Brevilin A, a novel LRRC15 inhibitor, exerts potent anti-rheumatoid arthritis effects by inhibiting the LRRC15/STAT3 signaling pathway

Abstract

Purpose

Leucine-rich repeat-containing 15 (LRRC15) is a transmembrane protein that is highly expressed in the synovium of patients with rheumatoid arthritis (RA). Brevilin A (BrA), an active compound isolated from Centipeda minima, exerts potent anti-inflammatory effects. However, the anti-RA effect of BrA and its underlying mechanism of action of BrA have not been fully elucidated.

Methods

Transcriptome analysis was performed to explore biomarkers of RA. An lipopolysaccharide (LPS)-induced RAW264.7 macrophage model, a TNF-α-stimulated RA fibroblast-like synoviocytes (RA-FLSs) model, as well as a collagen-induced arthritis (CIA) rat model were used to explore the anti-RA effects of BrA. Moreover, inhibition or overexpression of LRRC15 was performed to explore the role of LRRC15 signaling in the anti-RA effects of BrA.

Results

Transcriptome analysis of patients with RA revealed that LRRC15 expression was significantly upregulated in the synovial tissue of RA patients. BrA significantly downregulated the expression of inflammation-related markers in cell models, and inhibited their proliferation and migration; Moreover, it significantly reduced joint swelling and cartilage damage in CIA rats. Further mechanistic studies suggest that inhibition of LRRC15 inhibits cell proliferation and migration; and overexpression of LRRC15 increases the protein levels of STAT3’s downstream metastasis-related markers.

Conclusions

Our findings suggest that BrA, a novel LRRC15 inhibitor, has promising anti-RA activity and potently inhibits LRRC15/STAT3 signaling pathway both in vivo and in vitro. This study not only supports the development of BrA as a novel therapeutic agent for RA treatment, but also paves the way for the development of other LRRC15-targeting therapeutic strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13075-025-03629-1.

Introduction

Rheumatoid arthritis (RA) is a long-term autoimmune disease that predominantly affects the joints. It is a prevalent condition that affects approximately 1% of the global population [1]. The precise pathogenesis of RA is still unknown. Currently, it is believed to involve a combination of immunological, environmental and genetic factors. The pathological process of RA is typically divided into four stages: synovitis, pannus formation, cartilage damage, and bone erosion. During these stages, fibroblast-like synoviocytes (FLSs), which are located in the synovium, proliferate abnormally in an inflammatory environment and exhibit tumor-like invasiveness and migration. This is the major cause of cartilage damage in the joints. Additionally, FLSs release various adhesion molecules and inflammatory cytokines, which contribute to bone erosion and cartilage destruction. The migration of FLSs to unaffected cartilage also drives the progression of RA polyarthritis [2]. Without timely medical intervention, RA can cause significant disability and profoundly affects the quality of life of patients.

LRRC15 is a transmembrane protein that is highly expressed in the synovium of patients with RA [3]. Studies have reported that LRRC15 is activated in many types of cancer, including glioblastoma, sarcomas, ovarian cancer, etc [4]. The aberrant expression of LRRC15 is primarily found in cancer-associated fibroblasts (CAFs), which are related to cancer metastasis and immune response [5]. Targeting LRRC15 for drug development has emerged as a feasible strategy. The LRRC15-targeting drug ABBV-085 has been shown to inhibit tumor growth in clinical practice [6]. However, further investigations are needed to elucidate the relationship between LRRC15 and the progression of RA, and to explore whether LRRC15 signaling is involved in the anti-RA effects of BrA.

Currently, the first-line treatment options for RA include disease-modifying anti-rheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and other immunosuppressive medications. The aims of these drugs are to reduce inflammation, relieve pain, slow the progression of the disease, and improve joint function. Nevertheless, these drugs have various side effects, such as nauseaand potential damage to liver and kidney tissue. Hence, there is an urgent demand for the development of more effective and low-toxicity drugs for the treatment of RA.

Centipeda minima is a traditional Chinese herbal medicine frequently used to treat joint diseases. BrA is an active compound present in Centipeda minima that has been known to have several pharmacological activities, including antitumor, anti-inflammatory, immunosuppressive, and anti-liver fibrosis effects [7]. The inhibitory effect of BrA on the JAK/STAT3 pathway in various tumors has led to its recognition as a potent STAT3 inhibitor [8, 9]. BrA also exhibits excellent anti-inflammatory effects. It effectively alleviates LPS-induced lung damage, inflammatory infiltration, and the production of proinflammatory cytokines in LPS-induced acute lung injury [10]. In a DMM-induced osteoarthritis (OA) mouse model, BrA is reported to regulate the SIRT1/Nrf2/GPX4 signaling pathway to inhibit the production of MMP1 and MMP3 and alleviate joint damage in mice [11]. In this study, whether BrA exerts an anti-RA effect and its underlying molecular mechanism of action were investigated. We used an in vivo collagen-induced arthritis (CIA) model and in vitro TNF-α-induced FLS and LPS-induced RAW264.7 cell models to analyze the anti-RA effects of BrA and explore its underlying molecular mechanisms.

Methods and materials

Chemicals and reagents

BrA was purchased from Chengdu Desite Biotechnology Co. Ltd. (Sichuan, China; purity > 98%). LPS, dimethyl sulfoxide (DMSO) and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against COX-2, GAPDH and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against iNOS, MMP9 and LRRC15 were purchased from Abcam (Cambridge, MA, USA); and antibodies against MMP2, STAT3 and phospho-STAT3 (Tyr705) were purchased from Cell Signaling Technology (Danvers, MA, USA). The pEX1-LRRC15 overexpression plasmid and LRRC15-siRNA were obtained from Suzhou GenePharma Biotechnology Co., Ltd. (Suzhou, China). Enzyme-linked immunosorbent assay (ELISA) kits were obtained from Hangzhou MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China). TRizol, the Evo M-MLV RT Premix kit, SYBR Green Premix kit were purchased from Accurate Biology (Guangzhou, China). Recombinant Human TNF-α was obtained from PeproTech Inc. (Rocky Hill, NJ, USA). A hematoxylin-eosin (H&E) staining kit was obtained from Servicebio Technology Co., Ltd (Wuhan, China). Bovine type II collagen for CIA rat induction, incomplete Freund’s adjuvant (IFA) and complete Freund’s adjuvant (CFA) were purchased from Chondrex, Inc (Woodinville, WA, USA). Multimer anti-rabbit/mouse IgG HRP for immunohistochemistry (IHC) staining was purchased from Wuhan BOSTER Bioengineering Co., Ltd (Wuhan, China).

Cell culture

Human RA-FLSs and mouse RAW264.7 macrophages were purchased from Guangzhou Jennio Biotech Co., Ltd, and National Collection of Authenticated Cell Cultures (Shanghai, China), respectively. Cells were cultured in a humidified environment at 37℃ with 5% CO_2, they were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), and 1% penicillin/streptomycin (Gibco, USA).

Cell viability

The cytotoxicity of BrA to RA-FLSs and RAW264.7 cells was determined by the MTT assay. RA-FLSs (5 × 10³ cells/well) or RAW264.7 (8 × 10³ cells/well) were inoculated into 96-well plates. Cells were then treated with various concentrations of BrA (0.5, 1, 2, 4, 8, 16 and 32 µM) for 24 h. Then, the MTT solution (20 µL) was added to each well. After a 4-h incubation at 37℃, 150 µL of DMSO was added. Subsequently, the absorbance of each well was determined by utilizing a microplate spectrophotometer at 570 nm (BD Biosciences, USA).

Nitric oxide (NO) production

RAW264.7 cells were seeded in 12-well plates (1.5 × 10⁶ cells/well), incubated with LPS (1 µg/mL), or with both LPS and BrA (1, 2 and 4 µM) for 24 h. Then, Griess reagent was added to the cell supernatant after centrifugation to detect the NO level. The absorbance at 540 nm was measured using a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, USA).

Cell adhesion assay

The adhesion ability of RA-FLSs was analyzed using a cell adhesion detection kit (BestBio Science, Shanghai, China) according to the manufacturer’s protocol. In brief, the RA-FLSs (1 × 10⁵ cells/well) were inoculated into a 96-well plate pre-coated with Matrigel. Thirty minutes later, the non-adherent cells were discarded, and the absorbance of each well at 450 nm was detected. The cell adhesion rate was analyzed based on the absorbance.

Western blotting

Total protein of each sample was extracted and quantified, and equal amounts of the protein samples were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked in TBST for 2 h. After blocking, the membranes were incubated overnight with primary antibodies specific to the target protein. After washing, the membranes were incubated with the secondary antibodies. Finally, protein signals were determined using enhanced chemiluminescence (ECL) detection reagents (Genebase Bioscience Co., Ltd, Guangzhou, China), and the gray values of the bands were quantified using Image J software V1.44.

Reverse transcription-quantitative polymerase chain reaction (RT-PCR) analysis

Total RNA of RA-FLSs was extracted and reverse transcribed into cDNA using the TRizol and the Evo M-MLV RT Premix Kit. Finally, the mRNA expression level was determined using the SYBR Green Premix Kit in a QuantStudioTM 5 Real-time PCR system (Thermo Fisher Scientific Co., Ltd, MA, USA). Gene expression was normalized to GAPDH in each sample, and the results were analyzed via the 2^−ΔΔCT method.

ELISA

The levels of IL-6 and IL-1β released from LPS-induced RAW264.7 cells were detected using commercial ELISA kits according to the manufacturer’s protocol. Briefly, the cell culture supernatants were collected and incubated with the ELISA reagents. The absorbance was then measured to quantify the levels of cytokines.

EdU (5-ethynyl-2’-deoxyuridine) staining

According to our previous study [12], BrA at the indicated concentrations (1, 2 and 4 µM) was added to treat RA-FLSs for 24 h. Cells were seeded into 96-well plates (5 × 10³ cells/well) and stained with EdU for 12 h according to the experimental protocol (Beyotime Biotechnology Co., Ltd, Shanghai, China),after that,the EdU-positive cells were observed by fluorescence microscopy (Leica, Wetzlar, Germany) and counted using Image J software V1.44.

Wound healing assay

The migration of RA-FLSs was analyzed using a wound healing assay. RA-FLSs were seeded into a 6-well plate (2 × 10⁵ cells per well) and cultured until the cell density exceeded 90%. Scratches were created via the use of a 10 µL pipette tip. TNF-α (20 ng/mL) and different concentrations of BrA (1, 2 and 4 µM) were added to the wells. At 0, 12, 24 h, the scratch width was photographed with an inverted microscope (Leica, Wetzlar, Germany) and analyzed via ImageJ software V1.44. After 24 h of migration, the cells were fixed with paraformaldehyde and stained with crystal violet.

Small interfering RNA (siRNA) transfection

Negative control (NC) siRNA and LRRC15-siRNA were synthesized by Suzhou GenePharma Co., Ltd. (Suzhou, China). siRNA-NC or siRNA-LRRC15 was transfected into RA-FLSs using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, L3000015). Cells were seeded in a 6-well plate and transfected with 100 nM siRNA in 4 µL of Lipofectamine 3000 for 24 h. The transfection medium was discarded, and the cells were cultured in serum-free medium with or without 20 ng/mL TNF-α. Cells were harvested after incubation for an additional 36 h. The siRNA sequence targeting human LRRC15 was GCACAUCACUGAACUCAAUTT.

Plasmid transient transfection

The LRRC15 plasmid was transfected into RA-FLSs using Lipofectamine 3000 following the manufacturer’s instructions and preformed as previously described [13]. Briefly, RA-FLSs was plated in 6-well plates and transfected with plasmids for 48 h before functional assays were carried out.

Animal experiments

Male Wistar rats (6 weeks old, 160 g ± 20 g) were purchased from the Laboratory Animal Center of Southern Medical University [SCXK(GZ)2021-0041, Guangzhou, China] and housed in the animal laboratory at the International Institute for Translational Chinese Medicine [SYXK(GZ)2019 − 0144], Guangzhou University of Chinese Medicine (Guangzhou, China), and followed the WMA Statement on animal use in biomedical research. The registration number of ethical approval is 20,220,605.

Rats were randomly divided into five groups: control, CIA model, BrA-low dose (1.5 mg/kg, ip), BrA-high dose (3 mg/kg, ip), and positive control indomethacin (Indo) (2 mg/kg, ig) groups. Except for rats in the control group, others were used to establish a CIA model. Briefly, initial immunization involved an emulsion of CFA and bovine type II collagen at a 1:1 ratio. Rats in the five groups were given 0.2 mL of the CFA-containing emulsion by intradermal injection at the base of the tail. After 7 days, for the second immunization, IFA and bovine type II collagen were emulsified and injected in the same way as the first injection. Beginning on day 10, the rats in the treatment groups were treated with BrA or Indo. The rats in the control and CIA model groups received equivalent volumes of solvent. The body weight, hind paw volume, and arthritic indices were monitored every 3 days, and the scoring criteria and details for the arthritic indices were recorded as previously described [14]. All rats were subsequently sacrificed, and the blood, hindfoot, and synovial tissues of the knee joints were collected for subsequent analyses.

H&E and IHC staining

The ankle joints of each group were decalcified after fixation with 4% paraformaldehyde and subsequently embedded in paraffin. Synovial tissue was directly embedded in paraffin after fixation. All samples were sectioned into 4 μm slices, and stained with H&E to evaluate the condition of inflammatory cell infiltration and synovial hyperplasia. Histological-synovitis scores (HSS) and standardized microscopic arthritis scoring of histological (SMASH) were used to quantitatively assess the histological Sections [15, 16]. IHC staining was performed using anti-LRRC15 (1:200) or anti-pSTAT3 (1:200) antibodies at 4℃ for overnight. After incubation with the HRP-conjugated secondary antibody polymer for 2 h, the images were visualized with a histopathology slide scanner (Leica, Germany).

Radiological detection

X-rays can capture images of internal body structure. To assess the in vivo effects of BrA on joint inflammation and damage, X-ray images were performed with a SkyScan 1276 instrument (Bruker, Karlsruhe, Germany) with the following parameters: 75 mA of current and 65 kV of voltage.

Microcomputed tomography (Micro-CT)

Specimens of ankle joints were scanned using a Bruker Micro-CT SkyScan 1276 system (Kontich, Belgium) at 85 kV, 200 uA, Al 1 mm filter, 10.133345 μm voxel size and 384 ms integration time. Images were analyzed with CT Analyzer software 1.20.3.0. The resulting reconstructed images were subsequently conducted by NRecon software 1.7.4.2.

Radiographic assessments

The Sharp/van der Heijde modified Sharp score (mTSS) [17] was used to score structural damage in the joint by two experienced observers who were blinded to clinical data. Subjects with joint erosion > 0 or joint space narrowing > 0 were considered to have radiographic joint damage.

Statistical analysis

Data were shown as Mean ± SD. Data analyses were conducted using GraphPad Prism 8 (GraphPad Software, San Diego, CA, US). A T-test was used to compare the two groups. When three or more groups were compared, ANOVA tests with Tukey’s multiple comparisons tests to used to calculate adjusted P values. P < 0.05 was considered statistically significant in all tests.

Results

LRRC15 is involved in the proliferation, migration and adhesion of RA-FLSs

Analyses of RNA-seq data from RA patients in the GEO database (Fig. 1A) revealed that LRRC15 expression was significantly upregulated (Fig. 1B). To explore the role of LRRC15, we analyzed whether LRRC15 participated in the deterioration of RA by knocking down LRRC15 with siRNA (Fig. 1C-E) or overexpressing LRRC15 (Fig. 1H). Results showed that knockdown of LRRC15 significantly inhibited the adhesion (Fig. 1E), proliferation (Fig. 1F) and migration (Fig. 1G) of RA-FLSs. In contrast, overexpression of LRRC15 in RA-FLS significantly increased the mRNA levels of two metastasis markers including MMP9 (Fig. 1I) and MMP2 (Fig. 1J). These results suggest that LRRC15 promotes the proliferation and migration of RA-FLSs, and participates in the deterioration of RA.

Fig. 1.

LRRC15 is involved in RA-FLSs proliferation and migration. (A-B) Venn diagram depicts differential gene expression in the RNA-seq database from synovial tissues of four arthritis patients (A), and the expression levels of LRRC15 were shown in boxplots in each dataset (B). (C) The protein levels of LRRC15 were determined after siRNA of NC or LRRC15 in TNF-α-induced RA-FLSs using Western blotting; and the quantitative results were shown in the right panel. (D) The mRNA levels of LRRC15 LRRC15 were determined after siRNA of NC or LRRC15 in TNF-α-induced RA-FLSs using RT-qPCR analysis. (E-G) RA-FLSs adhesion (E), proliferation (F) and migration (G) were determined after siRNA of NC or LRRC15 in TNF-α-induced RA-FLSs; and the quantitative results were shown in the right panel. (H) The protein levels of LRRC15 were determined via RT-PCR analysis after overexpression of LRRC15 in TNF-α-induced RA-FLSs using Western blotting; and the quantitative results were shown in the right panel. (I-J) The mRNA levels of MMP2 and MMP9 were determined after overexpression of LRRC15 using the RT-qPCR analysis. Data are shown as Mean ± SD from three individual experiments. For B: ^**P < 0.01 vs. The corresponding control. For C-F: ^*P < 0.05, ^**P < 0.01 vs. control. ^#P < 0.05, ^##P < 0.01 vs. NC group. For G: ^##P < 0.01 vs. NC -pEX-1 group

BrA exerts anti-inflammatory effects in vitro

BrA is an active compound in Centipeda minima that has been known to have potent anti-tumor effects. Here, we determined whether it had anti-RA effects. Inflammation is the most prominent symptom of RA. First, to assess the in vitro anti-inflammatory effects of BrA, we screened the appropriate concentration of BrA and measured NO release in LPS-induced RAW264.7 cells. Results showed that 1, 2 and 4 µM of BrA (cell cytotoxicity < 15%, Fig. 2A) inhibited the NO production in a dose dependent manner (Fig. 2B). BrA also significantly inhibited the secretion of the inflammatory cytokines including IL-1β and TNF-α after BrA treatment (Fig. 2E-F). Moreover, the expression of the inflammatory markers COX-2 and iNOS was upregulated by LPS stimulation in RAW264.7 cells, and downregulated after treated with BrA (Fig. 2C-D). Similarly, 1, 2 and 4 µM of BrA (cell cytotoxicity < 15%, Fig. 2G) also dose-dependently decreased the protein levels of COX-2 and iNOS in the TNF-α-stimulated RA-FLSs model (Fig. 2H-I). These results suggest that BrA exerts potent anti-inflammatory effects.

Fig. 2.

BrA significantly inhibited the inflammatory response in RAW264.7 cells and RA-FLSs. (A, G) The cell viability of BrA in RAW264.7 (A) and RA-FLSs (G) were determined by using the MTT assay. (B) Measurement of NO production in the supernatant of RAW264.7 cells using the Griess reagent. (C, H) The protein levels of iNOS and COX2 in RAW264.7 cells (C) and RA-FLSs (H) were determined by Western blotting; and the quantitative results (D, I) were shown in the right panel. (E, F) The levels of IL-1β (E) and TNF-α (F) in supernatant of RAW264.7 cells were determined by the ELISA assay. Data are shown as Mean ± SD from three individual experiments. ^**P < 0.01 vs. The corresponding control. ^#P < 0.05, ^##P < 0.01 vs. LPS or TNF-α group.

BrA inhibits the proliferation, migration and adhesion of RA-FLSs

Studies have demonstrated that FLSs in the synovial lining develop aggressive phenotypes and produce pathogenic mediators, which results in the occurrence and progression of disease, and plays a major role in the pathophysiology of RA [18]. Activated RA-FLSs have “tumor-like” characteristics, such as promotion of inflammation, resistance to cell death, vigorous proliferation, enhanced migration, and invasion ability to joint cartilage and bone [19]. Herein, we assessed the proliferation, migration and adhesion of RA-FLSs using EdU assay, migration assay and cell adhesion assay, respectively. The results of the EdU assay revealed that BrA inhibited the TNF-α-enhanced RA-FLSs proliferation in a dose-dependent manner. (Fig. 3A). MMP-2 and MMP-9, two members of the MMP family, promoted the formation and invasion of pannus during RA progression [20]. As shown in Fig. 3B-C, we found that the protein levels of MMP-2 and MMP-9 were dramatically increased in the TNF-α-induced model group when compared to the control group. However, BrA significantly decreased the protein levels of these two markers. Additionally, cell adhesion assay revealed that BrA significantly suppressed TNF-α-induced RA-FLSs adhesion (Fig. 3D). Furthermore, BrA also effectively attenuated the enhanced migration caused by TNF-α in RA-FLSs (Fig. 3E-F).

Fig. 3.

BrA suppressed RA-FLSs proliferation and migration. (A) The proliferation of RA-FLSs was determined using the EdU assay. Representative images were shown in the left panel, and the numberof EdU-positive cells was shown in the right panel. (B-C) The protein levels of MMP-2 and MMP-9 in TNF-α-induced RA-FLSs were determined using Western blotting (B), and the quantitative results were shown in the right panel (C). (D) Cell adhesion of RA-FLSs after treated with BrA for 30 min was determined using a cell adhesion kit. (E-F) RA-FLSs migration ability was determined using wound healing assay after 24-h treatment, and photographed by microscopy. Data are shown as Mean ± SD from three individual experiments. ^*P < 0.05, ^**P < 0.01 vs. The corresponding control. ^#P < 0.05, ^##P < 0.01 vs. TNF-α group.

BrA ameliorates arthritis symptoms in CIA rats

Next, we established a CIA rat model to determine the anti-RA therapeutic effect of BrA. Compared with control group, a significant decrease in body weight was observed in the model group; whereas, the body weights of the rats in the BrA- or Indo-treated groups was lower than that of the rats in the model group (Fig. 4A). After immunizations for CIA modeling, the rats in all groups except for those in the control group displayed significant swelling of the paw (Figs. 4C-D). Following treatment with different concentrations of BrA, we found that the degree of feet swelling was significantly reduced in rats (Fig. 4C-D). In accordance with the paw swelling results, the arthritis scores also tended to decrease after the rats received BrA treatment (Fig. 4B). X-ray imaging and micro-CT revealed significant bone erosion in the ankles of CIA rats, an effect that was attenuated by the administration of BrA or Indo, respectively (Fig. 4E-F, Supplemental Fig. 1A). In addition, H&E staining (Fig. 4G) and HSS scoring (Supplementary Fig. 1B-C) of the synovial membrane revealed that BrA strongly alleviated synovial hyperplasia and monocyte infiltration and suppressed the angiogenesis. The mitigating effect of BrA on collagen-induced subchondral bone loss, inflammation in cartilage and synovium was subsequently evaluated by H&E staining of knee joints (Fig. 4H, Supplementary Fig. 1C). The results showed that BrA ameliorated the arthritis symptoms in CIA rats by inhibiting joint inflammation and bone erosion.

Fig. 4.

BrA relieved joint inflammation and joint damage in CIA rats. (A) Body weight, (B) arthritis indexes, as well as the (C, D) left and right paw volume of CIA rats were recorded. (E) X-ray and (F) micro-CT were used to observe the rat paws. (G-H) Representative H&E-stained images of synovial tissue (G), and knee joints (H) of CIA rats. Green arrows represent the angiogenesis in the synovial tissue, and theblue arrows represent the inflammatory infiltration in the knee joint tissues, respectively. Data are shown as Mean ± SEM, n = 6. ^**P < 0.01 vs. CTL group. ^#P < 0.05, ^##P < 0.01 vs. Model group

The LRRC15/STAT3 signaling pathway is involved in the anti-RA effects of BrA both in vitro and in vivo

To investigate whether BrA affects RA-FLSs via LRRC15, we examined the impact of BrA on LRRC15 expression. BrA significantly reduced the protein levels of LRRC15 in TNF-α-induced RA-FLSs (Fig. 5A). Recent studies have shown that LRRC15 expression in fibroblasts is linked to JAK/STAT3 pathway activation [5]. Another study suggested that microglia-derived LRRC15 can activate the JAK/STAT3 pathway by binding to CD248 on the surface of progenitor cells, thereby influencing astrogenesis [21]. It follows that JAK/STAT3 pathway activation has been well documented to play an important role in the progression of RA. Thus, we evaluated the protein levels of phosphorylated STAT3, and found that BrA significantly inhibited STAT3 phosphorylation without affecting total STAT3 protein levels (Fig. 5B). Moreover, IHC staining of the synovial tissues from rats confirmed these findings (Fig. 5C). Collectively, these results suggest that BrA has anti-RA effects via suppressing the LRRC15/STAT3 signaling pathway.

Fig. 5.

The LRRC15/STAT3 signaling pathway is involved in the anti-RA effects of BrA. (A-B) The protein levels of LRRC15 (A), p-STAT3 and STAT3 (B) after treatment with different concentrations of BrA were determined using Western blotting; and the quantitative results were shown in the right panel. (C) Representative images of LRRC15 and p-STAT3 in the synovial tissues of rats were stained using IHC staining. (D) The mechanism of the anti-RA action of BrA. BrA exerts anti-RA effects both in vitro and in vivo, and these effects are partially due to the inhibition of LRRC15/STAT3 signaling pathway. The red arrow represents the trend after BrA treatment. Data are shown as Mean ± SD from the individual experiments. *P < 0.05 vs. control. ^#P < 0.05, ^##P < 0.01 vs.TNF-α group

Discussion

RA is a chronic and systemic autoimmune disease that is characterized by persistent inflammation of the synovial joints, resulting in cartilage destruction, bone erosion, and progressive joint deformity. Despite significant advancements in understanding this disease, the pathogenesis of RA remains unknown. Current studies indicate that a complex interplay of environmental, genetic and immunological factors contributes to the initiation and progression of RA [22]. This multifactorial nature, combined with the involvement of multiple immune pathways, makes the identification of a singular cause or mechanism that drives the disease more complicated. One of the major challenges in the management of RA is the inability to achieve complete remission with conventional drug therapies. Traditional DMARDs such as methotrexate, sulfasalazine, and leflunomide, as well as NSAIDs and corticosteroids, are commonly prescribed to alleviate symptoms and slow the demonstrated that LRRC15 is highly expressed progression of the disease. Although these treatments can improve the quality of life of patients and reduce inflammation, they often fail to stop disease progression completely, leading to long-term joint damage and disability [23].

In recent years, natural compounds derived from plants and other natural sources have drawn increasing attention for their potential in the treatment of RA. BrA, a sesquiterpene lactone isolated from Centipeda minima, exhibits diverse pharmacological activities, including antitumor [24], anti-hepatic fibrosis [25] and anti-OA [11] effects. In addition, BrA is reported to have a broad inhibitory effect on acute and chronic inflammation; it can not only reduce the inflammatory status of the wound but also inhibit bacterial wound infection [26]. Recent studies have also highlighted the therapeutic potential of BrA in joint disorders. In models of OA, BrA inhibits osteoclast differentiation via mTOR and ERK signaling [27] or by modulating the SIRT1/Nrf2/GPX4 axis, thereby alleviating OA symptoms [11]. These findings support the role of BrA as a promising therapeutic candidate for the treatment of RA; However, the molecular mechanisms underlying RA pathogenesis remain to be fully elucidated. Here, it is found that BrA significantly reduced inflammatory response, inhibited cell proliferation and cell migration of RA-FLSs (Figs. 2 and 3). Moreover, in CIA mice, BrA can significantly reduce the joint swelling and cartilage damage. More importantly, no toxicity was observed after BrA treatment (Fig. 4A), suggesting that BrA has the potential to be an antiarthritic drug with no toxicity. In the future, the BrA content in the serum of animal models will be measured to further investigate its in vivo pharmacokinetics and biological activity. A thorough understanding of the anti-RA efficacy of BrA and its in vivo biological processes can facilitate its future clinical application for RA treatment.

FLSs are of vital importance for joint destruction in RA, and aggressive FLSs as well as pannus formation are crucial for progressive destruction. RA-FLSs also secrete various proinflammatory cytokines and MMPs, which activate local FLS and induce FLS hyperplasia and migration [28]. Recent studies have reported that LRRC15 can interacts with CD248 to activate the JAK/STAT3 signaling pathway and influences astrogenesis, inducing anxiety-like behaviors such as exploration disorders and social disorders [21]. JAK/STAT3 pathway activity has been shown to be critical in promoting inflammation and mediating the cellular responses that contribute to disease progression [29]. Persistent activation of the JAK/STAT3 pathway was shown to be an important symptom in maintaining chronic inflammation in RA patients. However, whether LRRC15 contributes to RA pathogenesis by influencing STAT3 activity requires further studies. Here, it is found that LRRC15 expression is obviously upregulated in RA patients’ synovial tissues (Fig. 1B) and in TNF-α-induced RA-FLSs model (Fig. 1C). Moreover, inhibition of LRRC15 decreased the protein level of STAT3, resulting in the significant inhibition of cell proliferation and migration. Future studies will be performed to screen Chinese medicinal plants, such as Centipeda minima, for LRRC15-targeted compounds and elucidate their molecular mechanisms of anti-RA action.

Conclusion

In summary, we demonstrated that LRRC15 is highly expressed in RA, and is contributes to the migration and proliferation of RA-FLSs. BrA significantly ameliorated arthritis symptoms in CIA rats by reducing joint cartilage damage and synovial inflammation, while it also inhibited the proliferation, adhesion and migration of RA-FLSs. Mechanistic studies demonstrated that BrA significantly inhibited the LRRC15/STAT3 signaling pathway. Moreover, overexpression of LRRC15 diminished the anti-RA effects of BrA. Our results indicate that BrA exerts anti-RA effects both in vitro and in vivo, primarily due to the inhibition of the LRRC15/STAT3 signaling pathway. Our findings not only provide molecular-level justification for the clinical application of BrA, but also suggest its potential as a complementary or alternative therapeutic agent for RA management.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BrA

Brevilin A

CIA

Collagen-induced arthritis

CRC

Colorectal cancer

CAFs

Cancer-associated fibroblast

CFA

Complete Freund’s adjuvant

DMARD

Disease-modifying anti-rheumatic drug

ELISA

Enzyme-linked immunosorbent assay

DMEM

Dulbecco’s modified Eagle medium

EdU

5-ethynyl-2’-deoxyuridine

FLS

Fibroblast-like synoviocytes

H&E

Hematoxylin-eosin

HSS

Histological-synovitis scores

Indo

Indomethacin

IHC

Immunohistochemistry

IFA

Incomplete Freund’s adjuvant

LPS

Lipopolysaccharide

LRRC15

Leucine-rich repeat-containing 15

Micro-CT

Microcomputed tomography

MMPs

Matrix metalloproteinases

NC

Negative control

NSAID

Non-steroidal anti-inflammatory drugs

NO

Nitric oxide

RA

Rheumatoid arthritis

OA

Osteoarthritis

PVDF

Polyvinylidene fluoride

RA-FLS

Rheumatoid arthritis-associated fibroblast like synoviocytes

SMASH

Standardized microscopic arthritis scoring of histological

TNF-α

Tumor necrosis factor-alpha

Author contributions

Q.ZP. and M.QQ. performed the experiments and analyzed the data, X.BX. and L.YL. participated in some experimental studies; S.T.and Q.ZP. wrote the original manuscript; S.T. and L.Q. conceived and supervised the study, and approved the final manuscript.

Funding

This work was supported by the Young Pearl River Scholar of Guangdong Province, Chinese Medicine Guangdong Laboratory (HOL2024PZ042), Guangzhou Basic and Applied Basic Research Foundation (2025A04J5398), Administration of Traditional Chinese Medicine of Guangdong Province (20241074) and Characteristic Innovation Projects of Universities in Guangdong Province (2023KTSCX023).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval

The ethical approval on humans was not required for the studies on humans, following the local legislation and institutional requirements because only commercially available established cell lines were used. The animal experiment was approved by the Ethics committee of the Guangzhou University of Chinese Medicine, Guangzhou, China (No. 20220605).

Consent for publication

Not applicable.

Clinical trial number

Not applicable.

Competing interests

The authors declare no competing interests.