The potential targets of RES were predicted using PubChem (pubchem.ncbi.nlm.nih.gov) and SwissTargetPrediction (swisstargetprediction.ch/). All potential disease targets were retrieved by searching databases including Genecards (genecards.org), Online Mendelian Inheritance in Man (OMIM) (omim.org), and Drugbank (drugbank.com) with the keyword 'osteoarthritis'. The standardized nomenclature annotation of the screened targets was performed using the UniProt database (uniprot.org/). The RES and OA targets predicted from the databases were uploaded to the Venny 2.1.0 platform (liuxiaoyuyuan.cn/) to obtain the common targets of RES and OA.

Overlapping targets shared between RES and OA were uploaded to the STRING database (cn.string-db.org) to construct a protein-protein interaction (PPI) network. The visualization of this PPI network was performed using Cytoscape 3.9.2 software (cytoscape.org). Overlapping targets were further analyzed using the Metascape database (metascape.org) to perform Gene Ontology (GO; geneontology.org) enrichment analysis, encompassing biological processes (BPs), molecular functions (MFs) and cellular components (CCs). Kyoto Encyclopedia of Genes and Genomes (KEGG; kegg.jp) enrichment analysis was conducted to identify relevant signaling pathways. The data were imported into the Bioinformatics Platform (bioinformatics.com.cn/login/) for visualization. P<0.05 was considered to indicate significant enrichment.

RES (purity >99%; cat. no. R5010) for in vitro assays was dissolved in dimethyl sulfoxide (cat. no. D8418; both Sigma-Aldrich; Merck KGaA). RES (purity >98%; cat. no. R8350; Beijing Solarbio Science & Technology Co., Ltd.) for oral gavage, due to its suitability for animal experiments, was diluted in 0.5% CMC-Na (dissolved in saline).

SW1353 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI-1640 (cat. no. SH30809.01; Cytiva) medium supplemented with 10% FBS (cat. no. ZQ500-A; Zhong Qiao Xin Zhou Biotechnology Co., Ltd.) and 1% penicillin/streptomycin (cat. no. SV30010; HyClone; Cytiva) at 37°C. The medium was refreshed every 2-3 days until cells reached ~80% confluence.

SW1353 cells were inoculated into 96-well plates at a density of 3×10³/well overnight. Subsequently, cells were treated with RES at 1, 2, 4, 16, 32, 64 and 128 μM for 24 or 48 h at 37°C. Following treatment, the culture medium was removed and 100 μl/well Cell Counting Kit-8 [CCK-8; cat. no. MI00615A; Mishu (Xi'an) Biotechnology Co., Ltd.] solution was added to the plates. The cells were incubated for an additional 2 h. Absorbance was determined by measuring the optical density (OD) at 450 nm using a microplate reader (BioTek Instruments, Inc.).

OA cell model was induced by IL-1β (10 ng/ml; cat. no. 200-01B; PeproTech, Inc.) as previously described (26). Briefly, SW1353 cells were inoculated into 6-well or 12-well plates at a density of 1×10⁴/cm² and incubated for 24 h at 37°C. Cells underwent serum starvation for 10-14 h before serum-free RPMI-1640 (Cytiva) was replaced with complete medium. Subsequently, cells were pretreated with 16 μM RES alone or a combination of RES and compound C (ComC) (1 μM; cat. no. ab120843; Abcam), a selective AMPK inhibitor, for 2 h at 37°C. This was followed by 24 h treatment with 10 ng/ml IL-1β at 37°C (27,28). The cells were harvested and centrifuged at 1,000 × g for 5 min at 4°C to obtain cell-free supernatant for subsequent experiments.

The cells were cultured at a density of 1×10⁴/cm² and fixed with 4% paraformaldehyde (cat. no. AR1069; BOSTER Biological Technology Co., Ltd.) for 30 min at room temperature. The cells were stained for 30 min with Alcian Blue (AB) staining solution (pH 0.2; cat. no. ST046; Heart Biological Technology Co., Ltd.) or Safranin O (SO) staining solution (cat. no. G1067; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature. Slides were sealed with mounting medium, and the cells were observed under a light microscope.

The cells were seeded into 6-well plates at a density of 1×10⁵/well and total RNA extraction was performed on ice using the SPARKeasy Cell RNA Quick Extraction kit (cat. no. AC0205-A; Shandong Sikejie Biotechnology Co., Ltd.). The RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). RNAs were reverse-transcribed into cDNA using the SPARKscript II RT Plus kit [with gDNA Eraser; cat. no. AG0304 (SY); Shandong Sikejie Biotechnology Co., Ltd.], according to the manufacturer's instructions. Primer sequences are listed in Table I. qPCR was performed using a Bio-Rad qPCR system (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and combined annealing/extension at 58°C for 60 sec. The expression levels of mRNAs were determined using the 2^−ΔΔCq method with β-actin as the internal reference (29).

The cells were washed with ice-cold PBS. Phosphatase inhibitors (cat. no. AR1183), protease inhibitors (cat. no. AR1182-1; both BOSTER Biological Technology Co., Ltd.) and RIPA buffer (cat. no. P0013C; Beyotime Biotechnology) were mixed at a ratio of 1:1:100 and added to the cells. The lysate was centrifuged at 12,000 × g for 10 min at 4°C and the supernatant was collected. Protein concentration was determined using the BCA protein assay kit (cat. no. ZJ102; Shanghai Epizyme Biomedical Technology Co., Ltd.). The denatured proteins (20 μg/lane) were separated using 8-12% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. Membranes were blocked using 5% skimmed milk for 1 h at room temperature and incubated with anti-MMP13 (1:2,000; cat. no. BA2204), anti-COL2A1 (cat. no. BA0533; both BOSTER Biological Technology Co., Ltd.), anti-AMPK (cat. no. CY5326), anti-phosphorylated (p-)AMPK (all 1:1,000; cat. no. CY5608), anti-PPARγ (cat. no. AY9445), anti-NLRP3 (both 1:750; cat. no. CY5651; all Shanghai Ebiwei Biotechnology Co., Ltd.), anti-cleaved caspase-1 (1:1,000; cat. no. ab179515; Abcam) and anti-β-actin (1:5,000; cat. no. AB0035; Shanghai Ebiwei Biotechnology Co., Ltd.) at 4°C overnight. The membrane was washed with 0.05% TBST, followed by incubation with horseradish peroxidase-conjugated anti-rabbit (1:10,000; cat. no. BA1054; BOSTER Biological Technology Co., Ltd.) secondary antibodies dissolved in TBST for 1 h at room temperature. The membrane was washed three times with TBST, followed by chemiluminescence band detection using the enhanced luminescence solution [cat. no. MI00607A; Mishu (Xi'an) Biotechnology Co., Ltd.]. The protein expression levels, normalized to β-actin, were quantified using ImageJ software (version 1.53a; National Institutes of Health).

C57BL/6 male mice (n=22; age, ~10 weeks; weight, ~20 g), were purchased from the Laboratory Animal Center of Xi'an Jiaotong University. Animals were housed at 25±2°C, 50±10% humidity with a 12/12-h light/dark cycle and ad libitum access to food/water. The animal procedures were approved by the Animal Care and Use Committee of Xi'an Jiaotong University (approval no. XJTUAE2024-1403; Xi'an, China). Mice were randomized into Sham (n=6), OA (n=8) and RES (n=8) groups. The OA and RES mice were established using the destabilization of the medial meniscus (DMM) method on the right knee (30). Mice were anesthetized via intraperitoneal injection of 3% sodium pentobarbital solution (50 mg/kg). The joint capsule was dissected along the medial margin of the patellar ligament and the tibial collateral ligament of the medial meniscus was transected. Finally, the joint cavity and skin were sutured. The mice in the Sham group were only incised in the joint capsule on the right knee and sutured. The DMM and Sham surgery success rates were 75-80 and 100%, respectively. Mice were excluded if surgical failure was defined, either by the incomplete transection of the medial meniscotibial ligament or by any accidental damage to the adjacent articular cartilage or meniscus during the surgical procedure. The final sample size was six mice/group. All exclusions were performed before outcome assessments and the investigators were blinded to group allocation. After 4 weeks, OA mice in the RES group received RES at 45 mg/kg through oral gavage once/day for 6 weeks. Oral RES exerts anti-OA effects at 45 mg/kg/day (31) and activates AMPK at 25-30 mg/kg (32). Generally, effective RES doses in animal studies range widely, with 40-50 mg/kg established as standard regimens (33,34). Thus, a midrange dose of 45 mg/kg/day was selected. The mice in the Sham and OA groups were concurrently administered an equal amount of the vehicle solution. At 10 weeks post-surgery, the mice were subjected to euthanasia using an overdose of sodium pentobarbital (150 mg/kg). Prior to euthanasia, the adequacy of anesthesia was verified by the absence of pedal withdrawal and corneal reflex, and then blood was collected via cardiac puncture. Final death was confirmed by the cessation of heartbeat and respiration. Liver and right knee joint tissue was harvested for subsequent experiments.

The cell culture supernatant and mouse blood samples were centrifuged at 3,000 × g for 20 min at 4°C to obtain the clarified cell supernatant and mouse serum, respectively. The IL-1β, TNF-α, IL-6, IL-10 or IL-18 ELISA kits (IL-1β: cat. no. RX203063M for serum; TNF-α: cat. no. RX104793H for supernatant; cat. no. RX202412M for serum; IL-6: cat. no. RX106126H for supernatant; cat. no. RX203049M for serum; IL-10: cat. no. RX103064H for supernatant; cat. no. RX203075M for serum; IL-18: cat. no. E10349 for supernatant; all Xi'an Shandezhiyuan Biotechnology Co., Ltd.) were used according to the manufacturer's instructions. The OD values were measured at 450 nm using a microplate reader (BioTek Instruments, Inc.), and protein concentration was determined using the standard curve method.

To evaluate liver function in the mice, serum levels of alanine aminotransferase (ALT)/glutamic-pyruvic transaminase (GPT) and aspartate aminotransferase (AST)/glutamic-oxaloacetic transaminase (GOT) were measured using the ALT/GPT and AST/GOT test kits (cat. nos. C009-2-1 and C010-2-1; both Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. The OD values were measured using a microplate reader (BioTek Instruments, Inc.) at a wavelength of 510 nm, with the corresponding values of ALT/GPT and AST/GOT determined using the standard curve method.

Mouse right knee joint and liver tissue were excised and fixed with 4% paraformaldehyde (cat. no. AR1069; BOSTER Biological Technology Co., Ltd.) for 48 h at room temperature. The joint tissues were decalcified. Tissue was embedded in paraffin and sectioned into 5 μm paraffin sections. The tissue sections were stained with hematoxylin and eosin (HE; hematoxylin for 6 min and eosin for 1 min) or SO-fast green (FG) staining (FG for 10 min, followed by SO for 30 sec) at room temperature. Stained sections were examined under a light microscope (Leica GmbH). The severity of synovitis was graded using a 9-point synovial score system (35). The severity of OA and cartilage degeneration was assessed using the Osteoarthritis Research Society International scoring system (36), which ranges from 0 to 24.

For IF, SW1353 cells were inoculated onto cover slides at a density of 1×10⁴/cm² and fixed with 4% paraformaldehyde (cat. no. AR1069; BOSTER Biological Technology Co., Ltd.) for 30 min at room temperature. For IHC, the 5 μm paraffin sections of the knee joint tissue were deparaffinized in xylene for 20 min and rehydrated through a graded ethanol series at room temperature. Subsequently, the sections were treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase at room temperature, and digested with 20 μg/ml proteinase K (cat. no. P6157; Macklin Biochemical Technology Co., Ltd.) for 20 min at 37°C. Next, both cell and tissue samples were blocked with 3% BSA for 30 min at room temperature. Samples were then incubated at 4°C overnight with the following primary antibodies: anti-COL2A1 (1:200; cat. no. BA0533; BOSTER Biological Technology Co., Ltd.), anti-p-AMPK (1:100; cat. no. CY5608) and anti-PPARγ (1:100; cat. no. AY9445) (both from Shanghai Ebiwei Biotechnology Co., Ltd.), anti-AMPK (for IF only; 1:100; cat. no. CY5326; Shanghai Ebiwei), anti-MMP13 (for IF: 1:300, cat. no. bs-0575R, Beijing Biosynthesis Biotechnology Co., Ltd.; for IHC: 1:100, cat. no. BA2204, BOSTER), and anti-NLRP3 (for IF: 1:100, cat. no. CY5651, Shanghai Ebiwei; for IHC: 1:50, cat. no. BA3677; BOSTER). Following PBS washes, IF samples were incubated with Alexa Fluor^® 594-conjugated goat anti-rabbit IgG (1:1,000; cat. no. ab150080; Abcam) in 3% BSA for 1 h at room temperature in the dark, and counterstained with DAPI for 5 min. Sections were incubated with HRP-conjugated goat anti-rabbit IgG (1:60; cat. no. RS0002; Immunoway Biotechnology, Inc.) for 30 min at room temperature, treated with streptavidin-peroxidase for 30 min, visualized with DAB, and counterstained with hematoxylin for 1 min at room temperature before being mounted with neutral gum. All images were captured using a microscope (fluorescence for IF and light for IHC; Leica GmbH). Image analysis was performed using ImageJ (version 1.53a; National Institutes of Health); specifically, the mean fluorescence intensity and the quantitative analysis of the PPARγ nuclear-to-cytoplasmic ratio were measured for IF samples.

All in vitro experiments were independently replicated ≥3 times and all in vivo animal studies were conducted with six mice per group. All data are presented as the mean ± SD. Prior to parametric testing, data normality and homogeneity of variance were verified using the Shapiro-Wilk test and Brown-Forsythe test, respectively. One-way ANOVA was used, followed by Dunnett's or Sidak's multiple comparisons test for post hoc pairwise analysis. All statistical analyses were performed using GraphPad Prism 9.5 software (GraphPad Software, Inc.; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

A total of 175 potential targets of RES were predicted using the SwissTargetPrediction database. In addition, 4,832 OA-related target genes were screened by searching the GeneCards, DrugBank, OMIM and DisGeNET databases. Finally, 123 overlapping targets were screened using the STRING database (Fig. 1A). A PPI network was constructed using a confidence threshold of ≥0.4, and the resulting data underwent topological analysis based on degree values. The nodes displayed a centripetal distribution pattern (Fig. 1B). Within this network, targets with a higher number of adjacent nodes were considered more critical and thus identified as core targets. These core targets were implicated in metabolism and inflammation, encompassing proteins such as multiple cytochrome P450 enzymes (CYP), prostaglandin-endoperoxide synthase 2 (PTGS2) and PPARγ.

BP terms were predominantly associated with functions such as 'monoterpenoid metabolic process', 'lipid hydroxylation' and 'epoxygenase P450 pathway' (Fig. 1C). The CC terms were primarily associated with structures, including 'basal part of cell', 'basal plasma membrane' and 'basolateral plasma membrane'. The MF terms were mainly enriched in 'estrogen 2-hydroxylase activity', 'caffeine oxidase activity' and 'oxidoreductases activity, acting on CH or CH₂ groups'. Among KEGG pathways, 'PPAR signaling pathway' and 'AMPK signaling pathway', both closely associated with metabolism, were significantly enriched (Fig. 1D).

The cytotoxicity of RES on SW1353 cells was assessed using the CCK-8 assay. RES was administered (0-128 μM) and cell viability was assessed 24 or 48 h after treatment. Based on the results, RES exhibited no cytotoxicity at concentrations up to 64, and 16 μM induced the most significant increase in cell viability at both 24 and 48 h; therefore, this concentration of RES was selected for subsequent experiments (Fig. 2A). Histological evaluation was performed to assess the impact of RES on the IL-1β-induced loss of GAG and proteoglycan in SW1353 cells. IL-1β treatment significantly diminished AB staining intensity compared with the control (Fig. 2B and C). Notably, co-treatment with RES significantly restored AB staining intensity, indicating its protective effect against the depletion of GAG and proteoglycan. Consistent with these findings, SO staining demonstrated that IL-1β significantly decreased the staining intensity compared to the control, whereas co-treatment with RES significantly reversed this decline (Fig. 2D and E), thereby confirming the protective capacity of RES on GAG and proteoglycan levels.

mRNA and protein expression of AMPK was not significantly different between the groups (Fig. 3A, C and D). Relative expression of p-AMPK was significantly decreased in the IL-1β group compared with that in the control group; pretreatment with RES partially restored its expression; cotreatment with ComC (RES+ IL-1β + ComC), an inhibitor of AMPK, suppressed p-AMPK expression, suggesting that RES activated AMPK and ComC effectively inhibited the activation (Fig. 3C and E). The mRNA and protein expression of PPARγ was significantly lower in the IL-1β group compared with the control group. This decrease was reversed in the RES+ IL-1β group, and diminished in the RES+ IL-1β + ComC group (Fig. 3B, C and F). These findings suggest that RES modulated the AMPK/PPARγ signaling pathway. Notably, the results of IF staining showed that the in situ protein expression of AMPK, p-AMPK and PPARγ was consistent with that in bulk cell lysate of western blotting (Fig. 3G-L). As evidenced by a higher nuclear-to-cytoplasmic PPARγ ratio, the nuclear translocation of PPARγ was enhanced in the RES+ IL-1β compared with that in the IL-1β group; inhibition of AMPK phosphorylation diminished this effect, leading to decreased nuclear PPARγ accumulation (Fig. 3M). Therefore, RES activated the AMPK/PPARγ pathway in SW1353 cells.

RES suppressed the IL-1β-induced upregulation of NLRP3 mRNA; inhibition of AMPK activation counteracted this effect, leading to increased NLRP3 expression (Fig. 4A). Western blot analysis of NLRP3 protein expression, along with IF staining, corroborated the qPCR findings (Fig. 4B, C, F and G). Western blot analysis of cleaved caspase-1 showed that RES effectively suppressed IL-1β-induced accumulation, which was counteracted by inhibition of AMPK activation (Fig. 4B, D and E). Furthermore, the secretion levels of IL-18, a product of cleaved caspase-1, mirrored the changes observed in NLRP3 expression across all groups (Fig. 4H). IL-1β significantly upregulated the expression of TNF-α and IL-6 and downregulated the expression of IL-10 at the mRNA level. Treatment with RES reversed the effects of IL-1β and inhibition of AMPK phosphorylation suppressed this effect of RES (Fig. 4I-K). The secretion levels of these inflammatory factors mirrored their intracellular expression patterns (Fig. 4L-N), indicating that RES blocked the NLRP3 inflammasome response in SW1353 cells by suppressing NLRP3 expression, inhibiting caspase-1 cleavage and reducing downstream IL-18 release via the AMPK-dependent signaling pathway.

The qPCR and western blotting results demonstrated that IL-1β decreased the mRNA and protein expression of COL2A1 and elevated the expression of MMP13. Pretreatment with RES significantly attenuated the effects induced by IL-1β, while cotreatment with ComC decreased the activity of RES (Fig. 5A-E). The IF staining results were consistent with those of qPCR and western blotting (Fig. 5F-I). RES-mediated restoration of COL2A1 and suppression of MMP13 were reversed by the AMPK inhibitor ComC, suggesting that RES could regulate ECM metabolism via the AMPK-dependent signaling pathway in SW1353 cells.

No significant differences were observed in the expression levels of ALT/GPT and AST/GOT among the groups of mice (Fig. S1A and B). The pathological examination of liver tissue revealed that the hepatocytes in the RES group maintained a relatively intact structure, devoid of notable inflammatory cell infiltration or degeneration. Furthermore, the overall liver architecture remained clear and well-preserved (Fig. S1C). The results indicated that RES, at a dose of 45 mg/kg, exerted no significant hepatotoxic effects on the liver of OA mice.

HE staining of cartilage tissue revealed a smooth and structurally intact cartilage in the Sham group. Conversely, the articular cartilage in the OA group exhibited notable damage and a decreased chondrocyte population. RES treatment attenuated this effect and restored the chondrocyte population (Fig. 6A and B). The SO-FG staining demonstrated that RES partly reversed proteoglycan loss induced via the DMM surgery (Fig. 6C). The OARSI score of the OA group was significantly higher than that of the Sham group, whereas the score for the RES group was significantly lower than that of the OA group (Fig. 6D), indicating that RES attenuated cartilage degeneration in OA mice.

The HE staining of the synovium revealed structural abnormalities in the OA group, including increased inflammatory infiltration, pannus formation and elevated cell density. RES attenuated these pathological changes and reduced synovial scores in OA mice (Fig. 6E and F). ELISA demonstrated that the expression levels of IL-1β, TNF-α and IL-6 were elevated, while IL-10 expression was decreased in the OA group; these alterations were reversed by RES (Fig. 6G-J). Therefore, RES suppressed the inflammatory response of synovium and serum in OA mice.

IHC staining was performed to detect the expression levels of proteins associated with the AMPK/PPARγ signaling pathway, NLRP3 (a key component of the inflammasome complex) and matrix metabolism. The staining showed a significant decrease in the protein expression levels of p-AMPK, PPARγ and COL2A1 in the OA compared with the Sham group. RES partially mitigated these effects (Fig. 7A, B and D). Conversely, the OA mice exhibited significantly elevated protein expression levels of NLRP3 and MMP13 compared with the Sham mice, with RES inhibiting these increases (Fig. 7C and E). Therefore, RES modulated the expression of AMPK, PPARγ and NLRP3, promoting ECM homeostasis in the articular cartilage of OA mice. These findings align with the in vitro results, suggesting that RES may exert protective effects in OA via the AMPK/PPARγ/NLRP3 signaling pathway.