Poricoic Acid A Attenuates Osteoarthritis Progression by Stabilizing PTEN and Suppressing PI3K/AKT Signaling

TL;DR

Poricoic acid A, a natural compound, may help treat osteoarthritis by stabilizing a protein called PTEN and reducing harmful signaling.

Contribution

The study reveals a new therapeutic mechanism of poricoic acid A in osteoarthritis through PTEN stabilization and PI3K/AKT suppression.

Findings

Poricoic acid A reduced cartilage degradation and inflammation in a mouse model of osteoarthritis.

Poricoic acid A suppressed PI3K/AKT signaling by stabilizing PTEN protein.

PTEN inhibition negated the protective effects of poricoic acid A in cartilage cells.

Abstract

Osteoarthritis (OA) is characterized by chronic inflammation, and progressive cartilage degradation. Poricoic acid A (PAA), a triterpenoid compound derived from Poria cocos, exhibits anti-inflammatory and anti-fibrotic activities, but its therapeutic potential in OA remains unknown. Here, we investigated the protective effects and mechanisms of PAA in IL-1β-stimulated chondrocytes and a destabilization of a medial meniscus (DMM) mouse model. PAA significantly restored cartilage matrix synthesis, reduced inflammatory catabolism, and alleviated cartilage degeneration in vivo. RNA-seq identified PI3K/AKT signaling as a major pathway regulated by PAA. Mechanistically, PAA stabilized PTEN protein, suppressed PI3K/AKT phosphorylation, and reversed IL-1β-induced cartilage catabolism. PTEN inhibition abolished the beneficial effects of PAA. These findings identify PAA as a promising therapeutic candidate for OA and reveal PTEN-PI3K-AKT as its major regulatory axis.

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1. Introduction

Osteoarthritis (OA), the most common degenerative joint disorder, exerts a significant socioeconomic burden globally as its prevalence escalates with an aging population [1,2]. OA is characterized by progressive cartilage degradation, osteophyte formation, subchondral bone sclerosis, and synovial inflammation, culminating in chronic pain and functional disability [3,4].

Increasing evidence indicates that persistent low-grade inflammation, excessive oxidative stress, and dysregulated cytokine signaling play central roles in driving disease progression [5,6]. Aberrant activation of pathways such as NF-κB [7], TGF-β/Smad [8], MAPK [9] and PI3K/AKT [10] accelerates extracellular matrix breakdown, promotes chondrocyte catabolism, and amplifies synovial inflammation. Recent evidence highlights the crucial involvement of the PI3K/AKT signaling pathway in the metabolic reprogramming and pathological transformation of chondrocytes during OA progression [11,12]. The activation of the Ras–PI3K/AKT/mTOR pathway promotes glycolysis and stabilizes HIF-1α, leading to lactate accumulation and subsequent histone lactylation. Metabolic and epigenetic alterations facilitate fibrotic reprogramming and the expression of catabolic genes, thereby expediting cartilage degeneration in OA [13]. Aberrant activation of the PI3K/AKT pathway disrupts mitochondrial quality control in chondrocytes, resulting in mitochondrial dysfunction that contributes to inflammation, apoptosis, and cartilage degeneration in OA [14]. The PI3K/AKT pathway regulates the inflammatory behavior of synovial macrophages by promoting a pro-inflammatory M1 phenotype, which enhances cytokine production and perpetuates synovitis [15]. Furthermore, the activation of PI3K/AKT exacerbates inflammation by activating the NF-κB and MAPK pathways [16], hence strengthening the pro-inflammatory cascade in OA.

Poricoic acid A (PAA), a bioactive triterpenoid derived from P. cocos, has emerged as a multifunctional regulator of inflammatory and metabolic homeostasis [17]. It has been primarily studied in kidney diseases, where it shows potent anti-inflammatory, antioxidant, and anti-fibrotic effects [18,19]. Evidence indicates that PAA efficiently inhibits NF-κB-mediated inflammatory responses, reduces pro-inflammatory cytokine production, and alleviates oxidative stress by curbing excessive ROS formation [20]. In addition to its anti-inflammatory and antioxidant properties, PAA regulates essential cellular processes-such as apoptosis, mitochondrial quality control, and autophagy—thus maintaining tissue integrity under stress circumstances [21,22]. Recent studies emphasize its capacity to modulate metabolic and signaling pathways, including PI3K/AKT and TGF-β/Smad, which are essential for sustaining extracellular matrix equilibrium and averting catabolic reprogramming [23,24]. These mechanistic insights suggest that PAA holds strong therapeutic potential in conditions characterized by chronic inflammation and structural tissue degradation.

Based on these findings, we hypothesized that PAA may exert chondroprotective effects by attenuating inflammatory responses in OA. To test this hypothesis, we conducted a series of in vitro and in vivo experiments to evaluate the protective role of PAA against IL-1β-induced chondrocyte injury and DMM-induced cartilage degeneration. Our study provides new insights into the therapeutic potential of PAA as a natural disease-modifying agent for OA.

2. Results

2.1. PAA Shows No Cytotoxicity to Chondrocytes

In P. cocos, glucose-derived acetyl-CoA enters the mevalonate pathway and undergoes a series of enzymatic reactions, including HMGR-mediated steps, to generate lanostane-type triterpenoids such as PAA [25] (Figure 1A). To determine the cytotoxicity profile of PAA, primary chondrocytes were treated with increasing concentrations of PAA (0–40 μM) for 24, 48, and 72 h. CCK-8 assays revealed that PAA treatment did not reduce cell viability at any tested concentration or time point, indicating good cellular tolerance (Figure 1B–D). Consistently, Live/Dead staining showed predominantly viable cells across all concentrations, with no notable increase in cell death or morphological abnormalities (Figure 1E). Quantitative analysis is presented in Figure S1. The findings demonstrate that PAA has favorable biocompatibility and does not induce cytotoxicity in chondrocytes within the examined concentration range.

2.2. PAA Attenuates IL-1β-Induced Extracellular Matrix Degradation In Vitro

To investigate whether PAA safeguards chondrocytes against inflammation-induced degeneration, IL-1β was used to create an in vitro OA-like environment. PAA dose screening (0–40 μM) revealed a significant induction of anabolic markers in chondrocytes, with both ACAN and COL2A1 reaching maximal expression at 20 μM (Figure S2A,B). Under IL-1β (10 ng/mL) stimulation, PAA treatment at 0–40 μM modulated chondrocyte metabolism. qPCR analysis showed that 20 μM PAA most effectively restored anabolic gene expression while suppressing catabolic markers (Figure S2C–F). Safranin O staining consistently demonstrated maximal preservation of proteoglycan content at this concentration (Figure S2G,H). Higher concentrations did not confer additional benefit. Accordingly, this concentration was used for subsequent experiments. Safranin O staining showed that IL-1β stimulation significantly decreased proteoglycan content, but PAA (20 μM) therapy considerably maintained matrix staining intensity (Figure 2A). Quantitative analysis of Safranin O staining is presented in Figure S2I. Treatment with PAA restored proteoglycan deposition, indicating improved matrix synthesis under inflammatory conditions. Consistent with histological findings, qPCR analysis demonstrated that IL-1β significantly downregulated anabolic genes Col2a1, aggrecan (Acan) and upregulated catabolic enzymes Mmp3 and Mmp13. PAA treatment largely reversed these alterations, enhancing Col2a1 and Acan expression while reducing Mmp3 and Mmp13 mRNA levels (Figure 2B–E). Western blot research also validated these effects at the protein level. IL-1β decreased COL2A1 and ACAN levels while significantly upregulating MMP3 and MMP13 expression. PAA restored the protein levels of COL2A1 and ACAN while inhibiting the IL-1β-induced overexpression of MMP3 and MMP13 (Figure 2F–K). Collectively, these data indicate that PAA substantially alleviates IL-1β-induced chondrocyte degeneration by enhancing matrix production and suppressing catabolic reactions.

2.3. PAA Alleviates DMM-Induced OA Progression In Vivo

To evaluate the protective role of PAA in vivo, a DMM mouse model was established and compared with sham-operated controls. OA-like pathological changes induced by DMM surgery were validated at the indicated post-operative time points by histological assessment and micro-CT analysis, including Safranin O–Fast Green staining, OARSI scoring, and subchondral bone evaluation. Safranin O–Fast Green staining revealed significant proteoglycan depletion and structural cartilage deterioration after DMM surgery, while intra-articular PAA therapy significantly maintained matrix staining intensity and enhanced cartilage surface integrity relative to the DMM group (Figure 3A). The OARSI scores were consistently and substantially decreased in PAA-treated mice, suggesting a deceleration of OA development (Figure 3B). Quantitative parameters including osteophyte size and osteophyte maturity score were significantly decreased after PAA treatment, indicating reduced aberrant bone remodeling (Figure 3C,D). Micro-CT analysis further supported these findings. Reconstructed 3D images and sagittal images of mice knee revealed significant subchondral bone sclerosis and osteophyte development in the DMM + Vehicle group. PAA treatment substantially reduced osteophyte development and mitigated subchondral bone thickening, restoring bone structure closer to the sham condition (Figure 3E). Compared with sham knees, DMM joints exhibited increased trabecular bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), accompanied by a reduction in trabecular separation (Tb.Sp), reflecting subchondral bone sclerosis and trabecular densification. Notably, PAA treatment effectively mitigated these pathological changes and partially restored the subchondral trabecular structure (Figure 3F–I). Immunohistochemical staining demonstrated that the expression of cartilage matrix proteins COL2A1 and ACAN was well-maintained in sham joints but significantly reduced in DMM mice. PAA treatment restored the expression of both markers in DMM cartilage. Conversely, DMM induction markedly increased the levels of catabolic enzymes MMP13 and MMP3, whereas PAA treatment suppressed their overexpression (Figure 3J–N). Together, these results demonstrate that PAA effectively protects articular cartilage and subchondral bone from OA-related structural deterioration in vivo.

2.4. RNA-Seq Identifies PI3K/AKT Signaling as a Major Pathway Regulated by PAA

To explore the molecular mechanisms by which PAA protects chondrocytes, RNA-seq analysis was performed on IL-1β-stimulated chondrocytes with or without PAA treatment. Volcano plot analysis identified 181 upregulated and 185 downregulated genes after PAA treatment, indicating a substantial transcriptomic shift induced by PAA (Figure 4A). Heatmap visualization further demonstrated that IL-1β induced strong upregulation of inflammatory and catabolic genes (Il1b, Nos2, Mmp3, and Adamts4), while suppressing anabolic markers (Col2a1, Acan) (Figure 4B). Gene Ontology (GO) molecular function analysis revealed that differentially expressed genes (DEGs) in the IL-1β + PAA group were enriched in protein regulatory and transcription-related functions (Figure S3A). KEGG enrichment analysis revealed that PI3K/AKT signaling was among the top significantly regulated pathways, together with MAPK signaling, and TGF-β signaling (Figure 4C). Consistently, gene set enrichment analysis (GSEA) demonstrated significant negative enrichment of the PI3K/AKT, NF-κB, and mTOR pathways in PAA-treated chondrocytes, indicating that PAA effectively suppresses IL-1β-induced activation of these inflammatory signaling cascades (Figure S3B–D). These findings suggest that PAA influences multiple pathways involved in inflammation, matrix homeostasis, and cell survival, with PI3K/AKT emerging as the dominant hub. PI3K/AKT pathway-related genes were significantly altered as shown in heatmap (Figure 4D). To validate the RNA-seq findings at the protein level, time-course Western blot analysis was performed. IL-1β markedly increased the phosphorylation of PI3K and AKT in a time-dependent manner. PAA treatment significantly reduced p-PI3K and p-AKT levels at all examined time points (30, 60, and 120 min), while total PI3K and AKT remained unchanged (Figure 4E–G). These results confirm that PAA inhibits IL-1β-induced activation of the PI3K/AKT pathway, consistent with transcriptomic predictions.

2.5. PAA Stabilizes PTEN and Suppresses PI3K/AKT Signaling

To further elucidate the downstream regulatory network involved in the action of PAA, a STRING-based protein–protein interaction (PPI) network study of PI3K/AKT-related DEGs revealed Pten as a prominent node with significant connectivity, indicating its possible essential regulatory function in the downstream regulatory network associated with PAA activity (Figure 5A). As a lipid phosphatase that antagonizes PI3K activity and suppresses AKT phosphorylation, PTEN functions as a critical negative regulator of the PI3K/AKT signaling pathway [26,27]. Western blot analysis showed that IL-1β markedly reduced PTEN protein levels in a time-dependent manner, whereas PAA treatment largely preserved PTEN expression under the same conditions (Figure 5B,C).

To determine whether PTEN is required for the chondroprotective effects of PAA, Pten expression was selectively silenced using siRNA. qPCR analysis confirmed the efficient knockdown of Pten expression in chondrocytes transfected with si-Pten compared with si-NC, regardless of IL-1β stimulation or PAA treatment (Figure S4A). Safranin O staining showed that PAA markedly attenuated IL-1β-induced proteoglycan loss in si-NC-transfected chondrocytes, whereas this protective effect was largely abolished in si-Pten-transfected cells (Figure 5D). Quantitative analysis of Safranin O staining is presented in Figure S4B. qPCR analysis revealed that PAA markedly suppressed IL-1β–induced catabolic gene expression (Mmp3 and Mmp13) and restored anabolic markers (Col2a1 and Acan) in control cells, whereas these protective transcriptional effects were largely abolished upon Pten knockdown (Figure 5E–H). Consistently, Western blot analysis demonstrated that the protein-level effects of PAA were markedly weakened in Pten-silenced chondrocytes (Figure 5I). Notably, parallel experiments using the PTEN inhibitor VO-OHpic [28] yielded comparable results, as VO-OHpic abrogated the protective effects of PAA on both Safranin O staining and gene expression profiles, consistent with the findings observed upon Pten knockdown (Figure S4C–H). These results indicate that the chondroprotective effects of PAA are PTEN-dependent.

To further evaluate PTEN protein stability, a thermal shift assay was performed. Increasing temperature caused progressive PTEN denaturation in both DMSO- and PAA-treated groups; however, PTEN exhibited markedly higher thermal stability following PAA treatment, indicating that PAA enhances PTEN conformational resistance (Figure 5J,K). These findings demonstrate that PAA exerts its chondroprotective effects predominantly by stabilizing PTEN and thereby restraining PI3K/AKT pathway activation.

3. Discussion

PAA has been reported to exert multiple biological effects, particularly in the kidney. It activates AMPK to alleviate tubular dilation and renal fibrosis [19] and promotes Sirt3 activity to inhibit renal fibroblast activation [18]. PAA also protects against high-glucose-induced podocyte injury by regulating the AMPKα/FUNDC1 pathway [29], and suppresses TGF-β1-mediated epithelial–mesenchymal transition (EMT) in tubular epithelial cells, thereby attenuating renal fibrosis [30]. In addition to its renoprotective actions, PAA also plays important roles in metabolic regulation. It exhibits hypoglycemic effects by inhibiting α-glucosidase activity [31]. Moreover, PAA demonstrates antitumor activity; recent studies have shown that it suppresses the progression of lung and colorectal cancers through modulation of the PI3K/AKT/mTOR pathway while exerting minimal toxicity on major organs in mice [23,32]. P. cocos also contains poricoic acid B (PAB), a structurally similar triterpenoid. Although both PAA and PAB influence AMPK and PI3K/AKT signaling, PAB is associated with fewer molecular targets and shows weaker links to inflammatory and metabolic regulation compared with PAA [33].

Despite significant advances in understanding the pathophysiology of OA, current therapeutic strategies remain largely palliative and fail to halt or reverse disease progression [34,35]. Conventional analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) provide short-term symptom relief but are often limited by gastrointestinal, cardiovascular, and renal adverse effects, especially with long-term use [36,37]. Intra-articular corticosteroid injections offer transient improvement yet may accelerate cartilage degeneration with repeated administration [38,39]. Hyaluronic acid supplementation shows variable efficacy and lacks consistent clinical benefits across patient populations [40,41]. Emerging cell-based therapies, including mesenchymal stem cell (MSC) injections, also show variable outcomes due to inconsistencies in cell quality, engraftment efficiency, and long-term safety [42,43], while gene therapies face challenges related to vector safety, durability, and high translational costs [44]. Critically, the absence of effective disease-modifying therapies that can reverse structural degeneration means that many patients ultimately progress to late-stage disease requiring joint replacement surgery [45,46].

In this study, we identified PAA as a potent chondroprotective compound capable of attenuating inflammation-induced cartilage degeneration in vitro and mitigating OA progression in vivo. Using IL-1β-stimulated chondrocytes and a DMM mouse model, we demonstrated that PAA restores matrix homeostasis, suppresses catabolic responses, and prevents structural deterioration of both articular cartilage and subchondral bone. Mechanistically, transcriptomic analysis and biochemical validation converged on the PI3K/AKT pathway as the principal signaling axis regulated by PAA. Among PI3K/AKT-associated genes, Pten emerged as a central hub, and subsequent experiments confirmed that PAA stabilizes PTEN protein and restrains PI3K/AKT phosphorylation under inflammatory stimulation. PTEN is a key lipid phosphatase that antagonizes PI3K activity and serves as a critical gatekeeper of inflammatory, metabolic, and survival pathways [47,48]. Aberrant PI3K/AKT signaling promotes chondrocyte catabolism, mitochondrial dysfunction, and epigenetic reprogramming, thereby accelerating cartilage degeneration in OA [49]. Our findings show that IL-1β rapidly reduces PTEN expression while activating PI3K/AKT signaling, consistent with the known role of inflammatory cytokines in destabilizing PTEN and driving downstream pathological cascades. PAA not only prevented the loss of PTEN but also enhanced its thermal stability, suggesting a direct or indirect effect on PTEN conformational resilience. Importantly, both genetic silencing of PTEN by siRNA and pharmacological inhibition of PTEN using VO-OHpic abolished the beneficial effects of PAA on anabolic gene expression, matrix synthesis, and catabolic suppression, confirming that PTEN is indispensable for PAA-mediated regulation of chondrocyte homeostasis.

The ability of PAA to stabilize PTEN provides mechanistic insight into how natural triterpenoids modulate intracellular signaling during inflammatory stress. Previous studies have shown that PAA exerts anti-inflammatory and antioxidant effects in kidney diseases by regulating NF-κB, TGF-β/Smad, and oxidative stress pathways [21,22]. Our data extend these findings to the joint environment and identify PTEN stabilization as a previously unrecognized mechanism underlying the chondroprotective actions of PAA. Given that PTEN is frequently downregulated in inflamed or fibrotic tissues, PAA may have broader therapeutic potential in conditions characterized by PI3K/AKT overactivation.

Despite these strengths, this study has several limitations. First, whether PAA directly binds PTEN or regulates its stability through upstream post-translational mechanisms remains unknown. Advanced proteomic or structural assays may be required to delineate these interactions. Second, although the DMM model recapitulates major features of human OA, long-term studies and additional models are needed to evaluate chronic efficacy and safety. Moreover, intra-articular injection of PAA represents an invasive delivery approach that carries procedural risks, such as injection-related joint irritation or infection, and its feasibility for repeated administration in clinical settings requires careful evaluation. Finally, the absence of a positive control treatment limits direct comparison with existing OA therapies which should be addressed in future translational studies.

In summary, in the inflammatory environment, PI3K/AKT signaling is aberrantly activated, contributing to downstream transcriptional reprogramming characterized by reduced anabolic markers (Col2a1, Acan) and increased catabolic enzymes (Mmp3, Mmp13). Our data indicate that PAA upregulates PTEN, a key negative regulator of the PI3K/AKT cascade, thereby restraining excessive AKT phosphorylation. By restoring PTEN-mediated inhibition, PAA effectively dampens PI3K/AKT overactivation and mitigates IL-1β-induced matrix-degrading gene expression. Our study identifies PTEN-dependent modulation of the PI3K/AKT pathway as a central mechanism by which PAA alleviates OA-associated cartilage degeneration (Figure 6). These results not only advance understanding of PAA biology but also highlight the PTEN-PI3K-AKT axis as a compelling target for OA treatment.

4. Materials and Methods

4.1. Reagents and Antibodies

Recombinant mouse IL-1β was obtained from R&D Systems (Minneapolis, MN, USA). Poricoic acid A was acquired from MedChemExpress (Monmouth Junction, NJ, USA). Anti-ACAN, COL2a1, MMP3, MMP13 and PTEN were acquired from Abcam (Cambridge, UK). Anti-PI3K, p-PI3K, AKT and p-AKT were supplied by Cell Signaling Technology (Beverly, MA, USA). Anti-Actin was purchased from Boster (Wuhan, China).

4.2. Isolation and Culture of Primary Chondrocytes

Primary chondrocytes were prepared from knee cartilage of 5-day-old C57BL/6 mice. Cartilage specimens were initially exposed to 0.2% trypsin (Boster Biotechnology, Wuhan, China) for 30 min, after which the tissue fragments were subjected to a second digestion using 0.25% collagenase II (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 6 h in a hybridization oven. The resulting cell suspension was filtered, collected, and transferred into DMEM/F12 medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 100 U/mL penicillin–streptomycin (Boster Biotechnology, Wuhan, China). Cells were maintained at 37 °C in a humidified incubator with 5% CO_2_. Once cultures reached approximately 80% confluence on 10 cm dishes, chondrocytes were harvested for subsequent experiments.

4.3. Cell Counting Kit-8 (CCK-8) Assay

Cell viability was evaluated using the CCK8 (TargetMol, Boston, MA, USA). Chondrocytes were seeded into 96-well plates at a density of 5 × 10^3^ cells/well and treated with PAA (0–40 μM) for 24–72 h. At the indicated time points, 10 μL of CCK-8 reagent was added to each well and incubated for 2 h at 37 °C. Absorbance at 450 nm was measured using a microplate reader (BioTek, Winooski, VT, USA). Cell viability was calculated relative to the control group after blank subtraction.

4.4. Live/Dead Staining

Cell viability and cytotoxicity were assessed using a Live/Dead Cell Staining Kit (Yeasen, Shanghai, China). After treatment with PAA, chondrocytes were washed twice with PBS and incubated with Calcein-AM (2 μM) and propidium iodide (4 μM) diluted in serum-free medium for 20 min at 37 °C in the dark. Fluorescent images were captured using a fluorescence microscope (EVOS FL Auto, Thermo Fisher Scientific, Waltham, MA, USA). Live cells appeared green, while dead cells were stained red.

4.5. Safranin O Staining

Safranin O staining was performed to assess proteoglycan content in chondrocytes. After treatment, cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and incubated with 0.1% Safranin O solution (Solarbio, Beijing, China) for 5 min. The excess stain was removed by rinsing briefly in 1% acetic acid. Stained cells were imaged under a light microscope.

4.6. RNA Isolation and qPCR

Total RNA was isolated from cultured cells using Trizol reagent (Takara, Otsu, Shiga, Japan) following the provided protocol. Reverse transcription was then carried out with the HiScript II Q RT SuperMix kit (Vazyme, Nanjing, China) to generate cDNA. Quantitative PCR was subsequently conducted using the ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China). Gene expression levels were normalized to Gapdh, and relative expression was calculated using the 2^−ΔΔCt^ method. Primer sequences are provided in Supplementary Table S1.

4.7. Protein Extraction and Western Blot

Chondrocytes were lysed on ice for 30 min using RIPA buffer (Boster, Wuhan, China) containing protease and phosphatase inhibitors. The resulting lysates were centrifuged at 10,000× g for 30 min at 4 °C to remove debris. Equal amounts of protein were separated on 10–15% SDS-PAGE gels and transferred onto PVDF membranes. Membranes were blocked in 5% non-fat milk prepared in TBST for 1 h and then incubated overnight at 4 °C with the indicated primary antibodies. After treatment with HRP-conjugated secondary antibodies (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h, protein bands were visualized using an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA) and imaged on a ChemiDoc XRS system. Densitometric analysis was performed using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA).

4.8. Destabilization of the Medial Meniscus (DMM) Surgery and Intra-Articular Injection

C57BL/6J male mice (10 weeks old) were randomly assigned into four groups (n = 6 per group): Sham + Vehicle, Sham + PAA, DMM + Vehicle, and DMM + PAA. The DMM surgery was performed on the right knee following established procedures. Briefly, after anesthesia, a medial parapatellar incision was made and the medial meniscotibial ligament was transected to induce joint instability. Sham-operated mice underwent arthrotomy without ligament transection. For intra-articular injection, PAA was initially dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted with sterile saline to a working concentration of 1 mg/mL (final DMSO ≤ 2%, v/v). Beginning one week after surgery, mice in the Sham + PAA and DMM + PAA groups received 10 μL of the PAA solution injected into the right knee joint once weekly for seven consecutive weeks. The Sham + Vehicle and DMM + Vehicle groups were administered an equivalent volume (10 μL) of vehicle containing 2% DMSO. All mice were euthanized at 8 weeks post-surgery, and knee joints were harvested for subsequent analyses.

4.9. Micro-Computed Tomography (Micro-CT) Imaging

Micro-CT analysis of the knee joints from sham and DMM mice was performed using a VivaCT 40 system (Scanco, Wangen-Brüttisellen, Switzerland) with a voxel size of 15 μm at 70 kVp and 112 μA. Three-dimensional reconstructions were generated according to the manufacturer’s procedures. The subchondral bone region of the proximal tibia was selected as the region of interest (ROI) for quantitative analysis of trabecular parameters.

4.10. Safranin O–Fast Green

Fixed knee joints were decalcified in 10% EDTA for 3–4 weeks, dehydrated, and paraffin-embedded. Serial sagittal sections (5 μm) were prepared and stained with Safranin O–Fast Green following standard protocols. Cartilage structure, proteoglycan loss, and surface integrity were evaluated. Degenerative changes in cartilage were evaluated using a semi-quantitative 0–6 grading scale [50]. Each sample was examined independently by two blinded observers, and the mean of their scores was used for subsequent statistical analysis. Osteophyte formation at the medial and lateral tibial plateau and femoral condyle was evaluated using a semi-quantitative scoring system (0–3). For each knee, osteophyte size and maturity were scored separately at four quadrants (medial/lateral femur and tibia), and the sum of the four sites was used for statistical analysis.

4.11. Immunohistochemistry

Paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer. Endogenous peroxidase activity was blocked using 3% H_2_O_2_. Sections were incubated with primary antibodies against COL2A1, ACAN, MMP13 and MMP3 at 4 °C overnight. After incubation with HRP-conjugated secondary antibodies, staining was visualized using DAB substrate and counterstained with hematoxylin. Images were captured under a light microscope, and positive staining areas were quantified using ImageJ.

4.12. RNA Sequencing and Functional Enrichment Analyses

Primary chondrocytes were divided into two groups: an IL-1β-treated group and an IL-1β + PAA-treated group, as described above. RNA purity was examined using a NanoDrop™ One/OneC spectrophotometer (Thermo Scientific), and RNA concentration was quantified with the Qubit™ RNA HS Assay Kit (Thermo Scientific). RNA integrity was assessed on an Agilent 4200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA). Libraries were generated with the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). Raw reads were filtered to remove adaptor sequences and low-quality reads, and the clean reads were aligned to the reference genome using HISAT2. Gene expression levels were quantified as counts using featureCounts, and differential expression between the IL-1β and IL-1β + PAA groups was analyzed with DESeq2. Genes with an adjusted p value < 0.05 and |log2(fold change)| ≥ 1 were defined as DEGs. GO enrichment analysis, KEGG pathway enrichment, and GSEA of DEGs were performed using the clusterProfiler package in R software (version 4.3.1). Pathways or terms with an adjusted p < 0.05 were considered significantly enriched.

4.13. Protein–Protein Interaction (PPI) Network Analysis

PPI network analysis was conducted based on DEGs enriched in the PI3K/AKT signaling pathway following PAA treatment, as identified by RNA-seq. These DEGs were uploaded to the STRING database (version 12.0) with the species specified as Mus musculus, and a protein interaction network was generated by integrating all available evidence types—including experimental data, curated databases, and text mining—at a medium confidence score threshold (≥0.400). The resulting network, in which edges represent functional associations between proteins, was visualized using the default STRING layout.

4.14. Cellular Thermal Shift Assay (CETSA)

The CETSA was utilized to evaluate the thermal stability of PTEN in chondrocytes following PAA treatment. Primary chondrocytes were treated with either PAA or DMSO for specified durations, harvested, and resuspended in phosphate-buffered saline (PBS) supplemented with protease inhibitors. The cell suspensions were divided into equal aliquots and subjected to a graded temperature series (50 °C, 54 °C, 58 °C, 62 °C, 66 °C, and 70 °C) for 3 min using a thermal cycler, followed by incubation at room temperature for an additional 3 min. Subsequently, cells were lysed through three freeze–thaw cycles in liquid nitrogen. After centrifugation at 12,000× g for 30 min at 4 °C, the supernatants containing soluble, thermostable proteins were collected. PTEN protein levels in each temperature-dependent fraction were analyzed by SDS-PAGE and Western blotting. Densitometric quantification of protein bands was performed to assess the thermal stability of PTEN under varying treatment conditions.

4.15. siRNA Transfection

Small interfering RNAs targeting mouse Pten (si-Pten) and a non-targeting negative control siRNA (si-NC) were purchased from RiboBio Co., Ltd. (Guangzhou, China). The sequence of Pten siRNA is 5′-UUUGUCUUCAAGUAGUAGC-3′. Chondrocytes were transfected with siRNAs using Lipofectamine 3000 reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, si-NC or si-Pten was applied at a final concentration of 50 pM, and cells were incubated for 24 h prior to subsequent treatments. The knockdown efficiency of Pten was verified by qPCR analysis.

4.16. Statistical Analysis

All quantitative data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 9.0 (Graphpad Software Inc., San Diego, CA, USA). Comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests, while multiple-group comparisons were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p value < 0.05 was considered statistically significant.

5. Conclusions

Collectively, this study demonstrates that PAA exerts potent chondroprotective and disease-modifying effects in OA. PAA effectively restored extracellular matrix homeostasis, suppressed inflammatory and catabolic responses in IL-1β-stimulated chondrocytes, and mitigated cartilage degeneration and subchondral bone abnormalities in the DMM mouse model. Transcriptomic profiling and mechanistic analyses revealed that PAA stabilizes PTEN protein and inhibits PI3K/AKT phosphorylation, thereby reversing inflammatory signaling activation and preventing cartilage matrix breakdown. Pharmacological inhibition of PTEN abolished these benefits, establishing PTEN as a critical mediator of PAA activity. Our findings identify PAA as a promising natural therapeutic candidate for OA and highlight the PTEN-PI3K-AKT axis as its primary regulatory mechanism.