1. Introduction
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by symmetrical multijoint swelling and pain [
1]. Its pathological features include synovial hyperplasia, inflammatory cell infiltration, and progressive joint destruction, affecting approximately 1% of the global population [
2]. The long-term progression of RA significantly increases the risk of osteoporosis, pulmonary interstitial fibrosis, and cardiovascular disease [
3,
4]. The current treatment strategy relies on nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and immunosuppressants such as celecoxib, prednisone, and methotrexate [
5,
6]. However, the efficacy of these drugs is limited and requires prolonged use, with severe adverse reactions, including gastrointestinal toxicity, bone marrow suppression, interstitial lung disease [
7], malignant tumors, cardiovascular disease, and liver toxicity [
8,
9]. These findings emphasize the urgent need to develop new drugs and targets with good therapeutic effects for treating RA. In recent years, researchers have gradually begun to explore active compounds derived from natural plant medicines for the treatment of RA. The overall goal is to develop a novel treatment approach based on the molecular origin of RA pathogenesis.
Obakulactone (OL), a natural tetracyclic triterpenoid isolated from
Phellodendri cortex (PC), has been used in traditional Chinese medicine for RA treatment for more than five centuries [
10,
11]. Our team’s prior investigations revealed that OL constitutes a core bioactive component of PC [
12,
13], exhibiting potent anti-inflammatory and analgesic properties [
14,
15]. Notably, while OL demonstrates therapeutic promise, its mechanistic underpinnings and systematic pharmacological evaluation remain understudied, hindering its translation into clinical practice. This study aims to address these gaps by elucidating the mechanisms underlying the efficacy of OL in treating RA.
Acyl coenzyme A (CoA) thioesterase 1 (ACOT1), a pivotal regulator of lipid metabolism that catalyzes the hydrolysis of acyl CoA into free fatty acids (e.g., arachidonic acid (AA)) and CoA, is the direct target of OL therapy in this study [
16]. In RA, proinflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin (IL)-6) drive synovial fibroblast (SF) activation through the nuclear factor (NF)-κB and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways [
17,
18], whereas ACOT1 amplifies inflammatory signaling via lipid mediators. Pathologically activated SFs exhibit tumor-like invasive phenotypes and secrete matrix metalloproteinases (MMPs) and proinflammatory cytokines that perpetuate cartilage erosion and neoangiogenesis [
19,
20]. Critically, ACOT1 serves as a metabolic switch in unsaturated fatty acid biosynthesis, making it a compelling therapeutic target. However, its role in RA pathogenesis and potential for pharmacological modulation remain poorly defined.
In this study, we used a complete Freund’s adjuvant (CFA)-induced RA rat model. Body weight, joint volume, histopathological analysis of joint/lymphoid organs (thymus and spleen), and changes in the expression of immune cells in the joints of RA rats before and after treatment were evaluated. The efficacy of OL was systematically evaluated. In addition, on the basis of metabolomics, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging technology, and proteomics, the ability of OL to correct the biosynthesis and metabolic disorders of unsaturated fatty acids in RA rats was determined. The potential molecular target of OL, ACOT1, was further identified. ACOT1 has been validated as a direct target for OL therapy of RA through molecular docking, cellular thermal shift assays (CETSA), surface plasmon resonance (SPR), microscale thermophoresis (MST), and small interfering RNA (siRNA) techniques. Our results indicate that OL enhances ACOT1 ubiquitination and reduces stearoyl-CoA desaturase 1 (SCD1) expression and the biosynthesis of unsaturated fatty acids. JAK-STAT/phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling is inhibited, thereby suppressing the secretion of inflammatory cytokines and inhibiting SF proliferation and fibrosis. Overall, these results establish ACOT1 as a potential drug target and position OL as a novel therapeutic agent for RA. Moreover, the importance of correcting the biosynthesis and metabolism of unsaturated fatty acids for the treatment of RA should be emphasized.
2. Materials and methods
2.1. Ethics statement
The male Sprague-Dawley (SD) rats used in this study were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (license No.: SCXK 2020-0001, batch No.: 210726231102617151; China). All animal experimental protocols were approved by the Institutional Experimental Animal Welfare Ethics Committee of the Heilongjiang University of Chinese Medicine, China, and all experiments were carried out in accordance with the Declaration of Helsinki. The rats were housed under specific pathogen-free (SPF) conditions with free access to sterile food (irradiated) and sterile water under a 12 h light/dark cycle.
2.2. Instruments
Acquity™ ultra-performance liquid chromatography (UPLC) liquid chromatograph (Waters, USA), an EASY-nLC 1200 liquid chromatograph (Thermo Fisher Scientific, USA), a Synapt™ G2-Si mass spectrometer (Waters), a Tims TOF™ pro mass spectrometer (Bruker, USA), an AP-SMALDI5 AF ion source (TransMIT, Germany), a Q Exactive™ Plus Orbitrap mass spectrometer (Thermo Fisher Scientific), a Freeze microtome (SMALDIPrep; TransMIT), a thermostatic drying oven (BPG-9056A; Shanghai Yiheng Technology Instrument Co., Ltd., China), a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus (DYY-6C; Six One Instrument Factory Beijing City, China), a Masslynx v4.1 workstation (Waters), an animal toe volume measuring instrument (YLS-7C; Jinan Yiyan Science & Technology Development Co., Ltd., China), an electronic analytical balance (XSE05; Mettler Toledo, Switzerland), a microplate reader (2030; Perkin Elmer, USA), a table centrifuge (Sorvall ST 16R; Thermo Fisher Scientific), and an ultralow temperature refrigerator (995; Thermo Fisher Scientific) were used.
2.3. Reagents and materials
OL (PubChem CID: 179651; purity ≥ 98%; batch No.: nkl-00255231102) was purchased from Chengdu Zhumo Technology Co., Ltd. (China). Other materials used included CFA (F5881; Sigma, USA), normal saline solution (0.9% NaCl; Harbin Sanlian Pharmaceutical Co., Ltd., China), leucine enkephalin (Sigma), MS grade acetonitrile and methanol (Thermo Fisher Scientific), distilled water (Guangzhou Watson’s Food & Beverage Co., Ltd., China), pentobarbital sodium (Beijing Bo’ao Tuoda Technology Co., Ltd., China), and 10% formalin stationary solution (Beijing Yili Fine Chemicals Co., Ltd., China). Additionally, IL-1β, IL-6, MMP-3, IL-17, anti-cyclic citrullinated peptide antibody (CCP-Ab), rheumatoid factor (RF), TNF-α, and c-reactive protein (CRP) enzyme-linked immunosorbent assay (ELISA) kits (batch No.: 20240222) were purchased from Nanjing Jiancheng Bioengineering Institute (China).
2.4. Animal handling
Six- to eight-week-old male SD rats weighing (180 ± 20) g were adaptively reared for one week in an environment with a temperature of (24 ± 2) °C, a humidity of (50 ± 5)%, and a 12 h light/dark cycle. After one week of adaptive feeding, 64 normal healthy rats were selected for the experiment. The rats were randomly divided into a control group (n = 16) and a RA model group (n = 48). On the first day, all the rats in the model group were injected with 0.1 mL of CFA at the ankle joint to prepare a rat model of RA. On Day 14 of model preparation, eight rats each were randomly selected from groups control and RA model. The rats were euthanized for RA model evaluation, including pathological, metabolomics, and proteomics studies.
After the RA rat model was successfully established, the remaining 40 RA rats were randomly divided into a model (Mod) group (
n = 8), a methotrexate (Mtx) group (1.1 mg∙kg
-1·d
-1;
n = 8) [
21], an OL low-dose (OL-L) group (50 mg∙kg
-1·d
-1;
n = 8), an OL medium-dose (OL-M) group (100 mg∙kg
-1·d
-1;
n = 8), and an OL high-dose (OL-H) group (200 mg∙kg
-1·d
-1;
n = 8) [
22,
23]. The rats in the treatment groups received daily oral gavages of OL at the specified doses, whereas those in the control (Con) group received an equal volume of distilled water. The treatment period lasted for 21 d.
2.5. Sample collection
On the 14th day of model preparation, eight rats were randomly selected from groups Con and Mod. Rat blood was collected after anesthesia with pentobarbital sodium. After the blood was maintained at room temperature for 30 min, it was centrifuged at 4 °C and 4000 r∙min-1 for 10 min. Three milliliters of methanol was added to 1.0 mL of serum and mixed thoroughly, and the sample was centrifuged at 4 °C and 13 000 r∙min-1 for 15 min. A 0.22 μm membrane was used to filter the sample, and the filtrate was used for UPLC-G2-Si-high-definition mass spectrometry (HDMS) analysis. Samples of the rat ankle joint, spleen, and thymus were simultaneously collected. Ankle joint samples were stored in a -80 °C freezer, while spleen and thymus samples were stored in 10% formalin stationary solution. On the 21st day of treatment, all serum, ankle joint, spleen, and thymus samples were collected and prepared using the same method after 60 min of treatment.
2.6. Evaluation of the efficacy of OL in RA
The daily state, weight, paw volume (tested every three days), immune organ index, ankle joint bone and surrounding tissue status, and immune organ pathological state of the rats were used to evaluate the RA rat model and the efficacy of OL. The body weight and paw volume of each group of rats were recorded every three days. In terms of skeletal status, X-ray examination was used to observe the skeletal status of the ankle joints of each group of rats before and after treatment, and pathological examination of the joints was conducted. In terms of immune function, after treatment, the thymus and spleen of all the rats were collected, the organ weights were recorded, and the immune organ index (organ index = organ weight (mg)/rat weight (g) × 100%) was calculated. Pathology was subsequently performed on all the collected thymus and spleen tissue samples. The tissue samples were fixed with 10% neutral buffered formalin fixative. After being embedded, sliced, and stained, the samples were subjected to pathological examination under an optical microscope. In addition, the expression levels of the inflammatory factors IL-1β, IL-6, IL-17, and TNF-α and the RA-specific biochemical indicators MMP-3, CRP, CCP-Ab, and RF in the serum of the rats in each group were detected. These indicators were integrated to evaluate the therapeutic effect of OL.
2.7. Metabolomics analysis
UPLC-G2-Si-HDMS analysis was performed on serum samples collected on the 14th day of RA model preparation. A MassLynx v4.1 workstation was used to establish the detection parameters. The specific UPLC and MS detection conditions are listed in Appendix A. In the detection process, leucine enkephalin ([M+H]+: m/z 556.2771, [M-H]-: m/z 554.2615) was used for real-time quality correction to ensure the accuracy of the MS data. The detected data were imported into Progenisis QI software, which performed peak alignment, peak extraction, and normalization. Next, the data were imported into Ezinfo2.0 software for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). According to the results of the analysis, ions with a variable importance in projection (VIP) ≥ 1, a maximum fold change (FC) ≥ 2, and an analysis of variance (ANOVA) p value ≤ 0.05 were defined as potential biomarkers of metabolic abnormalities in the blood of the RA model rats. By combining accurate MS data for the candidate ions, the Human Metabolome Database (HMDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used for identification, after which correlation analysis of the potential biomarkers was performed. Additionally, the same methods were used to detect and analyze serum samples collected on the 21st day of treatment to explore the regulatory effect of OL on the identified potential biomarkers and to conduct pathway analysis and mechanism research.
2.8. MALDI MS imaging research
The rat joints collected on the 21st day of treatment were embedded in 5% carboxymethyl cellulose solution, frozen fixed, and sliced on a cryosectioning machine. The slices (thickness: 30 µm) were placed on clean and sterilized glass slides and dried for 10 min in a constant-temperature environment of 25 °C. The sample surface was evenly sprayed with 30 mg∙mL-1 2,5-dihydroxybenzoic acid (DHB) methanol solution by a fully automatic matrix spray instrument, and the prepared sample was subjected to the AP-SMALDI5 AF&Q Active Plus imaging system for detection. The parameters were as follows: continuous detection mode; resolution: 70 000; pixel size: 30 µm; MALDI source parameters set to spray voltammetry: 4.00 kV; capillary temperature: 250 °C. On the basis of the detection results, the distribution status of differential substances in the joints of each group of rats was observed and analyzed.
2.9. Data-independent acquisition (DIA) proteomics analysis
After treatment, the frozen ankle joint tissue from each group was fully ground following the addition of liquid nitrogen, after which the protein was extracted. SDS-PAGE was used to inspect sample quality. After it was confirmed that the protein extract was suitable, it was enzymatically hydrolyzed into peptide segments and desalted. The specific detection parameter settings are shown in Table S1 in Appendix A. DIA proteomic analysis of the prepared sample was performed using EASY-nLC 1200 liquid chromatograph combined with a Q Exactive™ MS system. For liquid chromatography-tandem MS (LC‒MS/MS) identification, DIA technology was used to collect MS data for each sample. The analyzed DIA raw data were imported into “Spectraut Pulsar 18.4 (Biognosys)” software for analysis and protein identification. Peak matching was performed, quantitative information was extracted, and statistical analysis was performed. The FC and p value obtained through a t test were used to evaluate the differences in protein expression in the ankle joint tissues of the two groups of rats. Proteins with a p value < 0.05 and FC ≥ 2.0 or FC ≤ 1/2.0 were identified as differentially expressed proteins (DEPs) and subjected to Gene Ontology Enrichment Analysis (GO) and KEGG analysis to identify key pathways and targets of OL’s therapeutic effect on RA.
2.10. Molecular docking
On the basis of the results of the proteomics research, OL was subjected to D3Targets-2019-nCoV for molecular docking with potential anti-RA targets. The backend docking process was performed by Smina [
24], which is a fork of AutoDock Vina [
25].
2.11. 5-E thynyl-20-deoxyuridine (EdU) incorporation assay
Rat ankle joint synovial tissue was diced into small pieces and enzymatically digested in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1 mg∙mL-1 collagenase II (both from Thermo Fisher Scientific) at 37 °C for 2 h. After centrifugation, SFs were cultured in DMEM supplemented with 5 mmol∙L-1 glucose and 10% fetal bovine serum (FBS; Thermo Fisher Scientific) in a humidified 5% CO2 atmosphere at 37 °C. Cells were passaged upon reaching confluence, and fourth-generation SFs were used for experimentation. SFs were treated with various concentrations of OL and Mtx for 24 h, followed by a 3 h incubation with EdU in the culture medium. EdU incorporation was detected using a Click-iT™ EdU Alexa Fluor Imaging kit (Invitrogen, USA) according to the manufacturer’s protocol. EdU-positive cells (red) and 4′,6-diamidino-2-phenylindole (DAPI)-stained cells (blue) were visualized and quantified under a fluorescence microscope (Olympus, Japan) using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The percentage of EdU-positive cells relative to total DAPI-positive cells was calculated. The data were averaged from counts in five randomly selected fields per well and replicated using three independently pooled cell samples.
2.12. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for detecting apoptosis
The SF to be stained was placed in 4% paraformaldehyde for 15 min. Afterward, the cells were washed, stained, differentiated, and reacted in 0.6% ammonia solution. After the samples were cleaned, DNase-free protease K was added dropwise for coverage, TUNEL detection solution was added dropwise, and the samples were incubated at room temperature in the dark for 1 h. An anti-fluorescence quenching sealing agent was added, and the slides were covered with glass. The cells were observed under a dark-room fluorescence microscope, and the images were analyzed using ImageJ software.
2.13. Western blot assay
Joint tissue was lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase and protease inhibitors. The lysate was subsequently mixed with 2× loading buffer (Beyotime, China), boiled for 5 min, and quantified using a bicinchoninic acid (BCA) protein assay kit (Tanon, China). The protein samples were resolved by 10%-12% SDS‒PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Beyotime), and blocked with 5% nonfat milk (Sangon Biotech, China) for 1.5-2.0 h. PVDF membranes were then probed overnight at 4 °C with primary antibodies targeting ACOT1 (ab100915; Abcam, UK) and ACOT3 (orb592119; Biorbyt, UK). Following primary antibody incubation, the membranes were exposed to the corresponding secondary antibodies, and signal detection was performed using enhanced chemiluminescence (ECL) reagent (Tanon). Protein expression levels were quantified using ImageJ software and normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Cells were seeded at a density of 5 × 105 per well in six-well plates and treated with or without OL for 24 h. Following treatment, all the cells were collected for Western blot analysis. Total cellular proteins were extracted using RIPA lysis buffer (Beyotime). The proteins were separated by 10% SDS‒PAGE and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with the appropriate primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using an imaging system (Bio-Rad, USA).
2.14. Lentivirus-mediated short hairpin RNA (shRNA) infection
Lentivirus particles were generated through the transient transfection of shRNA lentiviral vectors, along with the packaging vectors (pVSV-G and pGag/pol), into 293T cells. As a control, a nontargeting shRNA vector containing a hairpin insert was used, which produces small interfering RNAs (siRNAs) with five base pair mismatches to any known human gene. Lentiviral supernatant stocks were collected 48 h post-transfection, filtered through a 0.45 μm filter, and either utilized for infection or stored at -80 °C. Vector titers were assessed by transducing cells with serial dilutions of the concentrated lentiviral supernatant in complete growth medium supplemented with 8 mg∙mL-1 polybrene (Invitrogen). After 48 h, the growth medium was supplemented with 2 mg∙mL-1 puromycin. Surviving colonies were counted under a microscope, and the titer of the lentiviral supernatant was calculated using the following formula: Transducing units = number of colonies × lentiviral dilution. All lentiviral stocks used in this study were selected at a multiplicity of infection (MOI) of 10.
2.15. CETSA
The CETSA experiment was performed as previously described [
26]. SFs were harvested and immediately lysed using liquid nitrogen. After centrifugation, the supernatant was collected and split into two equal portions. Each lysate was then incubated with OL or the solvent control for 2 h at room temperature. The samples were aliquoted into 100 μL per tube and heated at different temperatures (40, 45, 50, 55, 60, and 65 °C) for 5 min. Afterward, the samples were allowed to cool at room temperature for 3 min and kept on ice. All the samples were then centrifuged, and only the soluble fractions were collected. The supernatants were subsequently analyzed by Western blotting.
2.16. MST analysis
His6-tagged N-terminal ACOT1 protein was incubated with NT-647-NHS dye (Monolith Protein Labeling Kit RED-NHS; NanoTemper Technologies, Germany) at a 1:2 molar ratio for 30 min at room temperature in the dark. A serial dilution of OL was prepared in assay buffer, with the final dimethyl sulfoxide (DMSO) concentration maintained constant at < 5% to avoid solvent effects. Equal volumes of protein and compound solutions were mixed, yielding a total of 12 concentrations of OL (800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39 μmol∙L-1), followed by incubation at room temperature in the dark for 15 min. The samples were loaded into standard-treated capillaries and measured using a Monolith NT.115 instrument (NanoTemper Technologies) at 25 °C.
2.17. SPR analysis
SPR analysis was performed using a Biacore S200 instrument (GE Healthcare China, China) with a CM5 sensor chip (BR-1005-30). Recombinant human ACOT1 (CSB-YP001163RA; CUSABIO, USA) was immobilized on the CM5 chip via amine coupling using a commercial kit (BR-1000-50; GE Healthcare China). OL was dissolved in phosphate buffered saline with polysorbate 20 (PBS-P) buffer (28-9950-84; GE Healthcare China), and increasing concentrations of OL (0.78-50 μmol∙L-1) were injected into the flow system at a flow rate of 20 μL∙min-1. The association phase was set at 120 s, and the dissociation phase was set at 300 s. Binding kinetics were evaluated using Biaevaluation software version 2.0.
2.18. Cycloheximide (CHX) chase assay and immunoprecipitation
For inhibitor experiments, cells were treated with OL in the presence or absence of the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132). In the CHX chase experiments, cells were treated with OL for 12 h, and 25 µg∙mL-1 CHX was added at the indicated time points. The subsequent processing followed the same procedure as the Western blot experiment mentioned above.
For immunoprecipitation, the cells were treated with OL in the presence of MG132. The lysate was incubated overnight at 4 °C with rotation, either with immunoglobulin G (IgG) or an anti-ACOT1 antibody. Protein A/G beads (Santa Cruz Biotechnology, USA) were then added, and the mixture was incubated at 4 °C for 3 h with rotation. Subsequently, the protein A/G beads were washed five times with immunoprecipitation (IP) lysis buffer, and the samples were analyzed by immunoblotting.
2.19. Statistical analyses
Statistical analyses were performed using GraphPad Prism 8.0 software (USA). Data are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA and unpaired two-tailed t tests for comparisons between groups. Differences were considered to be statistically significant when the p value was < 0.05.
3. Results
3.1. OL significantly reduces joint tissue swelling in RA rats; improves the status of the cartilage, synovium, and immune organs; and reduces the inflammatory response
OL is a natural tetracyclic triterpenoid compound derived from the bark of the Chinese herb
Phellodendron amurense (
Fig. 1(a)). The animal experimental flowchart is shown in
Fig. 1(b). A RA rat model was induced by intra-articular injection of 0.1 mL of CFA, and the rats were subsequently treated with OL for three weeks. Compared with the Con group, the RA model group exhibited significant ankle joint swelling (
Fig. 1(c)). The joint bones showed obvious deformation, with pronounced swelling of the surrounding tissues (
Fig. 1(d)), along with irregular cartilage surfaces, synovial hyperplasia, connective tissue proliferation, fat accumulation, and scattered lymphocyte infiltration (
Fig. 1(e)). Analysis of immune organs revealed significant splenomegaly and an increased spleen index, accompanied by thymic atrophy and a decreased thymus index in RA rats (
Figs. 1(f)-(h)). Increased pigment deposition in the splenic white pulp, extensive congestion in the red pulp, and granulocyte infiltration were observed (
Fig. 1(i)). The thymus exhibited an irregular morphology with a blurred corticomedullary boundary (
Fig. 1(j)). On the basis of these results, an RA rat model was successfully established, effectively mimicking the joint swelling, deformation, and impaired immune function observed in patients with RA.
During the treatment period, compared with the Mod group, the OL groups showed a gradual recovery in body weight, with a significant difference detected by the end of the treatment (
p < 0.05;
Fig. 1(k)). A significant difference in hind paw joint volume was observed between the OL groups and the Mod group on day 7 of treatment (
p < 0.01), with the OL-H group showing the most pronounced effect (
Fig. 1(l)). Representative images of joint conditions from each group after treatment are shown in
Fig. 1(m). The radiographic improvements included dose-dependent restoration of the joint structure, with clearer bone contours and more regular tissue organization in the OL groups (
Fig. 1(n)).
Histopathological evaluation revealed smooth joint surfaces and a normal chondrocyte morphology in OL-treated RA rats. Synovial hyperplasia was significantly alleviated, fat deposition was reduced, and no obvious inflammatory cell infiltration was observed (
Fig. 2(a)). Moreover, we examined the effect of OL on immune cells in the joints of RA rats. OL significantly suppressed the abnormally high expression of CD3
+ T cells and CD68
+ macrophages in the joints and surrounding tissues (
Fig. 2(b)). Fluorescence detection of CD86 and CD206 indicated that OL inhibited M1 polarization and promoted M2 polarization, thereby enhancing the anti-inflammatory effects (
Fig. 2(c)).
Compared with control rats, RA model rats exhibited sustained splenomegaly and thymic atrophy. OL treatment effectively restored normal thymus and spleen morphology in RA rats, with a significant increase in thymus index and a significant decrease in spleen index (
p < 0.05;
Figs. 3(a) and
(b)). The OL-H group showing nearly complete recovery comparable to that of the Con group. Histological examination revealed clear thymic structure, distinct corticomedullary differentiation, abundant lymphocyte populations, and minimal pigment deposition in the OL groups (
Fig. 3(c)). Spleen sections revealed reduced granulocyte infiltration, normal sinus structure, and restored boundaries between the white and red pulp. The OL-M and OL-H groups achieved near-normal splenic histology (
Fig. 3(d)).
Serum analysis revealed that the levels of proinflammatory cytokines (IL-1β, IL-6, IL-17, and TNF-α) were significantly greater in the RA group than in the control group (
p < 0.01). OL treatment dose-dependently attenuated the expression of these inflammatory mediators (
Fig. 3(e)). Clinical RA biomarkers (RF, CCP-Ab, CRP, and MMP-3) were significantly increased in model rats, and these diagnostic parameters significantly decreased in all OL groups (
Fig. 3(f)).
Comprehensive evaluation based on body weight recovery, joint swelling indicators, radiographic results, and histopathological assessment demonstrated the multifaceted therapeutic effects of OL, establishing it as a promising candidate for RA intervention.
3.2. Metabolomics technology reveals that OL corrects the disordered biosynthesis and metabolism of unsaturated fatty acids in RA rats
To elucidate changes in the metabolic profile of RA rats, we performed a metabolomic analysis on serum samples via UPLC-G2-Si-HDMS. The PCA score plot revealed a clear separation trend between the Con and Mod groups (
Fig. 4(a); positive ion mode (POS): the first principal component (PC1) = 60.02%, PC2 = 10.13%; negative ion mode (NEG): PC1 = 71.54%, PC2 = 9.01%). The OPLS-DA permutation test plot revealed that the permuted
Q2 regression line intersected the
Y-axis in the negative region, and all permuted
R2Y values were significantly lower than those of the original model (
Fig. 4(b); POS:
R2Y = 0.955,
Q2 = 0.92; NEG:
R2Y = 0.986,
Q2 = 0.952). These results revealed significant differences in metabolic characteristics between the Con and Mod groups, suggesting the presence of metabolic disorders in RA rats. Accordingly, we conducted differential analysis on the metabolic data of the Con and Mod groups, selecting differentially abundant metabolites with
p < 0.05, a FC ≥ 2, and a VIP ≥ 1 as potential biomarkers for RA (
Fig. 4(c)). Ultimately, 34 distinct metabolites were identified as RA biomarkers, including AA, 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene A4, linoleic acid, palmitoleic acid, and 9-
cis-retinoic acid (Fig. S1 and Table S2 in Appendix A).
PCA of the metabolomic data obtained after the treatment period revealed that the metabolic profiles of the OL groups shifted in a dose-dependent manner. With increasing OL dose, the profiles progressively diverged from those of the Mod group and moved closer to those of the Con group, with the OL-H group profile showing the closest resemblance to that of the Con group (
Fig. 4(d)). Cluster heatmap analysis was performed on the levels of RA biomarkers in the serum of each group (
Fig. 4(e), Table S3 in Appendix A). The results revealed that the Con, OL-L, OL-M, and OL-H groups, and some data from the MTX group were clustered into one major category, with the OL-H and Con groups further grouped into a subcategory. These findings indicated that OL could significantly correct metabolic disorders in RA rats, with the OL-H group showing the best therapeutic effect. In addition, KEGG enrichment analysis was conducted on the corrected biomarkers after OL treatment. The results revealed that the therapeutic mechanism of OL for treating RA involved primarily regulation of the biosynthesis of unsaturated fatty acids, AA metabolism, linoleic acid metabolism, α-linolenic acid metabolism, primary bile acid biosynthesis, and other processes (
Fig. 4(f), Table S4 in Appendix A). These findings established the ability of OL to correct RA-related lipid disorders, particularly through the normalization of AA, linoleic acid, and α-linolenic acid metabolism, confirming its therapeutic targeting of RA pathogenesis.
Furthermore, to validate the ability of OL to correct unsaturated fatty acid biosynthesis in the joint tissues of RA rats, MALDI MS was performed on the joint tissues of rats from the Con, Mod, and OL groups. The results revealed that the levels of unsaturated fatty acids such as palmitelaidic acid, palmitic acid, and oleic acid increased in the ankle cartilage and bone of RA rats, whereas the level of linoleic acid decreased. Additionally, the levels of the proinflammatory substances AA and 5-HETE increased in the surrounding tissues. The above conditions significantly improved the ankle joints of OL-treated rats, with detection results similar to those observed in the Con group (
n = 3 biologically independent samples per group, randomly selected for analysis;
Fig. 4(g)).
In summary, on the basis of the metabolomics findings, OL alleviated RA mainly by regulating the abnormal biosynthesis and metabolism of unsaturated fatty acids such as AA, linoleic acid, and α-linolenic acid in RA rats.
3.3. Proteomics suggests that OL can downregulate the biosynthesis of unsaturated fatty acids, with ACOT1 and ACOT3 being key regulatory targets
On the basis of the results of pharmacological evaluation and metabolomics research, we selected the OL-H group rats, which showed the best therapeutic effect, and conducted proteomic studies to determine the effect of OL on protein expression levels in the joints of RA rats (using all five remaining biologically independent samples from the MALDI analysis). The PCA results revealed that the protein expression profiles in the joints of the Con and Mod groups were significantly separated, whereas the clustering of the OL and Mtx groups tended to approach that of the Con group (
Fig. 5(a)). Compared with those in the Con group, there were 1804 proteins with increased expression and 1022 proteins with decreased expression in the joints of the Mod group rats. These DEPs were involved mainly in lysosomal function, extracellular matrix (ECM)-receptor interactions, osteoclast differentiation, and B-cell signaling. The results revealed that proteins involved in the RA pathway, including proliferation, adhesion, antiapoptosis, and the release of proinflammatory factors, were abnormally activated and that SFs were abnormally expressed. This represents an important mechanism of RA disease (Fig. S2 in Appendix A).
Compared with those in the Mod group, the expression levels of 375 proteins in the OL treatment group significantly increased (
p < 0.05, log
2FC > 1), with 147 increasing and 228 decreasing (
Fig. 5(b), protein information is provided in Table S5 in Appendix A). Clustering analysis revealed that the OL, Mtx, and Con groups were clustered together and had different characteristics from the Mod group (
Fig. 5(c)). The functional annotation confirmed that OL alleviated inflammation and cell proliferation adhesion in RA through extracellular matrix components and enzyme activity, such as leukocyte migration, the inflammatory response, and monounsaturated fatty acid biosynthesis. KEGG enrichment analysis revealed the regulatory effects of OL on biosynthesis of unsaturated fatty acid, α-linolenic acid metabolism, and linoleic acid metabolism, while it inhibited the expression of the PI3K-AKT and JAK-STAT signaling pathways (
Fig. 5(d)).
The KEGG pathway priority identified the ten most significantly enriched pathways (with the lowest
p value). The top ten proteins with the highest |log
2FC| values in each pathway were designated as OL-regulated specific proteins (
Fig. 5(e)). OL significantly downregulated the expression of ACOT1, ACOT3, SCD1, SCD2, and fatty acid desaturase 2 (FADS2) in the biosynthesis of unsaturated fatty acids, as shown in the clustering heatmap analysis in
Fig. 5(f). SCD1/2 are downstream of ACOT1/3, and on the basis of the
p values, ACOT1 and ACOT3 were identified as key proteins in the treatment of RA with OL.
Molecular docking results indicated that OL bound to the asparagine-326 (ASN-326) site of ACOT1 via a hydrogen bond (binding energy: -14.8 kcal∙mol
-1) and to the glutamic acid-343 (GLU-343) and phenylalanine-344 (PHE-344) sites of ACOT3 via hydrogen bonds (binding energy: -13.6 kcal∙mol
-1). Generally, a binding energy of less than -9.0 kcal∙mol
-1 is defined as good binding affinity. OL strongly bound to the key proteins ACOT1 and ACOT3 (
Fig. 5(g)). Western blotting confirmed this regulatory effect, and OL inhibited the expression levels of ACOT1 and ACOT3 (
Fig. 5(h)). ACOT1 and ACOT3 are members of the acyl CoA thioesterase family, the core function of which is to catalyze the hydrolysis of acyl CoA to produce free fatty acids and CoA with a free sulfhydryl group (CoA SH). This reaction plays a crucial role in lipid metabolism, particularly by regulating the homeostasis of acyl CoA and affecting fatty acid oxidation, the inflammatory response, and energy metabolism balance. In recent years, the potential role of ACOT1 in autoimmune diseases such as RA and lipid metabolism disorders has gradually received attention.
In summary, the results of both the metabolomics and proteomics experiments indicated that OL mainly alleviated RA by correcting the biosynthesis and metabolism of unsaturated fatty acids in RA rats and that ACOT1 and ACOT3 were key therapeutic targets.
3.4. OL inhibits the PI3K-AKT and JAK-STAT signaling pathways, suppresses the proliferation of SFs, promotes apoptosis, and reduces the release of proinflammatory and damaging factors
To validate the metabolomics and proteomics results, we treated SFs with lipopolysaccharide (LPS; 1 μg∙mL
-1) to simulate the inflammatory state of RA rat joints. After 24 h of treatment with Mtx (5 μg∙mL
-1) and OL (1, 3, and 10 μg∙mL
-1), EdU staining revealed that OL inhibited abnormal SF proliferation in a dose-dependent manner (
Fig. 6(a)). The proportion of EdU-positive cells in the OL group was significantly lower than that in the Mod group (
p < 0.01;
Fig. 6(b)), and the effect of the high dose of OL was similar to that of the Mtx group. Similarly, TUNEL staining revealed significantly increased green fluorescence in OL-treated cells (
Fig. 6(c)), and the proportion of TUNEL-positive cells was significantly greater (
Fig. 6(d)), which indicated that OL significantly promoted SF apoptosis. With respect to cell viability, cell-counting kit-8 (CCK-8) assays conducted at 24, 48, and 72 h after OL treatment demonstrated that OL significantly reduced SF activity under inflammatory conditions (
Fig. 6(e)), suggesting that OL could inhibit inflammation-related stress responses induced by LPS stimulation. Consistent with the TUNEL staining results, flow cytometry analysis further confirmed the pro-apoptotic effect of OL (
Fig. 6(f)), with high-dose OL showing efficacy comparable to that of Mtx.
Western blot analysis revealed that OL treatment significantly inhibited the phosphorylation of PI3K and the JAK family members JAK1 and JAK3, reducing the phosphorylation ratio. These findings confirmed the results obtained from proteomics, indicating that OL significantly downregulated the overexpression of the PI3K-AKT/JAK-STAT signaling pathway in RA rats. OL also suppressed the overexpression of SCD1 (
Fig. 6(g)). SCD1 desaturates free fatty acids produced by AOCT1 and AOCT3 to generate unsaturated fatty acids, such as oleic acid and palmitic acid. Excessive accumulation of these fatty acids can cause damage to joints and bones.
Furthermore, ELISA detection of the culture medium after treatment with OL for 24 h revealed that OL significantly inhibited the release of the inflammatory factors IL-1β, IL-6, and TNF-α; reduced the level of the fibrotic factor, transforming growth factor-β1 (TGF-β1); and decreased the production of the key indicator MMP-3, thereby promoting the degradation of the articular cartilage and bone matrix (
Fig. 6(h)).
3.5. OL significantly inhibits macrophage M1 polarization, promotes M2 polarization, and reduces T helper 17 (Th17) cell activation
To investigate the immunomodulatory effects of OL on macrophages, Raw264.7 murine macrophages were subjected to polarization induction. For M1 polarization, cells were stimulated with LPS (1 μg∙mL
-1) and interferon-γ (IFN-γ; 20 ng∙mL
-1) for 12 h. To induce M2 polarization, cells were treated with IL-4 (20 ng∙mL
-1) for the same duration. OL was administered at the indicated concentrations with polarizing stimuli. The messenger RNA (mRNA) expression levels of polarization-associated markers, including
TNF-α and inducible nitric oxide synthase (
iNOS) for the M1 phenotype and
IL-10 and arginase 1 (
Arg1) for the M2 phenotype, were quantified using quantitative real-time polymerase chain reaction (qPCR). Our results demonstrated that OL treatment significantly attenuated the mRNA expression of the M1 markers
TNF-α and
iNOS in LPS/IFN-γ-stimulated macrophages (
Fig. 7(a)). Conversely, OL markedly upregulated the mRNA expression of the M2 markers
IL-10 and
Arg1 in IL-4-stimulated cells (
Fig. 7(b)). These findings collectively suggested that OL effectively suppressed M1 polarization while promoting M2 polarization in Raw264.7 macrophages. This finding was consistent with the results obtained from previous animal experiments.
In addition, owing to the stimulation of self-antigens or adjuvants, antigen-presenting cells were activated and secreted cytokines such as IL-6, IL-1β, and TGF-β. These cytokines collectively promoted the abnormal overactivity of Th17 cell differentiation, expansion, and function. These activated Th17 cells could directly participate in the processes of synovitis and bone destruction. To assess the direct impact of OL on Th17 cell differentiation, CD4
+ T cells were isolated from the spleens of naive mice using magnetic-activated cell sorting (MACS). After cytokine staining, the proportion of IL-17
+CD4
+ T cells was analyzed by flow cytometry. Additionally, the mRNA expression levels of key Th17-related genes, including
IL-17 and the master transcription factor retinoic acid receptor-related orphan receptor γt (
RORγt), were determined by qPCR. As illustrated in
Figs.7(c) and
(d), OL treatment dose-dependently reduced the proportion of IL-17
+ cells within the CD4
+ T-cell population and significantly inhibited the gene expression of both IL-17 and RORγt (
Fig. 7(e)).
3.6. ACOT1 is a direct target for the therapeutic effect of OL
To further elucidate the molecular targets of OL, we conducted CETSA experiments to verify that ACOT1/3 was a direct binding target for OL to exert its therapeutic effects. The OL (at a final concentration of 10 μg∙mL
-1) and SF lysis buffers were incubated at room temperature for 2 h. The protein stability was recorded at different temperatures (40, 45, 50, 55, 60, and 65 °C). Compared with the solvent control, OL increased the thermal stability of ACOT1 but did not significantly affect the thermal stability of ACOT3 (
Figs. 8(a) and
(b)). These findings confirmed the direct binding between OL and ACOT1, where OL altered the conformation and thermal stability of the ACOT1 protein, making it less prone to denaturation and precipitation upon heating. To quantify the direct binding affinity between OL and ACOT1, we performed MST assays. The fluorescently labeled ACOT1 protein was titrated with increasing concentrations of OL. MST analysis revealed a strong direct interaction, and we repeated the detection three times, with a dissociation constant (
Kd) of (6.18 ± 0.26) μmol∙L
-1 (
Fig. 8(c)). In addition, we also conducted SPR experiments to quantify the interaction between OL and ACOT1. The results revealed that OL bound to ACOT1 with an equilibrium
Kd of (6.34 ± 0.38) μmol∙L
-1 (
Fig. 8(d)). The binding kinetics, derived from fitting the sensorgrams obtained at OL concentrations ranging from 0.78 to 50 μmol∙L
-1, demonstrated a reversible interaction. These results unequivocally established OL as a bona fide ligand of ACOT1.
In addition, we conducted further experiments using lentiviral shRNA to knock out the
ACOT1 gene. The cells were treated with excipients or 10 μg∙mL
-1 OL. The knockout efficiency of
ACOT1 is shown in
Fig. 8(e). Compared with that in the short-hairpin negative control (shNC) group, the expression of
ACOT1 was significantly reduced (
p < 0.01). After
ACOT1 was successfully knocked out, the effect of OL on SF activity was reevaluated using a CCK-8 assay. Compared with that of the shNC group, the survival rate of the shRNA knockout group treated with the vector was reduced at 24, 48, and 72 h (
p < 0.01). Compared with that of the sh
ACOT1 group, the survival rate of the sh
ACOT1 + OL group did not further decrease or significantly differ (
Fig. 8(f)). The experimental results indicated that ACOT1 was the direct target of OL.
After the
ACOT1 was knocked out, an EdU proliferation assay was used to evaluate the effect of OL on SF proliferation. Compared with that in the shNC group, SF proliferation in the sh
ACOT1 group was significantly reduced. Treatment with OL did not further enhance the antiproliferative effect (
Fig. 8(g)). Similarly, TUNEL staining revealed a significant increase in apoptotic cells in the sh
ACOT1 group compared with the shNC group. However, compared with that in the sh
ACOT1 group, the proportion of apoptotic cells in the sh
ACOT1 + OL group did not significantly differ (
Fig. 8(h)). These findings indicated that ACOT1 was a direct target through which OL relieved RA. These findings confirmed the conclusion drawn from the CETSA, MST, SPR, and CCK-8 experiments that ACOT1 was the direct target protein through which OL exerted anti-RA efficacy and corrected unsaturated fatty acid biosynthesis and metabolism.
3.7. OL relies on ACOT1 to exert anti-inflammatory, antiproliferative, and proapoptotic effects
On the basis of the above experimental results, we preliminarily determined the key mechanism of OL treatment for RA: targeting ACOT1 and inhibiting its active expression. The biosynthesis and metabolism of unsaturated fatty acids were corrected, the production of AA and its proinflammatory metabolites was inhibited, and the subsequent cascade of inflammatory reactions was reduced. On the one hand, OL inhibited the production and release of proinflammatory and tissue-damaging factors, and on the other hand, it also inhibited synovial proliferation, antiapoptosis, inflammatory cell adhesion, immune cell infiltration, and synovial hyperplasia caused by activation of the PI3K-AKT and JAK-STAT signaling pathways (
Fig. 9(a)).
To determine whether the anti-inflammatory effects of OL were mechanistically dependent on its interaction with ACOT1, we performed a rescue experiment by supplementation with AA, the enzymatic product of ACOT1. Strikingly, exogenous AA supplementation (at a final concentration of 10 μmol∙L
-1) abrogated the proapoptotic and antiproliferative effects of OL (at a final concentration of 10 μg∙mL
-1) in stimulated cells, as determined by flow cytometry (
Fig. 9(b)), TUNEL (
Figs. 9(c) and
(d)), and EdU assays (
Figs. 9(e) and
(f)). Consistently, the suppression of PI3K and JAK-STAT pathway activation by OL, as evidenced by decreased phosphorylation of PI3K, JAK1, and JAK3, was also reversed upon AA addition (
Fig. 9(g)). This functional and biochemical rescue strongly suggested that ACOT1 was the direct functional target of OL.
Having linked the activity of OL to the subsequent inhibition of the PI3K-AKT and JAK-STAT pathways, we employed specific pharmacological inhibitors, LY294002 (LY) for PI3K and ruxolitinib (RUX) for JAK. If OL acted upstream by inhibiting these pathways, then cotreatment with the inhibitors should occlude any additional effect of OL. In the presence of LY (final concentration of 10 μmol∙L
-1) and RUX (final concentration of 1 μmol∙L
-1), OL (final concentration of 10 μg∙mL
-1) did not further enhance the pro apoptotic effect (
Figs. 9(h) and
(i)) and anti proliferative effect (
Figs. 9(j) and
(k)), with no significant difference (
p < 0.01). Compared with that in the LPS + LY + RUX group, SF apoptosis in the LPS + LY + RUX + OL group was not significantly different (
p > 0.05;
Fig. 9(l)). These findings demonstrated that OL conferred no additional benefit when these key downstream pathways were inhibited. This epistatic analysis confirmed that the PI3K-AKT and JAK-STAT signaling cascades were essential and nonredundant mediators of the anti-inflammatory and regulatory effects of OL on cell proliferation and apoptosis.
3.8. OL targets ACOT1 and enhances its ubiquitin (Ub) protease degradation to exert therapeutic effects
OL directly interacts with ACOT1 and suppresses its functional expression. To further investigate the mechanism underlying the OL-mediated downregulation of ACOT1, we performed CHX chase assays. As shown in
Fig. 10(a), OL significantly reduced the protein stability of ACOT1 (
p < 0.05), which suggested that OL regulated ACOT1 expression through posttranslational mechanisms. On the basis of these findings, we hypothesized that OL might activate intracellular proteasomal pathways to facilitate ACOT1 degradation. This hypothesis was supported by our observation that the proteasome inhibitor MG132 effectively blocked OL-induced ACOT1 degradation (
Fig. 10(b)). Furthermore, endogenous coimmunoprecipitation assays revealed a marked increase in ACOT1 ubiquitination following OL treatment (
Fig. 10(c)). Collectively, these findings demonstrated that OL promoted ACOT1 degradation through the Ub-proteasome pathway.
On the basis of the experimental results mentioned above, we hypothesized that OL could bind to ACOT1, reduce its expression activity, and decrease the inflammatory response and joint damage caused by the accumulation of proinflammatory unsaturated fatty acids and their metabolites (such as AA, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), and 5-HETE). Moreover, the expression of ACOT1 decreased, reducing the production of free fatty acids, thereby inhibiting the activity of the desaturase SCD1 and reducing the high contents of palmitic acid, oleic acid, and long-chain unsaturated fatty acids in the joints of RA rats. OL corrected the biosynthesis and metabolism of unsaturated fatty acids by targeting ACOT1, which not only reduced the release of proinflammatory and tissue-damaging factors, thereby reducing inflammatory responses, but also inhibited the activation of the PI3K-AKT and JAK-STAT signaling pathways, preventing further inflammatory reactions and joint deformities caused by abnormal SF proliferation (
Fig. 10(d)).
4. Discussion
The current therapeutic landscape for RA relies on NSAIDs, glucocorticoids, and disease-modifying antirheumatic drugs [
27], which exhibit limited efficacy and severe adverse effects, such as infections, osteoporosis, and hypertension [
28,
29]. To address these gaps, our study investigated OL, a bioactive compound derived from PC, a traditional Chinese medicine. OL can significantly alleviate joint inflammation and swelling in RA rats and restore overactivated T cells and macrophages. The therapeutic mechanism involves targeting the key enzyme ACOT1 in fatty acid metabolism and correcting the abnormal biosynthesis and metabolism of unsaturated fatty acids, thereby inhibiting the release of proinflammatory fatty acids such as AA and its metabolites. Inhibiting the PI3K-AKT and JAK-STAT signaling pathways suppresses inflammation and SF proliferation. OL has shown outstanding therapeutic effects in the treatment of RA in rats.
Unsaturated fatty acids (UFAs), such as AA, linoleic acid, and α-linolenic acid, are closely related to chronic inflammatory diseases such as RA [
30,
31]. ACOT1 hydrolyzes acyl-CoA esters to release unsaturated fatty acids, including AA, which are precursors of proinflammatory mediators. In joints, elevated ACOT1 expression exacerbates AA-derived inflammation, driving joint swelling, pain, and bone erosion. The release of metabolites such as PGE2, prostaglandin I2 (PGI2), thromboxane A2 (TXA2), and leukotriene C4 (LTC4) from AA can further activate the PI3K-AKT and JAK-STAT signaling pathways [
32,
33], causing inflammation and SF proliferation and thereby promoting the occurrence of RA. Our findings revealed that OL suppressed ACOT1 by enhancing its ubiquitination and proteasomal degradation, thereby reducing the levels of UFAs and their inflammatory metabolites. This mechanism was consistent with the metabolomics data showing that OL corrected aberrant lipid profiles in RA joints, including AA accumulation.
The PI3K-AKT signaling pathway is crucial for maintaining the integrity of basic cellular processes, cell growth, survival, death, and metabolism. After being activated by inflammatory factors, the PI3K-AKT signaling pathway can regulate the proliferation of fibroblast-like synoviocytes and blood vessels; activate inflammatory cytokine-related signaling pathways; release large amounts of proinflammatory factors, such as TNF-α, IL-1β, and IL-6; and continuously induce abnormal proliferation and inflammatory responses in SFs, thereby promoting the deterioration of RA [
34]. The JAK-STAT pathway is an important signal transduction pathway in cells that mainly mediates the signal transmission of cytokines (such as interleukins and interferons); regulates gene expression; and affects cell proliferation, differentiation, apoptosis, and the immune response. The core components of the JAK family include four nonreceptor tyrosine kinases: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). Among them, JAK1/JAK3 is involved mainly in immune regulation (signal transduction of cytokines such as IL-6 and IL-2), whereas JAK2 is involved in hematopoietic functions (such as erythropoietin) [
35]. RA is an autoimmune disease that is characterized by chronic synovitis and bone destruction, with abnormal activation of the PI3K-AKT and JAK-STAT pathways as its core pathogenesis. In the synovial tissue of RA patients, proinflammatory cytokines such as IL-6, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are overexpressed, continuously activating the JAK-STAT pathway and leading to abnormal proliferation of SFs and the formation of invasive vascular opacities [
36]. JAK inhibitors (JAKis) have become important for the treatment of RA because they inhibit kinase activity by blocking the ATP binding domain, such as through multitarget inhibition by tofacitinib and baritinib (mainly JAK1/JAK3), widely blocking inflammatory factors such as IL-6 and IL-17 [
37,
38]. Inhibiting the overexpression of ACOT1 in RA, thereby suppressing the generation and release of proinflammatory fatty acids and metabolites, achieved the upstream inhibition of the activation of the PI3K-AKT and JAK-STAT signaling pathways. Controlling the activity of ACOT1 is highly important for the future clinical treatment and drug development of RA.
Before specific RA-related markers were detected in rat serum, we consulted a large number of clinical diagnostic research reports on RA. Ultimately, RF, CCP-Ab, MMP-3, and CRP, four commonly used clinical diagnostic indicators for RA, were selected to evaluate the efficacy of OL in the RA model [
39,
40]. Among them, RF is an autoantibody that targets the Fc fragment of denatured IgG. RF is produced in peripheral lymph nodes, the synovium of joints, the lymphoid follicles of tonsils, and the bone marrow. It is an RA-related autoantibody [
41,
42]. CCP-Ab is an autoantibody that targets synthesized CCP as an antigen and has high sensitivity and specificity for the treatment of RA. The level of CCP-Ab in the blood of RA patients is high, making it a highly specific indicator for the early diagnosis of RA [
43,
44]. MMP-3 is a matrix-soluble enzyme. After stimulation, fibroblasts, chondrocytes, and endothelial cells can generate MMP-3, which can destroy collagen and proteoglycans, causing the abnormal dissolution of extracellular matrix proteins dependent on chondrocytes and leading to cartilage degeneration [
45,
46]. The level of CRP rapidly increases (acute protein) in the blood during infection or tissue damage. It is also an immune modulator that plays an important role in the inflammatory pathways associated with RA and is a commonly used biomarker for diagnosing RA [
40,
47]. In addition, the combination of four proinflammatory cytokines, namely, IL-1β, IL-6, IL-17, and TNF-α, can reflect the therapeutic effect of OL on RA, ensuring the reliability of the experimental results and conclusions [
48].
While the CFA model robustly elicits innate immune-driven synovitis, articular cartilage destruction, and bone resorption—pathological features highly relevant to human RA—it may not fully encompass the complex involvement of the adaptive immune system, a hallmark of human disease. However, the primary objective of our study was to investigate the direct anti-inflammatory and antifibrotic effects of OL on synovial hyperplasia and joint destruction—a hallmark pathology in RA that is robustly represented in the CFA model. This model is characterized by rapid onset, high incidence, and severe synovitis and bone erosion, making it exceptionally suitable for the initial screening of therapeutic agents targeting SF activation and osteoclastogenesis, which are central to our mechanistic inquiry. In addition, as this study represents a foundational, early-stage mechanistic investigation, our initial strategy was to utilize well-controlled in vitro systems and a standardized animal model to precisely delineate the signaling pathways targeted by OL. We are subsequently collecting clinical datasets and samples from RA patients for correlation studies, which will significantly increase the translational impact of our research.
In this study, metabolomics, MS, and proteomics techniques were integrated to establish a mutually validated and complementary relationship between the metabolomic and proteomic data. This technique can more scientifically and realistically elucidate the mechanism of drug efficacy. It is an effective technique for drug screening, drug development, pathogenesis, and the discovery of disease treatment targets. This study provides a research idea and a technical model for research on drug efficacy mechanisms. In addition, the pathogenesis of RA and the therapeutic targets and signaling pathways of OL discovered in this study provide new ideas and research directions for the treatment of arthritis-related diseases.
5. Conclusions
OL exerted significant therapeutic effects in a rat model of RA. It markedly inhibited the proliferation of SFs and reduced the release of inflammatory and tissue damage factors. The underlying therapeutic mechanism involved targeting ACOT1 to increase its ubiquitination and protease degradation, thereby reducing the biosynthesis and metabolic levels of unsaturated fatty acids such as arachidonic acid, linolenic acid, α-linoleic acid, and 5-HETE. This phenomenon reduced the release of proinflammatory and tissue-damaging factors caused by the stimulation of unsaturated fatty acids and their metabolites. The overexpression of the PI3K-AKT and JAK-STAT signaling pathways was inhibited, preventing excessive proliferation, adhesion, joint deformities, and inflammatory reactions in SF. The results of this study emphasize OL as a promising candidate for RA treatment and highlight the importance of metabolic reprogramming of unsaturated fatty acids in RA management.
CRediT authorship contribution statement
Hongda Liu: Writing - review & editing, Writing - original draft, Visualization, Validation, Software, Investigation, Formal analysis, Data curation, Conceptualization. Le Yang: Writing - review & editing, Writing - original draft, Validation, Investigation, Formal analysis. Yu Yang: Visualization, Validation, Software, Formal analysis. Huan Tang: Writing - review & editing, Supervision, Methodology, Investigation, Conceptualization. Junling Ren: Writing - review & editing, Methodology, Investigation. Hui Sun: Supervision, Project administration, Methodology. Xin Sun: Software, Investigation. Songyuan Tang: Software, Investigation, Formal analysis. Chong Qiu: Writing - review & editing, Software, Methodology, Conceptualization. Ye Sun: Software, Investigation. Jigang Wang: Writing - review & editing, Methodology, Conceptualization. Guangli Yan: Resources, Project administration, Methodology, Data curation, Conceptualization. Ling Kong: Validation, Software. Ying Han: Writing - review & editing, Supervision, Methodology. Xijun Wang: Writing - review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by the International (Regional) Cooperation and Exchange Program of the National Natural Science Foundation of China (U23A20501), the National Natural Science Foundation of China (32141005), the Youth Science Foundation of China (82404816 and 82204690), the State Key Laboratory of Dampness Syndrome of Chinese Medicine (SZ2021ZZ49 and SZ2024QN06), the Heilongjiang Provincial Natural Science Foundation—Joint Guidance Program (LH2023H069), the Heilongjiang Provincial Key Research and Development Program (2022ZX02C04), and the Guangdong Guangzhou Joint Fund Youth Fund Project (2023A15151110703). Thanks to all authors and their respective organizations for their efforts and support in this research work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2025.10.029.
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