Arctiin alleviates the progression of osteoarthritis by regulating the cholesterol metabolic pathway

Abstract

Osteoarthritis (OA) is a multi-factorial degenerative joint disease with unclear pathogenesis. Conservative treatments, primarily aimed at pain relief, fail to halt disease progression. Metabolic syndrome has recently been implicated in OA pathogenesis, underscoring the need for novel therapeutic strategies. Arctiin (ARC), a lignan known for its anti-inflammatory and anti-osteoporotic properties, has potential effects on OA that merit exploration. We assessed ARC’s impact on chondrocyte viability using the Cell Counting Kit-8 and toluidine blue staining for glycosaminoglycan presence. Gene and protein expression were analyzed via RT-PCR, Western blotting, and immunofluorescence. An OA rat model was employed for in vivo evaluations through histological assessments and micro-CT scanning. ARC reversed IL-1β-induced upregulation of MMP3, MMP13, and COX-2 and the downregulation of collagen II and SOX9. It modulated cholesterol metabolism in IL-1β-stimulated chondrocytes by inhibiting the CH25H-CYP7B1-RORα axis, reducing cartilage damage and proteoglycan loss in OA rats, and effectively inhibiting subchondral bone osteolysis. ARC inhibits IL-1β-induced inflammatory responses and ECM degradation, suggesting its potential as a therapeutic agent for OA. It acts partly by modulating cholesterol metabolism and suppressing the CH25H/CYP7B1/RORα axis in chondrocytes.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-83993-1.

Introduction

Osteoarthritis (OA) ranks among the most prevalent musculoskeletal disorders, primarily manifesting as joint pain, minimal morning stiffness, and functional impairment¹. This condition commonly affects diverse joints, including the fingers, hips, knees, and ankles. The incidence of OA is on the rise, driven largely by factors such as population aging, increasing prevalence of obesity, and joint injuries².

The articular cartilage, composed of chondrocytes and an extracellular matrix, plays a crucial role in joint functionality. Initial stages of OA are marked by a reduction in proteoglycan content and degradation of the type II collagen network, eventually involving the entire joint surface in more advanced stages³. OA is recognized as a complex, multi-factorial degenerative disease with a pathogenesis that remains to be fully elucidated. Contributing factors include aging, inflammation, and mechanical stress. Recent studies have implicated metabolic syndrome as a significant contributor to OA pathogenesis^4,5. For instance, apolipoprotein A1 (APOA1), a lipid transport protein, is notably elevated in the serum of OA patients compared to healthy individuals⁶, and higher levels of LDL cholesterol are known to exacerbate synovial inflammation and ectopic bone formation, independent of body weight⁷.

Current conservative pharmacological treatments, such as non-steroidal anti-inflammatory drugs (NSAIDs) and steroids, predominantly provide palliative care focused mainly on pain alleviation rather than slowing the disease’s progression². Consequently, many patients with advanced OA ultimately require joint replacement surgery.

Arctiin (ARC), a lignan extracted from the seeds of Arctium lappa, exhibits a range of biological activities, including anti-inflammatory and anti-osteoporotic effects. It has been demonstrated that ARC can significantly reduce LPS-induced iNOS and COX-2 expression in Raw264.7 cells⁸. Furthermore, ARC has been shown to decrease cholesterol and triglyceride levels in HepG2 cells at concentrations ranging from 0.01 to 0.1 µM⁹. Despite these findings, the specific effects of ARC on OA chondrocytes and cartilage are poorly understood, thus meriting further investigation into its potential to mitigate OA progression both in vitro and in vivo.

Results

Impact of arctiin on chondrocyte viability

Figure 1A illustrates the molecular structure of Arctiin (ARC). We employed the Cell Counting Kit-8 (CCK-8) to evaluate its cytotoxic impact on chondrocytes. This analysis was conducted at 24, 48, and 72-hour intervals. Results indicated that at 20 µM, ARC notably decreased cell viability at the 24 and 72-hour marks. In contrast, ARC showed no cytotoxic effects at concentrations from 0.01 to 1 µM, consistent with prior research (Fig. 1B). Based on these findings, subsequent experiments utilized 0.01 and 0.1 µM non-toxic concentrations.

Fig. 1.

Arctiin’s Effects on Chondrocyte Viability. (A) Chemical structure of ARC. (B) Viability of chondrocytes exposed to varying concentrations of ARC (0.01, 0.1, 1, 10, 20 µM) was measured at 24, 48, and 72 h using the CCK-8 assay. Data are represented as means ± SD. Statistically significant deviations from the control (0 µM) are indicated: *p < 0.05, **p < 0.01; n = 5. (C) Morphological evaluation of chondrocytes stained with Toluidine blue, showcasing cellular structure.

Mitigation of inflammatory responses and ECM degradation by Arctiin

The efficacy of Arctiin (ARC) in modulating IL-1β-induced inflammatory responses and extracellular matrix (ECM) degradation was evaluated through various assays, including toluidine blue staining, real-time PCR, Western blotting, and immunofluorescence. Toluidine blue staining illustrated a significant diminution in staining intensity upon IL-1β exposure, indicative of glycosaminoglycan depletion, notably lessened by treatment with ARC at 0.1 µM (Fig. 1C). Immunohistochemical findings revealed that ARC curbed the expression of MMP13 and augmented that of Collagen II relative to the control.

The Immunofluorescence assay was performed to assess the Collagen II and MMP13 expression in each group (Fig. 2A, B). and our results showed that IL-1β treatment significantly upregulated MMP13 expression while downregulating Collagen II expression, which were partially reversed by ARC treatment (Fig. 2C, D). Quantitative real-time PCR revealed that ARC markedly diminished COX-2, MMP9, and MMP13 mRNA expressions while also reversing the decline in Collagen II and SOX9 expressions caused by IL-1β (Fig. 2E, F). These molecular changes were substantiated by Western blot analysis, which aligned with the gene expression data, showing similar trends in protein levels (Fig. 2G, H, I).

Fig. 2.

Arctiin’s role in counteracting IL-1β-induced inflammatory and ECM deterioration in chondrocytes. (A, B) Immunofluorescence imaging of Collagen II and MMP13 post-treatment; scale bar: 100 μm. (C, D) Fluorescence intensity quantification for Collagen II and MMP13 using ImageJ. (E, F) mRNA levels of COX-2, MMP3, MMP13, Collagen II and SOX9quantified via RT-PCR. (G, H, I) Protein expression and quantitative analysis of COX-2, MMP3, MMP13, Collagen II and SOX9, performed by Western blotting. Data are presented as means ± SD. Significant differences indicated by ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to control, and ^*P < 0.05, **P < 0.01, ***P < 0.001 compared to the IL-1β-treated group; n = 3.

ARC’s role in cholesterol metabolism via the CH25H/CYP7B1/RORα Axis in chondrocytes

To elucidate the underlying mechanisms by which ARC mitigates osteoarthritis progression, we explored its impact on cholesterol metabolism regulation in chondrocytes. RT-qPCR analysis demonstrated that IL-1β treatment significantly upregulated LOX-1 expression while downregulating LXR-α and ABCA1 expressions. In contrast, ARC treatment partially reversed these effects (Fig. 3A). Consistent with previous findings, the CH25H/CYP7B1/RORα axis—a known regulatory pathway in chondrocyte cholesterol metabolism—was also assessed. RT-PCR and Western blot analyses showed increased expressions of CH25H, CYP7B1, and RORα following IL-1β stimulation, effectively suppressed by ARC treatment (Fig. 3B, C, D).

Fig. 3.

ARC regulation of cholesterol metabolism via the CH25H/CYP7B1/RORα axis in chondrocytes. (A, B) mRNA expressions of LOX-1, LXR, ABCA1, CH25H, CYP7B1, RORα in chondrocytes determined by RT-qPCR. (C, D) Protein expression levels of CH25H, CYP7B1, and RORα analyzed by Western blotting; protein band densities were quantified using ImageJ. The values represent the means ± S.D. ^#P < 0.05 vs. the control, ^##P < 0.01 vs. the control, ^###P < 0.001 vs. the control, ^*P < 0.05 vs. the IL-1β treated group, ^**P < 0.01 vs. the IL-1β treated group, ^***P < 0.001 vs. the IL-1β treated group, n = 3.

ARC’s ameliorative effects on OA Development in a rat model

The in vivo efficacy of ARC was evaluated using a rat model of osteoarthritis induced by ACLT + MMx surgery. Post-operative administration of ARC for eight weeks resulted in noticeable preservation of hyaline cartilage thickness, significantly reduced in untreated OA rats, as depicted in Fig. 4A. Comparative histological assessments using Safranin O and Fast Green staining showed substantial ECM component loss in the OA group, which was mitigated by ARC treatment (Fig. 4B). Immunohistochemistry findings indicated decreased Collagen II deposition and increased MMP13-positive cells in OA rats, both positively modulated by ARC treatment (Fig. 4C, D). Quantitative analysis using the OARSI scoring system further validated the protective effects of ARC against cartilage degeneration (Fig. 4E).

Fig. 4.

ARC preservation of articular cartilage and amelioration of OA in rats. (A, B) H&E and Safranin O staining of cartilage. (C, D) Immunohistochemical staining for Collagen II and MMP13 expressions in cartilage samples. (E) OARSI scores for cartilage damage at 8 weeks post-operation. The values represent the means ± S.D. ^#P < 0.05 vs. the sham group, ^##P < 0.01 vs. the sham group, ^###P < 0.001 vs. the sham group, ^####P < 0.0001 vs. the sham group, ^*P < 0.05 vs. the OA group, ^**P < 0.01 vs. the OA group, ^***P < 0.001 vs. the OA group, ^****P < 0.001 vs. the OA group, n = 5.

ARC’s effects on bone resorption in OA rats

The impact of ARC on subchondral bone remodeling was assessed using micro-CT scanning of rat knees post-ACLT + MMx surgery. The analysis revealed pronounced bone resorption and osteolysis in the OA group, which were significantly inhibited by ARC treatment, demonstrating its effectiveness in preventing subchondral bone degradation (Fig. 5A). The microstructural parameters of the tibial plateau subchondral bone, including bone mineral density (BMD) and trabecular metrics, were significantly improved in the ARC-treated group compared to the OA group (Fig. 5B, C).

Fig. 5.

Impact of ARC on subchondral bone structure in OA rats via Micro-CT analysis. (A) 3D Micro-CT images of knee joints. (B) Coronal views of tibial subchondral bone. (C) Quantitative analysis of bone microstructure parameters. The values represent the means ± S.D. ^#P < 0.05 vs. the sham group, ^##P < 0.01 vs. the sham group, ^###P < 0.001 vs. the sham group, ^*P < 0.05 vs. the OA group, ^**P < 0.01 vs. the OA group, ^***P < 0.001 vs. the OA group, n = 5.

Discussion

(OA) is a prevalent joint disease characterized by the involvement of articular cartilage, subchondral bone, and synovium. The progression of OA is influenced by multiple factors, such as aging, inflammation, and mechanical stress, yet its pathogenesis remains incompletely understood. Recent literature suggests that systemic metabolic disorders play a significant role in OA development. Current clinical treatments primarily offer symptomatic relief without achieving satisfactory long-term outcomes. Notably, the frequent use of (NSAIDs) in OA management is associated with a heightened risk of adverse effects. Consequently, there is a compelling need to explore alternative therapeutics derived from natural sources that can mitigate OA progression with minimal side effects.

(ARC), a lignan extracted from the seeds of Arctium lappa has been shown to possess a range of pharmacological properties, including anti-inflammatory and antioxidant effects. However, the specific mechanisms by which ARC affects OA treatment have not been fully elucidated. Our study contributes novel insights into the protective role of ARC against OA, as evidenced both in vitro and in vivo.

The integrity of the articular cartilage’s extracellular matrix (ECM), primarily composed of type II collagen and proteoglycans, is crucial for joint function. IL-1β has been identified as a potent inducer of pro-inflammatory and pro-catabolic responses in chondrocytes, promoting the release of COX-2—a key mediator in the inflammatory pathway of OA¹⁰. Moreover, IL-1β stimulation increases the expression of matrix metalloproteinases (MMPs), such as MMP3 and MMP13, which are critical in the catabolic process that degrades type II collagen fibrils¹¹. This enzymatic activity is a pivotal step in ECM deterioration. Additionally, SOX9, a key transcriptional factor, is essential in maintaining ECM homeostasis and is vital for chondrogenesis, regulating the expression of genes such as Col2a1 and Acan.

In our research, we established that IL-1β markedly increased MMP3, MMP13, and COX-2 levels while simultaneously reducing the expression of collagen II and SOX9. These alterations were effectively countered by the administration of Arctiin (ARC), which restored the balance in gene expression. These results emphasize ARC’s efficacy in moderating IL-1β-driven inflammatory responses and degradation of the extracellular matrix, reinforcing its viability as a potential treatment modality for osteoarthritis.

Our study consolidates the pathophysiological understanding of osteoarthritis (OA) and positions Arctiin (ARC) as a promising natural agent for advancing OA treatment strategies. This compound shows potential to enhance clinical outcomes and reduce the side effects typically associated with traditional medications.

Cholesterol regulation within chondrocytes is crucial for OA progression, governed by the dynamics of cholesterol absorption and elimination^12,13. Dysregulated cholesterol metabolism, especially through the actions of oxidized low-density lipoprotein (ox-LDL), not only contributes to atherosclerosis but also plays a significant role in OA development¹⁴. The lectin-like ox-LDL receptor 1 (LOX-1) on chondrocytes mediates cholesterol intake and aggravates inflammatory processes by increasing MCP-1 production, reducing chondrocyte viability and accelerating OA progression^15,16. In contrast, Liver X receptors (LXRs), as nuclear overseers, enhance cholesterol outflow by triggering the ATP-binding cassette transporter A1 (ABCA1)⁶, a key channel for cholesterol efflux, thus helping to prevent excessive cholesterol accumulation within cells^17,18.

Our findings indicate that (ARC) modifies the expression of key cholesterol metabolism genes such as LOX-1, LXR-α, and ABCA1 in IL-1β-stimulated chondrocytes, suggesting a protective mechanism against cholesterol-induced chondrocyte dysfunction. This study further elucidates the involvement of the CH25H/CYP7B1/RORα axis in chondrocyte degeneration during OA progression. Specifically, ARC has been shown to inhibit the overexpression of CH25H, CYP7B1, and RORα induced by IL-1β, aligning with previous findings that underscore the axis’s regulatory role in cholesterol metabolism^19,20.

In vivo experiments using an OA rat model reinforced the therapeutic potential of ARC. The treatment notably mitigated severe cartilage damage and proteoglycan loss, conditions typically exacerbated by ACLT + MMx surgery. The suppressive effect of ARC on MMP13 expression and its enhancement of Collagen II synthesis within cartilage highlight its capacity to modify key pathogenic processes in OA. Furthermore, the micro-CT analysis revealed that ARC treatment substantially reduced bone resorption, a critical factor in OA progression, confirming the dual therapeutic action of ARC on both cartilage and subchondral bone structures^21–25.

Despite these promising outcomes, our study has limitations. The molecular mechanisms by which ARC influences cholesterol metabolism in chondrocytes were not fully delineated, and further research is needed to explore these pathways in detail. Future studies should aim to clarify the specific interactions between ARC and the molecular mediators of cholesterol metabolism to fully harness ARC’s potential as a comprehensive therapeutic agent for OA.

Conclusions

Our findings demonstrate that Arctiin (ARC) effectively mitigates IL-1β-induced inflammatory responses and extracellular matrix (ECM) degradation by modulating the CH25H/CYP7B1/RORα axis within chondrocytes. The detailed mechanism underlying this modulation is illustrated in (Fig. 6), providing a visual summary of the interactions and pathways involved.

Fig. 6.

Schematic illustration of the molecular mechanism underlying arctiin’s anti-osteoarthritic effects. This figure provides a visual representation of how (ARC) modulates key biochemical pathways to mitigate the effects of osteoarthritis. Specifically, ARC suppresses the activation of the CH25H/CYP7B1/RORα axis within chondrocytes, thereby inhibiting IL-1β-induced inflammatory responses and extracellular matrix degradation. This diagram underscores the potential of ARC as a natural therapeutic agent, targeting fundamental pathogenic processes in osteoarthritis at the molecular level.

Materials and methods

Reagents

Arctiin, with a purity of at least 98%, was obtained from Weikeqi Biological Technology Co., Ltd., in Sichuan, China. Recombinant Human IL-1β was procured from PeproTech, based in Rocky Hill, NJ, USA. The cell culture media and supplements, including Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F-12), Penicillin-Streptomycin Solution, and 0.25% Trypsin-EDTA, were acquired from Gibco, Life Technologies Corporation, Carlsbad, CA, USA. The study utilized primary antibodies targeting MMP3, MMP13, COX-2, Collagen II, and SOX9, which Affinity Biosciences, OH, USA provided. Additionally, antibodies against CYP7B1 and RORα were purchased from PeproTech, and those against CH25H were supplied by Abcepta Co. in Suzhou, China. The antibody for GAPDH was purchased from Abcam, located in Cambridge, UK.

Preparation of Arctiin

Arctiin (molecular formula: C27H34O11, molecular weight: 534.6). According to the instructions, the dried seeds of Arctium lappa were extracted by alcohol and the extract underwent a series of processes including filtration, evaporation, chromatography, elution, purification, and recrystallization to obtain Arctiin. The analyses were performed with a Thermo BDS Hypersil C18 column (250 mm × 4.6 mm, ,5 μm) at a column temperature of 35 ℃. The mobile phase composed of acetonitrile-water (30:70, v/v) was isocratically eluted at a flow rate of 1.0 ml/min and the effluent was monitored at 280 nm.

Culture of human chondrocytes

Human chondrocytes were obtained from Pricella Biotechnology Company (Wuhan, China) and cultured in a DMEM/F-12 medium enriched with 10% fetal bovine serum and 1% Penicillin-Streptomycin, maintained in a humidified incubator with 5% CO₂ at 37℃. The medium was replaced every two to three days. Cells were subcultured upon reaching 80–90% confluence, using 0.25% Trypsin-EDTA for detachment. To preserve cellular phenotype, only cells from the second or third passages were utilized in subsequent experiments.

Cell viability assay

The impact of the Arctiin (ARC) on the viability of chondrocytes was evaluated using the Cell Counting Kit-8 (CCK-8, Fude, China). Initially, 5,000 chondrocytes per well were seeded into 96-well plates and allowed to attach for 24 h. The cells were subsequently exposed to ARC at concentrations ranging from 0.01 to 20 µM for 24, 48, and 72 h. At the end of each treatment period, 100 µL of 10% CCK-8 solution, diluted in serum-free DMEM/F12, was administered to the wells. The plates were then incubated for three hours at 37℃. Optical density was measured at 450 nm using a spectrophotometer (Sunnyvale, CA) to assess cell viability.

Glycosaminoglycan detection by toluidine blue staining

To evaluate extracellular matrix secretion, toluidine blue staining was employed to identify glycosaminoglycans within chondrocytes. Chondrocytes, approximately 50,000 per well, were cultured in 24-well plates and subsequently fixed using 4% paraformaldehyde. The cells were then stained with toluidine blue for one hour. After staining, the chondrocytes were examined under an Olympus IX73 microscope to visually assess glycosaminoglycans’ distribution and density.

Real-time polymerase chain reaction (RT-PCR) analysis

Gene expression in chondrocytes was quantified using real-time polymerase chain reaction (RT-PCR). Total RNA was isolated from the chondrocyte samples utilizing TRIzol reagent (Invitrogen) as per the provided instructions. This RNA served as a template to synthesize complementary DNA (cDNA) using 1000 ng of RNA with the Evo M-MLV RT Kit. Quantitative assessments were conducted using Power SYBR^® Green PCR Master Mix (Applied Biosystems, UK). Gene expression levels were determined by the 2^−ΔΔCt method, with GAPDH serving as the normalization reference. Details on the primer sequences used are provided in the associated Table 1.

Table 1.

Sequences of primers for quantitative real-time PCR analyses.

Gene	Forward primer	Reverse primer
COL2	5’-GGACGATCAGGCGAAACC-3’	5’-GCTGCGGATGCTCTCAATCT-3’
SOX9	5’-AGGAAGCTCGCGGACCAGTAC-3’	5’-GGTGGTCCTTCTTGTGCTGCAC-3’
MMP3	5’-CACTCACAGACCTGACTCGGTT-3’	5’-AAGCAGGATCACAGTTGGCTGG-3’
MMP13	5’-ACTGAGAGGCTCCGAGAAATG-3’	5’-GAACCCCGCATCTTGGCTT-3’
LOX-1	5’-GAAACCCTTGCTCGGAAGCTGA-3’	5’-CAGATCCAGTCTTGCGGACAAG-3’
LXRα	5’-ACTGATGTTCCCACGGATGC-3’	5’-CACAGTGTTAGCGAGGGCT-3’
ABCA1	5’-GCGACCATGAGAGTGACACG-3’	5’-GCATCCACCCCACTCTCTTC-3’
CH25H	5’-ATCACCACATACGTGGGCTTT-3’	5’-GTCAGGGTGGATCTTGTAGCG-3’
CYP7B1	5’-TCTCTTTGCCGCCACCTTAC-3’	5’-AGGCTTTCGCTGATAATCGG-3’
RORα	5’-ACTCCTGTCCTCGTCAGAAGA-3’	5’-CATCCCTACGGCAAGGCATTT-3’
GAPDH	5’-GTCTCCTCTGACTTCAACAGCG-3’	5’-ACCACCCTGTTGCTGTAGCCAA-3’

Western blot analysis

Following treatment, chondrocyte lysates were prepared using ice-cold RIPA buffer enhanced with 1% phenylmethanesulfonyl fluoride (PMSF) from Beyotime, China, for 20 min. The lysates were then centrifuged at 13,000 g for 20 min at 4 °C. Protein content was quantified using the BCA Protein Assay Kit from Beyotime. 8% SDS-PAGE separated proteins (20 µg) and subsequently transferred onto PVDF membranes (Millipore). The membranes were blocked with QuickBlock™ Blocking Buffer from Beyotime for 15 min at room temperature and incubated overnight at 4 °C with primary antibodies against MMP3, MMP13, COX-2, Collagen II, SOX9, CH25H, CYP7B1, RORα, and GAPDH at specified dilutions. Following primary antibody incubation, membranes were washed with TBST and exposed to a secondary antibody (1:2000) for 1.5 h at room temperature. Protein bands were visualized using ECL detection reagents and a Bio-RAD scanner, with band densities quantified using Image Lab 3.0 software.

Immunofluorescence analysis

Chondrocytes were plated at a density of 50,000 cells per well on 75-mm glass coverslips within 24-well plates. Following 24 h to allow for cell adhesion, the cultures were treated with either IL-1β (10 ng/mL) alone or with Arctiin (ARC) at 0.1 µmol/L and IL-1β for 48 h. After treatment, cells were fixed using 4% paraformaldehyde for 20 min and thoroughly rinsed three times with PBS. Permeabilization was achieved by treating the cells with a solution containing 10% fetal bovine serum and 0.1% Triton X-100 in PBS for one hour. Primary antibodies directed against collagen II and MMP13, both diluted 1:200, were applied to the cells and left to incubate overnight at 4℃. This was followed by incubation with Cy3-tagged secondary antibodies (1:200) and DAPI for nuclear staining, each for one hour and five minutes, respectively, under ambient conditions. Fluorescence microscopy was performed using an Olympus IX73 microscope to visualize and capture the stained samples.

Rat models of osteoarthritis

We obtained fifteen eight-week-old male Sprague-Dawley rats from the Animal Experiment Center of Guangzhou University of Chinese Medicine, housing them in a specific pathogen-free environment. The Animal Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine approved the study protocols, including all animal handling procedures. To induce osteoarthritis, we performed the Anterior Cruciate Ligament Transection plus Medial Meniscectomy (ACLT + MMx) surgery on the right knees of the rats using a well-established method. This involved anesthetizing the animals with 2% pentobarbital, depilation, disinfection with povidone-iodine, and surgical joint exposure. The medial meniscus was excised, and the ACL was sectioned, after which the joint capsule was sutured.

In contrast, the control (sham) group only underwent arthroscopy without any ligament or meniscus removal. Post-operative management included dividing the rats into three groups: sham, (OA), and OA treated with (ARC), dosed at 10 mg/kg. The OA and sham groups were administered 200 µL of PBS intraperitoneally every other day, while the ARC group received the same volume of PBS containing ARC. Eight weeks after surgery, the knees were collected for detailed structural and histological evaluation.

Micro-CT scanning evaluations

Rats were anesthetized by intraperitoneal injection of pentobarbital (100 mg/kg) and cervical dislocation after treatment. Right knee joints were harvested, fixed in 4% paraformaldehyde for 24 h, and subsequently preserved in 75% ethanol. These samples were then imaged using a Skyscan1172 micro-CT scanner (Bruck, Belgium), configured to a 9 μm resolution with an 80 kV voltage, 100µA current, and 0.4° rotation step. Three-dimensional images were generated using CT vox software. The entire subchondral bone of the tibial plateaus was designated as the region of interest (ROI). Structural parameters including bone volume (BV), bone volume to total tissue volume ratio (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), and trabecular spacing (Tb. Sp) were quantitatively analyzed using CTAn software (Skyscan).

Histological assessment

Joint tissues were decalcified using 0.5 M EDTA for five weeks at room temperature, followed by dehydration through a graded alcohol series and embedding in paraffin. Sagittal sections, 5 μm in thickness, of the medial joint compartment were prepared and stained with hematoxylin & eosin (H&E) and safranin O/fast green. These sections were imaged with a Pannoramic MIDI digital slide scanner (3DHISTECH Ltd, Hungary). Immunohistochemical analysis was conducted on additional sections to detect Collagen II and MMP13, monitoring their expression changes within the joint tissue.

Statistical analysis

Data were analyzed using GraphPad Prism 8.0, expressed as mean ± standard error of the mean (SEM). Statistical differences were determined through analysis of variance (ANOVA) with subsequent Tukey’s multiple comparison test for inter-group comparisons and the Student’s t-test for comparisons between two groups. A significance threshold was set at p < 0.05. All experiments were performed in triplicate to ensure robustness and reproducibility.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

J.L. participated in the design of the study. J.M. performed the experiments, analyzed the data and composed the manuscript. J.X., Y.M., D.G. participated in animal experiments. All authors reviewed and approved the final manuscript.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All animal handling procedures were approved by the Animal Welfare Ethics Committee of First Affiliated Hospital of Guangzhou University of Chinese Medicine (K2020-131). We state that all experiments in this study were conducted in accordance with National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Meanwhile, we confirm that our work was carried out in compliance with the ARRIVE guidelines.