, and CD206 in green). Data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 100 μm

JAK1 is a direct binding target for SBR

The molecular structure of SBR is presented in Fig. 4A. Using the GeneCards website, we identified 5319 RA-related target genes of SBR. We subsequently used the SwissTargetPrediction website and the Venny2.1.0 tool to predict potential SBR targets. Following a comparison of the identified targets using these programs, we finally obtained 60 common targets (Fig. 4B). We then analyzed these intersecting targets using the STRING database and employed the Cytoscape platform to construct a PPI network diagram illustrating the role of SBR in RA treatment (Fig. 4C). Furthermore, we conducted GO and KEGG analyses to assess the functional and enrichment aspects of the 60 predicted SBR targets that were closely related to RA. We selected the top 20 KEGG signaling pathways for mapping, revealing that the differentially expressed pathways were mainly associated with the immune response, tumors, viral and bacterial infections, inflammation, and endocrine metabolism (Fig. 4D). The top ten terms in the three GO categories, including biological processes, cellular components, and molecular functions, are illustrated in a bar chart (Fig. 4E). Additionally, we used the TargetNet website for SBR target prediction, which indicated the high binding affinity of SBR with JAK1. Therefore, we performed molecular docking of SBR and JAK1 using the COACH-D website (Fig. 4F). To validate the predicted anti-RA mechanism based on network pharmacology, we performed western blotting to assess the effect of SBR on the expression of JAK1 and its downstream target protein, STAT3. We found that the expressions of total and phosphorylation of JAK1 and STAT3 proteins in the ankle tissue of CIA mice were significantly higher than that in the normal control group. However, this increase was significantly ameliorated after treatment with SBR (0.5 mg/kg/d) (Fig. 4G-H).

Fig. 4.

JAK1 was identified as a direct binding target for SBR. (A) Molecular structure of SBR. (B) Venn diagram of the target genes of SBR and RA-related genes. (C) PPI network of critical SBR targets. (D) Top 20 terms of KEGG pathway enrichment analysis. (E) Top 10 enriched categories in biological processes, cellular components, and molecular functions from the GO analysis. (F) Docking results of SBR and the RA-related key target JAK1. (G) Western blot analysis of p-JAK1, JAK1, p-STAT3, STAT3, and GAPDH protein expression in ankle joint tissues. (H) Quantitative analysis of the levels of p-JAK1/JAK1 and p-STAT3/STAT3. Data are presented as the mean ± SD, *p < 0.05, **p < 0.01

SBR modulates the JAK/STAT pathway activation in RA-FLS and BMDMs

We investigated whether the inhibition of the inflammatory response in RA-FLS by SBR involves the JAK1/STAT3 signaling pathway. We used 25 ng/mL TNF-α to activate the JAK1/STAT3 pathway (Fig. 5A). Western blot analysis demonstrated that SBR inhibited the phosphorylation of JAK1 at 60 and 120 min and the phosphorylation of STAT3 at 30 and 60 min (Fig. 5B-C). We further used JAK1 inhibitor (Itacitinib) or STAT3 inhibitor (S3I-201) to test the effect of JAK1/STAT3 inhibition on the production of inflammatory cytokines and MMPs (Fig. 5D-F). The results showed that SBR could further reduce the production of inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) and MMPs (MMP-1, MMP-3, MMP-9 and MMP-13) in TNF-α-stimulated RA-FLS. Together, the inhibitory effect of SBR on the production inflammatory cytokines and MMPs is dependent on JAK1/STAT3 signaling pathway.

Fig. 5.

SBR modulates the JAK1/STAT3 pathway activation in RA-FLS. (A) Representative western blots of phosphorylation levels of JAK1 and STAT3 in RA-FLS stimulated with TNF-α at different time points. (B-C) Quantitative analysis of p-JAK1/JAK1 and p-STAT3/STAT3 levels in RA-FLS. (D) qRT-PCR was performed to test the mRNA expression levels of TNF-α, IL-1β, IL-6 and IL-8 in RA-FLS after stimulation with TNFα (25 ng/ml) in the presence or absence of SBR for 24 h. (E) qRT-PCR was performed to test the mRNA expression levels of MMPs in RA-FLS after stimulation with TNFα (25 ng/ml) in the presence or absence of SBR for 24 h. (F) ELISA was detected the levels of IL-1β, IL-6 and MMPs in RA-FLS after stimulation with TNFα (25 ng/ml) in the presence or absence of SBR for 24 h. Data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Furthermore, we investigated the involvement of the JAK/STAT pathway in the regulation of BMDM polarization by SBR. Western blot analysis revealed that LPS + IFN-γ significantly increased the phosphorylation of JAK1 and STAT3 in BMDMs (Fig. 6A). Conversely, we observed a dose-dependent suppressive effect of SBR on JAK1 and STAT3 phosphorylation (Fig. 6C). Similarly, we observed that the phosphorylation levels of JAK1 and STAT6 significantly increased after the addition of IL-4, a crucial factor for M2 polarization (Fig. 6B). Simultaneously, SBR promoted the expression of p-JAK1 and p-STAT6 in a dose-dependent manner (Fig. 6D), explaining the facilitation of M2 polarization by SBR. We further examined the iNOS and IL-6 mRNA expression in M1 macrophage after treatment with JAK1 inhibitor (Itacitinib) or STAT3 inhibitor (S3I-201) (Fig. 6E), and examined the CD206 and Arg1mRNA expression in M2 macrophage after treatment with JAK1 inhibitor (Itacitinib) or STAT6 inhibitor (AS1517499) (Fig. 6F). Then, TNF-α and IL-6 levels were detected in M1 macrophage, IL-10 level was detected in M2 macrophage using ELISA (Fig. 6G). The results showed that JAK1 inhibitor and STAT3 inhibitor could suppress the M1 macrophage polarization, JAK1 inhibitor and STAT6 inhibitor suppress the M2 macrophage polarization. SBR could further inhibit the polarization of M1 macrophage while rescue the polarization of M2 macrophage. Conclusively, the inhibitory effect of SBR on the M1 macrophage polarization is dependent on JAK1/STAT3 signaling, whereas the promoting effect of SBR on the M2 macrophage polarization is dependent on the JAK/STAT6 signaling pathway.

Fig. 6.

SBR modulates the JAK/STAT pathway activation in BMDMs. (A) Representative western blots of JAK1 and STAT3 phosphorylation levels in BMDMs stimulated with LPS + IFN-γ. (B) Representative western blots of JAK1 and STAT6 phosphorylation levels in BMDMs stimulated with IL-4. (C-D) Quantitative analysis of p-JAK1/JAK1, p-STAT3/STAT3, and p-STAT6/STAT6 levels in BMDMs. (E) The expression levels of the M1-related genes iNOS and IL-6 in M1 macrophage after treated with Itacitinib or S3I-201 in the presence or absence of SBR for 24 h. (F) The expression levels of the M2-related genes Arg1 and CD206 in M2 macrophage after treated with Itacitinib or AS1517499 in the presence or absence of SBR for 24 h. (G) The levels of TNF-α and IL-6 in M1 macrophage, the level of IL-10 in M2 macrophage using ELISA. Data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Discussion

RA is a chronic systemic autoimmune disease primarily characterized by joint lesions manifesting as synovial hyperplasia, immune cell infiltration, and pannus formation [21]. Current therapeutic approaches for RA include the use of glucocorticoids, disease-modifying antirheumatic drugs, nonsteroidal anti-inflammatory drugs, and biologics [22]. Nonsteroidal anti-inflammatory drugs and glucocorticoids often serve as transitional treatments, providing rapid symptom relief without slowing disease progression. Although biologics can delay disease progression, their high cost and risk of non-responsiveness to treatment or increased infection susceptibility pose additional challenges [23, 24]. Therefore, research and development of alternative and complementary drugs for RA are imperative. P. zeylanica, an herb plants, which has been documented in Chinese traditional medicine to treat RA, but the mechanism is unclear. In this study, SBR, as the important extract of P. zeylanica, we explored the therapeutic effect of SBR on RA, the result showed SBR could inhibit the inflammation of RA-FLS and regulate macrophage polarization, attenuate joint inflammation in CIA mice. After combining network pharmacology and target prediction, we demonstrated that JAK-STAT signal pathway involved the treatment effect of SBR on RA.

FLSs are widely recognized as pivotal effector cells that drive disease progression in RA. Unlike normal synovial tissue, FLS proliferates wildly in the synovium of RA patients, and RA-FLS can secrete various proinflammatory cytokines (such as TNF-α, IL-1β and IL-6) and MMPs family (such as MMP-1, MMP-3, MMP-9 and MMP-13) [2, 25], they actively contribute to various pathological processes, including synovial hyperplasia, pannus formation, articular cartilage destruction and bone erosion [7]. RA-FLS not only serve as target cells downstream of the inflammatory environment, but also actively participate in the regulation of immune inflammation independent of the inflammatory milieu through promoting the abnormal activation and differentiation of immune cells (such as macrophage, B cell and T cell), thus lead to the disease continues to deteriorate [1, 26]. Therefore, targeted FLS therapy has been proposed as promising therapeutic approach for RA [7, 27]. In our study, SBR could inhibit the expression of proinflammatory cytokines from RA-FLS stimulated by TNF-α, and decrease the expression of MMP-1, MMP-3, MMP-9 and MMP-13. in vivo, SBR also decreased the serum level of MMP-3, TNF-α, IL-1β and IL-6, regulated the expression of these pro-inflammatory and anti-inflammatory cytokines, thus relieved the joints inflammation and bone erosion.

Additionally, macrophage is the crucial immune cell in synovium of patients with RA. Previous studies showed that macrophage produce various proinflammatory cytokines, and recruit other immune cells and promote FLS activation, thus drive disease progression of RA. In this process, macrophages also different into various types in synovial microenvironment [28], M1 macrophages exhibit proinflammatory characteristics and are marked by the high expression of MHC Class II molecules CD80, CD86, CD38, and toll-like receptor 4. They also release cytokines such as TNF-α, IL-1β, and IL-6, thereby amplifying synovial inflammation. Conversely, a higher proportion of M2 macrophages is correlated with more effective control of RA progression and improved prognosis [29, 30]. So, the imbalance of M1/M2 polarization is confirmed to be increasing important in RA progression. Numerous studies have proposed macrophage repolarization maybe a new therapeutic insight for RA [28, 31–33]. Here, we focused the effect of SBR on polarization of macrophage. Our investigation revealed that SBR induced the polarization of M1 macrophages toward the M2 phenotype at the cellular level. Immunofluorescence results from mouse knee joints demonstrated that SBR treatment impeded the aggregation of proinflammatory M1 macrophages in the knee joints, whereas it promoted that of anti-inflammatory M2 macrophages. Notably, our study targeted both reducing RA-FLS inflammation and macrophage repolarization simultaneously to identify more effective RA treatments. Thus, SBR maybe a multifunctional treatment for RA, which could reduce the side effects of drugs combination.

TNF-α is a central pro-inflammatory cytokine in the pathogenesis of RA, which can derive other cytokines overproduction, including IL-1β, IL-6, and MMPs. These cytokines active various signal pathway. JAK/STAT pathway is one of pivotal signal pathway in RA through involving in the immune and inflammation response of synovium. Stimulation of TNF-α or IL-6 could activate this pathway and exacerbate the inflammatory and immune response [34–36]. JAK/STAT signal pathway can regulate the proliferation, invasion and inflammation of RA-FLS [34, 37]. Aurélie et al. reported that the expression of JAK1 and STAT3 is increased in RA-FLS stimulated by TNF-α [36]. Zhong et al. reported that JAK2/STAT3 signal pathway involve in RA-FLS proliferation, migration and secretion of inflammatory cytokines, periplogenin could reverse synovial proliferation and infiltration by suppressing JAK2/STAT3 signal pathway [34]. Zhang et al. found that quercetin could inhibit JAK1/STAT3 signal pathway, and thus decrease the inflammation, proliferation, migration and invasion of FLS [38]. Moreover, JAK/STAT signal pathway influences the immune cell activation and differentiation, leading to the dysregulation of immune response [39–41]. The development of anti-RA drugs for the JAK/STAT signaling pathway dysregulation is also one of hot spots in the field of RA therapy. Herein, combining the result of network pharmacology and target prediction, JAK1was found to be a candidate target for SBR. Given that TNF-α is a key role of RA pathogenesis, we initially focused on the mechanism of SBR on TNF-α-stimulated RA-FLS and macrophage polarization. Our results demonstrated that phosphorylated expression of JAK1and STAT3 in TNF-stimulated RA-FLS is increased, and SBR could inhibit this pathway activation. Treating RA-FLS with JAK1 inhibitors or STAT3 inhibitors can reduce the expression of inflammatory factors and MMPs, and SBR can enhance this inhibitory effect. Similarly, LPS and IFN-γ activated the JAK1/STAT3 signaling pathway in macrophage, JAK1 inhibitors or STAT3 inhibitors promote M1 macrophage polarization, whereas SBR dose-dependently inhibited this activation. JAK1 inhibitors or STAT6 inhibitors suppress the polarization of M2 macrophage, while SBR could rescue the inhibitory effect of M2 macrophage polarization. So, SBR facilitated macrophage polarization towards the M2 phenotype by upregulating the JAK1/STAT6 signaling pathway. In vivo, SBR inhibited the protein level of JAK1, p-JAK1, STAT3 and p-STAT3 in synovium, and promote the mRNA expression of anti-inflammatory meditors IL-10 and TGF-β.

SBR is a coumarin extracted from P. zeylanica which an herb plant. Previous studies reported that SBR have anti-tumor, anticoagulant and anti-inflammatory, anti-ferroptosis functions [15, 42–44]. In this study, we found that SBR have anti-arthritis ability through inhibiting RA-FLS inflammation and regulating macrophage polarization by JAK/STAT signal pathway. However, we found that the anti-arthritis ability did not increase with dose of SBR. Drug dose-effect relationship is various, one of common curves is inverted U-shaped curve [45], means that the therapeutic effect would decrease with the drug dosage beyond the maximum efficacy dosage. And we will further explore the relationship of concentration of SBR and anti-arthritis effect in CIA mice, and find the best dosage to avoiding potential adverse effect.

While our study provides novel insights into the RA treatment strategy, several limitations should be acknowledged. Firstly, the safety of SBR in CIA model did not observe. Secondly, the effects of SBR on other cells like T cell, balance of osteoclasts and osteocytes involved in the pathology of RA are unclear. Lastly, we examined the prophylactic treatment of SBR in CIA mice, but did not assess the effectiveness of SBR after the onset of arthritis. All these limitations highlight the need for additional research to comprehensively address the underlying complexities of SBR treatment for RA.

Conclusion

In summary, SBR ameliorated inflammation in RA-FLS and regulated macrophage polarization in RA. SBR also significantly mitigated clinical symptoms, joint pathological damage, and the expression of inflammatory cytokines in the synovium and serum of CIA mice. The mechanism underlying the anti-arthritic effects of SBR potentially involves the regulation of the JAK1/STAT3 and JAK1/STAT6 signaling pathways, thereby inhibiting synovial tissue inflammation, suppressing M1 macrophage polarization, and promoting M2 macrophage polarization. Our study revealed the potential pharmacological mechanisms of SBR, indicating its potential as a novel therapeutic agent for RA treatment.

Electronic supplementary material

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Abbreviations

RA

Rheumatoid arthritis

IL-1

Interleukin-1

IL-6

Interleukin-6

JAK

Janus kinase

FLS

Fibroblast-like synoviocytes

MMPs

Matrix metalloproteinases

TNF-α

Tumor necrosis factor-alpha

RA-FLS

RA-derived FLS

M1

Classical activation macrophage

M2

Alternative activation macrophage

IL-10

Interleukin-10

TGF-β

Transforming growth factor-β

SBR

Suberosin

BMDMs

Bone marrow-derived macrophages

DMEM

Dulbecco’s modified Eagle’s medium

FBS

Fetal bovine serum

RPMI

Roswell Park Memorial Institute

LPS

Lipopolysaccharide

IFN-γ

Interferon-γ

M-CSF

Macrophage colony-stimulating factor

ACR

American College of Rheumatology

H&E

Hematoxylin and eosin

PBS

Phosphate-buffered saline

ELISA

Enzyme-linked immunosorbent assay

RIPA

Radioimmunoprecipitation assay

STAT

signal transducer and activator of transcription

qRT-PCR

Quantitative real-time-PCR

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

PPI

Protein-protein interaction

Author contributions

HL was responsible for writing – review and editing, investigation, conceptualization, data curation, formal analysis, and funding acquisition. QL contributed to writing – original draft, methodology, project administration, validation, and visualization. YC contributed to methodology and supervision. MD provided supervision, methodology, and project administration. HL and JZ both participated in supervision and project administration. LY also contributed to supervision and project administration. GY and QX were involved in supervision and funding acquisition.

Funding

This study was supported by the Sichuan Science and Technology Program [grant numbers 2021JDRC0169]; Clinical Research Incubation Project of West China Hospital, Sichuan University [grant numbers 2021HXFH018]; and the 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University [grant number ZYJC21024].

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of West China Hospital, Sichuan University and the Animal Ethics Committee of the West China Hospital, Sichuan University. Informed written consent was secured from all participating individuals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.