Targeting mitochondrial fission-autophagy axis in rheumatoid arthritis: leflunomide promote apoptosis of fibroblast-like synoviocytes

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia and joint destruction, driven by aberrantly activated fibroblast-like synoviocytes (RA-FLS). Mitochondrial dysfunction, particularly excessive mitochondrial fission, contributes to RA-FLS activation and apoptosis resistance, yet the impact of disease-modifying antirheumatic drugs (DMARDs) on mitochondrial dynamics remains unclear. Here, we examined the correlation between mitochondrial dynamics proteins and RA disease activity and investigated the effects of leflunomide and methotrexate (MTX) on mitochondrial dynamics, autophagy, and apoptosis in RA-FLS and collagen-induced arthritis (CIA) mice. Mitochondrial dynamics proteins in synovial fluid correlated more strongly with disease activity than those in peripheral blood, and were partially normalized in RA patients receiving leflunomide or MTX. Both leflunomide and MTX attenuated TNF-induced mitochondrial fragmentation and decreased mitochondrial membrane potential in RA-FLS. Notably, leflunomide, but not MTX inhibited DRP1 phosphorylation at Ser616, increased reactive oxygen species, and induced apoptosis via the BCL-2/BAX/caspase-3 pathway. Additionally, leflunomide influenced autophagy by promoting LC3B II/I and enhancing p62 expression. In CIA mice, leflunomide reduced the expression and phosphorylation of DRP1 on synovium, increased the expression of OPA1, and alleviated joint inflammation and destruction. These findings identify mitochondrial dynamics as a therapeutic target in RA and suggest that leflunomide promotes apoptosis of RA-FLS by modulating the mitochondrial fission-autophagy axis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13075-026-03828-4.

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation, progressive joint destruction, and pain, primarily affecting synovial joints [1]. Fibroblast-like synoviocytes (FLS) are key effector cells in RA pathogenesis, driving synovial hyperplasia, cartilage degradation, and bone erosion [2]. RA-FLSs actively promote inflammation through cytokine secretion and exhibit aggressive, tumor-like behaviors, including apoptosis resistance and excessive proliferation, which together contribute to joint destruction and disease progression [3, 4]. Despite extensive investigation, the molecular mechanisms underlying RA pathogenesis remain incompletely understood.

Accumulating evidence has linked mitochondrial dysfunction to the pathogenic activation of RA-FLS [5–7]. Our previous work demonstrated that excessive mitochondrial fission plays a critical role in regulating cell survival, autophagy, and inflammatory responses in RA-FLS, and that inhibition of mitochondrial fission alleviates arthritis severity in collagen-induced arthritis (CIA) mice [8]. However, whether current disease-modifying anti-rheumatic drug (DMARD) therapies modulate mitochondrial dynamics in RA has not been fully elucidated.

Mitochondrial dynamics, the coordinated balance between mitochondrial fission and fusion—are central to mitochondrial function and cellular homeostasis [9]. DRP1 (dynamin-related protein 1) is the primary mediator of mitochondrial fission, whereas MFN1/2 and OPA1 are essential regulators of mitochondrial fusion [10]. Disruption of this balance leads to mitochondrial fragmentation, oxidative stress, and impaired cellular function, contributing to the development of diverse pathological conditions, including cardiovascular disease, cancer, and metabolic disorders [11–13]. Modulation of mitochondrial dynamics has been implicated in the regulation of metabolic homeostasis and organismal lifespan. Aerobic exercise has been reported to suppress DRP1 activation and mitochondrial fission, thereby improving fatty acid oxidation and insulin sensitivity [14]. Irisin is a hormone-like factor secreted by muscle cells, and exercise promotes its production. In our previous study, the exercise-induced myokine irisin inhibited proliferation, migration, invasion, and inflammatory signaling in RA-FLS through modulation of the YAP–DRP1 pathway [15]. We also found that inhibiting FASN-mediated fatty acid synthesis in RA reduced phosphorylation of DRP1 on Ser616 [16].

DRP1 activity is tightly regulated by post-translational modifications, including phosphorylation, SUMOylation, ubiquitination, and S-nitrosylation [17]. During mitosis, DRP1 is activated through phosphorylation at Ser616 by cyclin-dependent kinase 1 (CDK1) and protein kinase C-δ (PKCδ), promoting mitochondrial fission. Beyond regulating mitochondrial morphology, mitochondrial fission participates in multiple cellular processes, including mitophagy, cell division, and apoptosis [18]. In contrast, mitochondrial fusion facilitates the mixing and exchange of mitochondrial contents, preserving mitochondrial integrity and function [19]. Mitophagy is essential for mitochondrial quality control and for preventing the release of pro-apoptotic factors from dysfunctional mitochondria.

A tightly regulated balance exists between mitophagy and apoptosis in maintaining cellular homeostasis. Autophagic capacity and mitochondrial heterogeneity critically determine how BNIP3-mediated mitophagy modulates apoptotic signaling [20]. Disruption of this mitophagy–apoptosis crosstalk leads to cellular injury, as evidenced by brain damage in chickens resulting from impaired mitochondrial quality control [21]. Moreover, restoring the balance between mitophagy and apoptosis has been shown to attenuate H₂O₂-induced oxidative stress and apoptosis in alveolar epithelial cells [22].

Current RA treatment strategies rely primarily on DMARDs, such as methotrexate (MTX) and leflunomide, which effectively suppress inflammation and slow joint damage. However, the mechanisms underlying their therapeutic effects remain incompletely defined. Given the emerging importance of mitochondrial dynamics in RA pathogenesis [8, 15, 23], it is critical to determine whether these conventional therapies exert their effects, at least in part, through modulation of mitochondrial function.

In this study, we investigated the effects of leflunomide’s active metabolite A771726 and MTX on mitochondrial dynamics, autophagy, and apoptosis in RA-FLS, focusing on key mitochondrial regulators including DRP1, MFN1, and OPA1. We hypothesize that these agents modulate the mitochondrial fission-fusion balance, thereby influencing RA-FLS pathogenic behavior. By elucidating the relationship between mitochondrial dynamics and RA therapy, this study aims to provide new mechanistic insights into DMARD action and to highlight mitochondrial dynamics as a potential therapeutic target in RA.

Materials and methods

Patient samples and demographics

Serum and synovial fluid samples were collected from participants at the Department of Rheumatology and Immunology, Second Affiliated Hospital of Harbin Medical University. A total of 42 participants were enrolled, including 8 healthy controls and 34 RA patients. Synovial biopsies were obtained from RA patients undergoing knee arthroplasty. All RA patients fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA and provided written informed consent. Ethical approval (Ethics Number: Ky2020-151) was obtained from the Second Affiliated Hospital of Harbin Medical University. Patient demographics are summarized in Table 1.

Table 1.

Demographic and clinical characteristics of RA patients

Item	early-DMARD naïve RA (n = 13)	Leflunomide-treated RA (n = 11)	MTX-treated RA (n = 10)
Age (y)
Range	26–82	40–71	36–68
Mean ± SD	52.85 ± 16.29	55.1 ± 9.87	51.09 ± 10.01
disease duration (y), Median (IQR)	11 (3–15)	11 (6.5-14.75)	11 (7-12.5)
Female, No.(%)	8 (61.53%)	9 (90%)	8 (72.73%)
DAS28-ESR, mean ± SD	5.52 ± 1.41	3.22 ± 1.24**	4.04 ± 1.74*
HAQ Score	0.40 ± 0.42	0.41 ± 0.24	0.15 ± 0.12
C-Reactive protein (mg/L), Median (IQR)	34.3 (10.1–66.1)	7.36 (4.08–16.4)	10.7 (3.88–37.9)
IgM-rheumatoid factor-positive, No. (%)	11 (84.62%)	7 (70%)	8 (72.73%)
Anti-cyclic citrullinated peptide-positive, No. (%)	11 (84.62%)	8 (80%)	10 (90.91%)
Erythrocyte sedimentation Rate, mm/hour	158.08 ± 32.64	40.2 ± 14.27***	41.73 ± 22.32***

DAS28-ESR  Disease Activity Score-28 for Rheumatoid Arthritis with Erythrocyte sedimentation Rate, IQR interquartile range, SD standard deviation. Categorical data are expressed as the number (%); continuous data distributed normally are expressed as the mean ± SD; continuous data distributed non-normally are expressed as the median (IQR). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to early-DMARD naïve RA, the p values were determined by one-way ANOVA test

Enzyme linked immunosorbent assay (ELISA)

Protein levels in the serum and synovial fluid for DRP1 and MFN1 were quantified using human ELISA kits (Wuhan Boster Bioengineering Co., Ltd.), following the manufacturer’s instructions.

Cell preparation

Synovial tissue was minced and digested with 1 mg/ml type I collagenase at 37 °C for 3 h to isolate FLSs. Collagenase digestion was neutralized with DMEM/F12 containing 10% FBS, and the cell suspension was filtered through a 70 μm strainer. Cells were cultured in DMEM/F12 with 10% FBS at 37 °C and 5% CO2. Only cells from passages 3 to 6 were used in the experiments. Additionally, the MH7A human RA-FLS cell line, derived from primary FLS cultures, was used in some experiments.

Cell viability assay

Cell viability was assessed using the CCK-8 assay kit. RA-FLSs (6 × 10³ cells/well) were seeded into 96-well plates and incubated overnight. The cells were then treated with either the control vehicle or various concentrations of A771726 (1, 10, 100 µg/mL) or MTX (0.1, 1, 10 µM) in triplicate for 24 h. During the final 2 h of incubation, 100 µL of F12 medium containing 10 µL of CCK-8 solution was added to each well. The absorbance at 450 nm was measured using a microplate reader to determine cell viability.

RT-qPCR

Total RNA was extracted from RA-FLS using the Transcriptor First Strand cDNA Synthesis Kit (Roche LifeScience), following the manufacturer’s instructions. Reverse transcription quantitative PCR (RT-qPCR) was performed using the Bio-Rad CFX96 system. Primer sequences used for amplification are listed in Table 2. Gene expression levels were quantified by normalizing Ct values to the endogenous reference gene ACTIN (ΔCt = Ct_target - Ct_ACTIN). Relative expression was calculated using the ΔΔCt method, with the calibrator serving as a reference (ΔΔCt = ΔCt_sample - ΔCt_calibrator). Each experiment was performed in at least three independent replicates to ensure reproducibility.

Table 2.

The sequences of primers

β-ACTIN	forward: 5’-CTCCATCCTGGCCTCGCTGT-3’ reverse: 5’-GCTCTCACCTTCACCGTTCC-3’
DNM1L	forward: 5’-TAGCAGCAGTGACAGCGAGGA-3’
	reverse: 5’-GGGAGTAAGCCCTGAACCAAT-3’
MFN1	forward: 5′-CAGTGGGAAGAGCTCTGTTATC-3′ reverse: 5′-TCTCCATCAGTTCCTTCAACAC-3′
MFN2	forward: 5′-AGATTACGGAGGAAGTGGAGA-3′ reverse: 5′-GGTGGAAGTCCATCTGGTAATC-3′
OPA1	forward: 5′-CCACAGACAAACTTGGGTTCTA-3′ reverse: 5′-GAGGAAGTGCTGATTCCTGTC-3′

Cell stimulation and drug treatment

RA-FLS under basal conditions exhibit a relatively low activation state, to maintain the inflammatory phenotype of RA-FLS and mimic the in vivo pathological microenvironment, cells were stimulated with TNF (10 ng/mL) in functional and mechanistic assays. A771726 and MTX were administered followed TNF stimulation to evaluate their therapeutic effects under inflammatory conditions.

Western blot analysis

RA-FLSs (2.5 × 10⁵ cells/well) were treated with A771726 (50 µg/ml) and MTX (10µM) for 48 h, followed by treatment with TNF (10 ng/ mL) for 30 min. Cells from different experimental groups were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors, followed by centrifugation to remove cell debris. Protein concentration was quantified using the Thermo Fisher Scientific protein assay kit. Equal amounts of protein (30 µg per lane) were resolved on 10% or 12.5% SDS-PAGE gels, then transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon) by electrophoresis. Proteins were denatured by mixing with Protein Loading Buffer (TRANS) and heating at 95 °C for 10 min. Membranes were incubated overnight at 4 °C with primary antibodies targeting MFN1 (14739, Cell Signaling Technology), MFN2 (11925, Cell Signaling Technology), OPA1 (80471, Cell Signaling Technology), DRP1 (8570, Cell Signaling Technology), Phospho-DRP1 (Ser616) (4494 S, Cell Signaling Technology), Actin (4970, Cell Signaling Technology). Primary antibodies were diluted in antibody dilution buffer (Beyotime). Following primary antibody incubation overnight, membranes were incubated with appropriate secondary antibodies at room temperature for 1 h. Protein bands were visualized using Enhanced Chemiluminescence (ECL) detection (GE Healthcare). Each experiment was performed in at least three independent replicates to ensure reproducibility.

Mitochondrial morphology assessment by confocal microscopy

Mitochondrial morphology in different RA-FLS groups was analyzed using confocal microscopy (Zeiss 800) following MitoTracker Green fluorescent staining, performed according to the manufacturer’s protocol. Specifically, 1 × 10⁴ FLSs were seeded into each well of a chamber slide and cultured for 24 h. Cells were then incubated with MitoTracker Green for 40 min at 37 °C in the dark. After staining, mitochondrial morphology was visualized and documented using a confocal microscope. Mitochondrial membrane potential was assessed using a JC-1 detection kit (Beyotime, C2006) according to the manufacturer’s protocol. Cells from different groups were stained with JC-1, and fluorescence microscopy was used to visualize and evaluate the mitochondrial membrane potential based on the fluorescence signal.

Apoptosis and ROS detection

RA-FLSs (1 × 10⁵ cells/well) were treated with A771726 (50 µg/ml) or MTX (10 µM) for 48 h, in the presence of TNF (10 ng/mL). Apoptosis was assessed by flow cytometry using Annexin V-Alexa Fluor 647 and DAPI staining. Briefly, cells were incubated with 0.5 µL Annexin V-Alexa Fluor 647 and 4 µL DAPI at 4 °C for 15 min in the dark. The percentage of apoptotic cells was then determined by flow cytometry. Cellular ROS levels were assessed using the fluorescent probe 2’,7’-Dichlorodihydrofluorescein diacetate (H2DCF-DA). RA-FLS were incubated with 0.5 µM H2DCF-DA at 37 °C in the dark for 30 min. The average FITC fluorescence intensity of cells in each group was then measured by flow cytometry.

Induction of CIA and treatment

DBA/1 mice (GemPharmatech Co., Ltd.) were housed in a specific pathogen-free environment with a 12-hour light/dark cycle at 22–26 °C and 40–65% humidity. CIA was induced by subcutaneous injection of an emulsion containing bovine type II collagen (CII, 2 mg/ml in 0.05 M acetic acid) mixed with complete Freund’s adjuvant (Chondrex, Redmond, WA, USA) on day 0. A booster injection of CII (0.1 ml) emulsified in Freund’s incomplete adjuvant was administered on day 21. Concurrent drug treatments include MTX (1 mg/kg) by gavage weekly and leflunomide (10 mg/kg) by gavage every other day. All procedures were approved by the Ethics Committee of Guangdong Provincial People’s Hospital.

Clinical and histomorphometric analysis of arthritis

Mice were scored every other day post-booster injection until sacrifice on day 37. Arthritis severity was graded on a scale of 0 to 4, and paw thickness was measured using a vernier caliper. On day 37, mice were euthanized, and their ankle joints were collected and fixed in 10% formalin. Joint tissue sections were stained with H&E, Safranin O, and TRAP as previously described. Ankle joints from CIA mice and healthy controls were fixed in 4% paraformaldehyde overnight and decalcified for 12 days in EDTA decalcification solution (Servicebio, G1105) at 22–24 °C. The joints were embedded in paraffin, sectioned into 4 μm slices, and stained. H&E staining was used to evaluate inflammatory cell infiltration and synovial angiogenesis, Safranin O (Sigma-Aldrich, S8884) to assess cartilage damage, and TRAP staining was performed for 40 min at 22–24 °C, followed by hematoxylin counterstaining.

Immunohistochemistry (IHC) analysis

Joint Sect.  (4 μm) were rehydrated and treated with 3% H₂O₂ for 5 min. Antigen retrieval was performed by boiling the sections in 0.01 M sodium citrate buffer (pH 6.0). After blocking with 10% goat serum, sections were incubated overnight at 4 °C with primary antibodies: anti-DRP1 (1:200, ab184247, Abcam), anti-phospho-DRP1 (Ser616) (1:200, Affinity, AF8470) and anti-OPA1 (1:200, ab157457, Abcam). The sections were then incubated with species-specific HRP-conjugated secondary antibodies for 1 h at room temperature. After staining with 3,3’-diaminobenzidine (DAB substrate kit # ab64238) and counterstaining with hematoxylin, images were captured using a Leica pathology slide scanner (Aperio CS2).

Statistical analysis

Data are presented as the mean ± SEM from three or more independent experiments. Comparisons between two groups were made using the independent t-test, while differences among three or more groups were analyzed by one-way or two-way ANOVA. Pearson correlation coefficients were used to assess correlations. Statistical significance was set at p < 0.05. All analyses were conducted using GraphPad Prism version 10.0.

Results

Leflunomide and methotrexate reduce DRP1/MFN1 ratio in the serum and synovial fluid of RA patients

RA synovial tissue is typically hypoxic. Based on our previous observation that RA-FLSs display enhanced mitochondrial fission and that DRP1 inhibition ameliorates arthritis in CIA mice [8], we aimed to examine whether standard anti-rheumatic therapies modulate mitochondrial dynamics. Accordingly, DRP1, a mitochondrial fission mediator, and MFN1, a mitochondrial fusion protein, were assessed in serum from healthy controls (HC) and RA patients, and in synovial fluid from early-DMARD naïve RA patients or patients receiving DMARDs. Demographic and clinical characteristics of the patients are presented in Table 1. There were no significant differences in age, disease duration, or other baseline characteristics among the three groups. Compared with early DMARD-naïve RA patients, both the leflunomide-treated group and the MTX-treated group exhibited significantly lower erythrocyte sedimentation rate (ESR) and DAS28-ESR scores.

Compared with HC, early-DMARD naïve RA patients exhibited a pronounced increase in mitochondrial fission. Serum DRP1 levels were increased approximately 8-fold, while MFN1 levels were reduced to ~ 20% of those in HC (Fig. 1A-B). Notably, treatment with leflunomide or MTX partially restored the imbalance between DRP1 and MFN1. DRP1 levels were reduced by 73.83% and 63.15%, respectively, whereas MFN1 levels increased by approximately 2.3-fold and 2.2-fold compared with levels in early DMARD-naïve RA patients. However, these changes did not fully normalize mitochondrial dynamics to levels observed in HC (Fig. 1A-B), suggesting that DMARD therapy partially restores mitochondrial homeostasis in RA patients.

Fig. 1.

Leflunomide and methotrexate reduce DRP1/MFN1 ratio in the serum and synovial fluid of RA patients. (A-B) Quantification of the concentration of DRP1 (A) and MFN1 (B) in serum from HC (n = 8), early-DMARD naïve RA patients (n = 8), patients treated with MTX (n = 7) or LEF (n = 6) were performed using ELISA. (C) Correlations of serum DRP1 and MFN1 levels with DAS28 and ESR (n = 21). (D-E) Quantification of the concentration of DRP1 (D) and MFN1 (E) in synovial fluid from early-DMARD naïve RA patients (n = 5), patients treated with MTX (n = 4) or LEF (n = 4) was performed using ELISA. (F) Correlations of DRP1 and MFN1 levels in the synovial fluid of RA patients with DAS28 and ESR (n = 13). *p < 0.05, **p < 0.01, and ***p < 0.001. The p values were determined by one-way ANOVA test (A-B, D-E), nonparametric Spearman correlation (C, F). LEF, leflunomide

DAS28 and ESR are established indicators of RA disease activity. In serum, levels of DRP1 and MFN1 were significantly correlated with ESR, but showed only weak associations with DAS28. Notably, DRP1 displayed a positive correlation with ESR, whereas MFN1 was negatively correlated with ESR (Fig. 1C).

Compared with early-DMARD naïve patients, DRP1 levels in the synovial fluid of RA patients treated with leflunomide or MTX were reduced by 72.60% and 83.41%, respectively, whereas MFN1 levels were increased by 1.87-fold and 1.70-fold (Fig. 1D-E). Both DAS28 and ESR were positively correlated with DRP1 expression levels, whereas negatively correlated with MFN1 expression levels (Fig. 1F). Notably, stronger correlations were observed in synovial fluid than in serum, indicating that mitochondrial dynamics in the joint microenvironment more closely reflect RA disease activity.

A771726 and MTX inhibited excessive mitochondrial fission in RA-FLS

RA-FLS are key drivers of synovial hyperplasia in RA. Our previous studies demonstrated that elevated DRP1 expression in RA-FLS promotes excessive mitochondrial fission, increases ROS production, and contributes to apoptosis resistance [8]. To evaluate whether DMARDs modulate mitochondrial function in RA-FLS, we examined the effects of leflunomide’s active metabolite A771726 and MTX on mitochondrial dynamics.

CCK-8 assays showed that both A771726 and MTX reduced RA-FLS viability in a dose-dependent manner, with non-cytotoxic concentrations of 1–100 µg/mL and 0.1–10 µM, respectively (Fig. 2A-B). Analysis of mitochondrial dynamics-related genes revealed that A771726 dose-dependently increased OPA1 mRNA, while MTX upregulated MFN2 mRNA, neither drug affected DRP1 or MFN1 mRNA expression (Fig. 2C-D). However, at the protein level, the expression of MFN2 and total OPA1, as well as the S-OPA1/L-OPA1 ratio, remained unchanged following treatment. (Fig. 2E).

Fig. 2.

A771726 and MTX inhibited excessive mitochondrial fission in RA-FLS. (A-B) The CCK-8 assay was used to determine the cell viability of RA-FLSs following treatment with various concentrations of A771726 (A, n = 3) or MTX (B, n = 4) for 24 h. (C-D) qPCR analysis of mRNA levels of DNM1L, OPA1, MFN1, and MFN2 in RA-FLSs treated with A771726 (C) or MTX (D) for 48 h (n = 3). (E) Western blot analysis of protein levels of DRP1 (n = 3), p-DRP1 (Ser616) (n = 3), OPA1 (n = 6), MFN1 (n = 5), and MFN2 (n = 5) in RA-FLSs treated with A771726 or MTX for 48 h. (F) Representative confocal images (left) and statistical analysis (right) of mitochondria stained with MitoTracker Green in RA-FLSs following treatment with A771726 or MTX for 24 h (n = 6). Scale bar represents 20 μm. *p < 0.05, **p < 0.01, and ***p < 0.001 versuscontrol. The p values were determined by one-way ANOVA test (A-F)

TNF-induced DRP1 phosphorylation at Ser616 was suppressed by A771726 but not by MTX (Fig. 2E). Consistently, confocal microscopy showed pronounced mitochondrial fragmentation in TNF stimulated RA-FLS, whereas both A771726 and MTX preserved mitochondrial length and network integrity (Fig. 2F).

Together, these results indicate that both A771726 and MTX attenuate TNF-induced excessive mitochondrial fission in RA-FLS, with A771726 acting through inhibition of DRP1 phosphorylation at Ser616.

A771726 and MTX influenced autophagy in RA-FLS

Mitochondrial membrane potential (ΔΨm) reflects mitochondrial functional integrity and was assessed using JC-1 staining. At high ΔΨm, JC-1 forms red-fluorescent aggregates in the mitochondrial matrix, whereas mitochondrial depolarization favors green-fluorescent monomers. Both A771726 and MTX significantly decreased ΔΨm, as indicated by a reduced red/green fluorescence ratio (Fig. 3A).

Fig. 3.

A771726 and MTX influenced autophagy in RA-FLS. (A) The representative fluorescence diagram (left) and statistical analyses (right) of mitochondrial membrane potential of RA-FLSs treated with A771726 or MTX for 24 h determined by JC-1 staining (n = 6). (B) Western blot analysis of protein levels of LC3B and P62 in RA-FLSs treated with A771726 or MTX for 48 h (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. The p values were determined by one-way ANOVA test (A-B)

Loss of ΔΨm is an early event in mitophagy and apoptosis. p62 (SQSTM1) is a selective autophagy adaptor protein that binds ubiquitinated cargo and delivers it to autophagosomes through interaction with LC3 for subsequent lysosomal degradation. Treatment with A771726 increased the LC3B II/I ratio, whereas MTX had no significant effect (Fig. 3B). Both A771726 and MTX markedly elevated p62 levels (Fig. 3B). These findings suggest that A771726 may promote autophagosome formation, while the increased p62 levels observed with both treatments indicate accumulation of autophagic cargo, which could reflect enhanced p62 expression or altered autophagic degradation.

A771726, but not MTX, induces apoptosis in RA-FLS

Resistance to apoptosis in RA-FLS contributes to synovial hyperplasia in RA. A771726 significantly induced apoptosis in RA-FLSs, whereas no significant effect on apoptosis was observed with MTX at 1 or 5 µM (Fig. 4A). In line with these findings, A771726 significantly elevated intracellular ROS levels in RA-FLSs, whereas no significant change was observed following MTX treatment (Fig. 4B), indicating a potential role of oxidative stress in A771726-induced apoptosis.

Fig. 4.

A771726, but not MTX, induces apoptosis in RA-FLS. (A) Representative flow cytometry plots (left) and quantitative analysis (right) of apoptosis in RA-FLSs treated with A771726 or MTX for 48 h (n = 3–4). (B) Representative flow cytometry plots (left) and quantitative analysis (right) of intracellular ROS levels measured by DCFH-DA staining after 48 h of stimulation (n = 3–4). (C) Representative Western blot images (left) and quantitative analysis (right) of caspase 3 (n = 4), cytochrome c (Cyt-c) (n = 3), Bax (n = 3), and Bcl-2 (n = 5) expression in RA-FLSs treated with A771726 or MTX. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. The p values were determined by one-way ANOVA test (A-C)

Apoptosis is tightly regulated by the BCL-2/BAX/caspase-3 signaling pathway. Treatment with A771726 significantly downregulated the anti-apoptotic protein BCL2 and upregulated the pro-apoptotic protein BAX, resulting in enhanced cytochrome C release and activation of caspase-3 (Fig. 4C). In contrast, MTX did not significantly affect components of the BCL-2/BAX/caspase-3 pathway (Fig. 4C).

Collectively, these results suggest that A771726 induces apoptosis in RA-FLSs at least in part through ROS-mediated activation of the BCL-2/BAX/caspase-3 signaling pathway.

Leflunomide and MTX restore mitochondrial dynamics and alleviate arthritis in CIA mice

To clarify the in vivo effects of leflunomide and MTX on mitochondrial dynamics, we analyzed the expression of DRP1, phospho-DRP1 (Ser616), and OPA1 in the synovial tissues of CIA mice. Administration of leflunomide or MTX significantly decreased arthritis scores, paw thickness, and joint swelling in CIA mice (Figs. 5A–D), which was accompanied by reduced inflammatory cell infiltration and preservation of cartilage and bone integrity (Fig. 5E).

Fig. 5.

Leflunomide and MTX restore mitochondrial dynamics and alleviate arthritis in CIA mice. (A-C) Statistical analyses of CIA clinical scores, mean paw thickness (mm), and number of affected paws. (D) Representative images of paws from different groups of mice. (E) Representative histological sections of the ankle joint: H&E staining (scale bar = 400 μm), Safranin O/Fast Green staining (scale bar = 100 μm), and TRAP staining (scale bar = 100 μm). n = 3. (F) Representative histological staining of DRP1, p-DRP1 (Ser616), and OPA1 in ankle joints. Regions indicated by black boxes are shown at higher magnification. Scale bar = 20 μm. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. The p values were determined by two-way ANOVA test (A-C)

In the synovial tissue of CIA mice, the expressions of DRP1 and phospho-DRP1 (Ser616) were markedly increased. Leflunomide and MTX treatment significantly decreased DRP1 and phospho-DRP1 levels, with the effect being more pronounced for leflunomide (Fig. 5F). In contrast, OPA1 expression, which was downregulated in CIA mice, was significantly upregulated following leflunomide administration (Fig. 5F).

Collectively, these results suggest that leflunomide suppresses mitochondrial fission by inhibiting DRP1 activation and enhances mitochondrial fusion through upregulation of OPA1 in the synovial tissue of CIA mice.

Discussion

Aberrant activation of FLS drives joint destruction in RA and is associated with excessive mitochondrial fission [8, 24]. Our data reveal that dysregulated mitochondrial dynamics, particularly excessive DRP1-dependent fission, underlie pathogenic activation, impaired autophagy, and apoptosis resistance in RA-FLS, are closely associated with disease activity and inflammatory status, and are differentially modulated by leflunomide therapy.

Our clinical data revealed that DRP1 levels were positively correlated with RA disease activity, whereas MFN1 showed an inverse association, particularly in synovial fluid. These findings suggest that mitochondrial dynamics within the joint microenvironment more closely reflect local inflammation than systemic circulation. The stronger correlations observed in synovial fluid are likely attributable to the hypoxic and inflammatory milieu of the RA joint [25], which exacerbates mitochondrial stress and metabolic reprogramming in resident synovial cells. Notably, treatment with MTX or leflunomide partially restored the balance of mitochondrial fission–fusion–related proteins, suggesting that modulation of mitochondrial dynamics may represent an additional downstream effect of DMARD therapy.

Under basal culture conditions, RA-FLS gradually lose their pathogenic characteristics, which limits the observation of their pathogenic functional phenotypes. In this study, we employed TNF stimulation to maintain the activated phenotype of RA-FLS, which is essential for mimicking the inflammatory microenvironment of RA in vitro. However, future studies will focus on performing additional mechanistic experiments, including single-agent treatment paradigms, to further validate our findings.

Mechanistically, our in vitro data demonstrate that A771726 and MTX both attenuate TNF–induced mitochondrial fragmentation in RA-FLS, albeit through distinct molecular pathways. A771726 potently suppressed DRP1 phosphorylation at Ser616, a key activation site that promotes mitochondrial fission, whereas MTX exerted a weaker effect on DRP1 activation and instead appeared to favor mitochondrial fusion through upregulation of MFN2. These findings highlight mechanistic divergence between DMARDs in regulating mitochondrial dynamics, despite their shared clinical efficacy in RA.

Interestingly, although MTX increased MFN2 mRNA expression, this effect was not observed at the protein level. This discrepancy may be explained by post-transcriptional regulatory mechanisms, including limited translation efficiency, altered protein turnover, or enhanced degradation of MFN2. It is also possible that the time point selected for protein analysis did not capture transient changes in MFN2 expression. In addition, MTX may preserve mitochondrial morphology and function through MFN2-independent mechanisms.

Consistently, while both MTX and leflunomide were associated with decreased DRP1 and increased MFN1 expression in synovial fluid, MTX did not significantly affect these proteins in RA-FLS in vitro. These discrepancies likely reflect fundamental differences between in vitro and in vivo conditions. In vivo, the joint microenvironment is highly complex, involving interactions among multiple cell types, inflammatory mediators, and systemic factors, all of which can influence mitochondrial dynamics. In contrast, isolated RA-FLS represent a simplified system lacking these regulatory inputs.

We further show that both A771726 and MTX reduce mitochondrial membrane potential in TNF-induced RA-FLS. While TNF has been reported to induce mitochondrial depolarization in certain cell types [26, 27], its effects are highly context-dependent. In our model, TNF primarily serves to maintain the inflammatory phenotype of RA-FLS rather than directly inducing mitochondrial dysfunction.

Autophagy, including mitophagy, is increasingly recognized as a double-edged process in RA, exerting both cytoprotective and pathogenic effects depending on disease stage and cellular context [28]. While basal autophagy facilitates the clearance of damaged organelles and limits inflammation, excessive or dysregulated autophagy may promote the survival and persistence of FLS, thereby contributing to synovial hyperplasia and joint destruction. Consistent with this complexity, pharmacological modulation of autophagy—such as inhibition of the mTOR signaling pathway using everolimus in combination with MTX—has been shown to provide clinical benefit in RA patients with an inadequate response to MTX monotherapy [29].

In the present study, leflunomide increased the LC3B-II/I ratio accompanied by p62 accumulation, whereas MTX primarily elevated p62 without significantly affecting LC3B. Importantly, concurrent accumulation of LC3B-II and p62 does not necessarily indicate enhanced autophagic flux, but may instead reflect impaired autophagic degradation [30]. Given the dynamic and multistep nature of autophagy, assessment based solely on LC3B and p62 is insufficient to distinguish between increased autophagosome formation and defective lysosomal turnover. A limitation of the present study is the lack of autophagic flux analysis using lysosomal inhibitors such as bafilomycin A1 or chloroquine. Future studies incorporating these approaches will be necessary to clarify whether leflunomide and MTX modulate autophagy through induction or blockade of autophagic flux, and to further define the functional role of autophagy in RA pathogenesis.

Furthermore, autophagy and apoptosis are tightly interconnected processes that collectively regulate cell fate through complex signaling networks. Their dynamic interplay is critical in determining the balance between survival and death in RA-FLS, thereby influencing disease progression and therapeutic responses. MTX exhibited a weaker effect on autophagic degradation and did not significantly induce apoptosis. This observation appears to differ from previous reports showing that MTX can induce apoptosis in RA-FLS [31]. Such discrepancies may be attributed to differences in MTX concentration, exposure duration, and experimental conditions, as well as variability in cellular context. Notably, low-dose MTX, as commonly used in vitro to mimic clinical exposure, may preferentially exert anti-proliferative or immunomodulatory effects rather than directly inducing apoptosis.

These findings support a model in which excessive mitochondrial fission sustains RA-FLS survival by facilitating mitochondrial quality control and limiting apoptotic signaling. Disruption of DRP1-mediated fission impairs mitophagic clearance, leading to accumulation of dysfunctional mitochondria, oxidative stress, and activation of intrinsic apoptosis pathways. Thus, mitochondrial fission-mitophagy coupling may function as a critical checkpoint governing RA-FLS fate. Targeting this axis, particularly through inhibition of DRP1 activation, may therefore represent an effective strategy to overcome apoptosis resistance and synovial hyperplasia in RA.

Our in vivo data further corroborate this mechanism. In CIA mice, both MTX and leflunomide reduced synovial DRP1 and p-DRP1 (Ser616) levels and alleviated joint inflammation and destruction, with leflunomide exerting a more pronounced effect. Notably, leflunomide also restored OPA1 expression, suggesting coordinated suppression of mitochondrial fission and enhancement of fusion in the inflamed synovium. However, leflunomide did not significantly alter OPA1 protein levels in RA-FLS in vitro, whereas increased OPA1 expression was observed in the paw tissues of CIA mice. This discrepancy may reflect the differences between in vitro and in vivo conditions. Moreover, differences in drug exposure, treatment duration, and systemic metabolic or inflammatory cues in vivo may further influence OPA1 expression. Therefore, the upregulation of OPA1 observed in CIA mice may result from indirect or microenvironment-dependent effects of leflunomide rather than a direct action on RA-FLS.

Collectively, our study identifies mitochondrial dynamics as a critical mediator of RA-FLS pathogenic behavior and reveals that DMARDs exert previously unrecognized effects on mitochondrial fission–fusion balance, autophagy, and apoptosis. These findings expand the current understanding of RA therapy and highlight mitochondrial dynamics, particularly DRP1 activation, as a potential therapeutic target in RA.

Conclusions

Our results suggest that mitochondrial dynamics, particularly the regulation of DRP1, play a pivotal role in RA pathogenesis and treatment response. Leflunomide and MTX effectively modulate mitochondrial fission and fusion in RA-FLS and synovial tissue, with leflunomide showing a more robust effect, particularly in reducing the phosphorylation of DRP1. These findings highlight the potential of targeting mitochondrial dynamics as a therapeutic strategy in RA. By restoring the balance between mitochondrial fission and fusion, leflunomide may help mitigate the mitochondrial dysfunction that underlies synovial inflammation and tissue damage in RA. Furthermore, mitochondrial dynamics markers, such as DRP1 and MFN1, could serve as potential biomarkers for monitoring RA disease activity and treatment efficacy.

Authors’ contributions

Conceptualization, X.W., J.S. and S.F.; methodology, X.W., J.S., S,F, and J.C.; software, J.S., X.W., S.F., and J.C.; validation, J.S., X.W., S.F., J.Q., W.J., J.C., X.F., Y.Y. and J.L.; formal analysis, J.S., X.W., S.F., and J.C.; investigation, X.W., J.S.; resources, J.S., X.W., J.Q., J.C., X.F., Y.Y.; data curation, J.S., X.W. and Y.L.; writing-original draft preparation, X.W. and J.S.; writing-review and editing, Y.L., J.S., S.F. and J.C.; visualization, S.F., J.Q. and W.J.; supervision, Y.L., J.S.; project administration, Y.L., J.S.; funding acquisition, Y.L., J.S. and X.W.

Funding

This work was supported by the National Natural Science Foundation of China (82271822, 82502152), Medica Scientific Research Foundation of Guangdong Province, China (A2026276), and Heilongjiang Provincial Natural Science Foundation of China (LH2020H054).

Data availability

All data analyzed during the current study are included within this article.

Declarations

Ethics approval and consent to participate

All animal procedures were approved by the Ethics Committee of Guangdong Provincial People’s Hospital (KY2023-496-01) and Ethics Committee of Second Affiliated Hospital of Harbin Medical University (Ky2020-151).

Consent for publication

All authors have read and approved the final manuscript and consent to its publication.

Competing interests

The authors declare no competing interests.