Dulbecco’s Modified Eagle Medium: F12 (DMEM/F12), 1× phosphate-buffered saline (PBS), 100× penicillin-streptomycin, and trypsin (without EDTA) were obtained from Thermo Fisher Scientific (Waltham, MA, United States). Fetal bovine serum (FBS) was sourced from HyClone (Suzhou, China). Sprague Dawley (SD) rats were procured from Envigo Biotechnology Co., Ltd. (Chengdu, China).

SD rats aged 33 days were euthanized by inhaling isoflurane after being wiped and disinfected with 75% alcohol throughout the body. Subsequently, the joint cartilage was isolated and placed in 1.5 mL sterile EP tubes, then minced using micro-scissors. 1 mL of 0.25% trypsin was added to suspend the cartilage tissue, followed by incubation at 37°C in a cell culture incubator for 30 min with agitation every 5 min. Following digestion termination, the mixture was centrifuged at 300 g for 10 min to remove the trypsin supernatant. Subsequently, 3 mL of 1.5% collagenase type II was added to the pellet, and the resulting cell suspension was transferred into 15 mL centrifuge tubes. The samples were then incubated in a cell culture incubator for 6–8 h, followed by a second centrifugation step at 300 × g for 10 min.Then, the resulting cell suspension was transferred to a T25 flask (passage 1) and cultured at 37°C in a cell culture incubator with 5% CO₂. The cells were cultured in DMEM/F12 medium supplemented with 10% FBS and 1× penicillin-streptomycin. Once 90% confluency was reached, the culture was expanded at a 1:2 ratio or cryopreserve the cells. In this study, only chondrocytes within passage 3 were utilized to ensure their inherent characteristics.

Passage 1 chondrocytes were digested with trypsin, centrifuged, and seeded onto a 6-well plate pre-coated with coverslips. The plate was incubated at 37°C with 5% CO₂ in a cell culture incubator. After incubation, the cells were fixed with 95% ethanol at 4°C for 30 min, washed with 1×PBS twice, and then stained with 1% toluidine blue for 20 min. Post-staining, cells were washed with absolute ethanol, allowed to air dry, mounted with neutral resin, and observed under an inverted fluorescence microscope for subsequent photography.

Similarly, cells seeded on the aforementioned 6-well plate were fixed with 4% paraformaldehyde for 20 min, followed by incubation with 3% hydrogen peroxide at room temperature for 10 min. The cells were then washed three times with 1×PBS for 5 min per wash. After incubating with normal non-immune animal serum at room temperature for 10 min, the serum was removed, and cells were incubated overnight at 4°C with 1:100 rabbit anti-collagen II primary antibody. Then, washing three times with PBS for 5 min per each, a biotinylated secondary antibody was added, and the cells were incubated at room temperature for 10 min. Following additional washes with 1×PBS three times for 5 min per each, streptavidin-biotin peroxidase was added at room temperature for 10 min. Finally, DAB staining was performed, and the cells were counterstained with hematoxylin, dehydrated, and mounted with coverslips for observation.

Cultured chondrocytes were divided into three experimental groups: (A) control group (n = 5), (B) model group (n = 5), and (C) EGCG treatment group (n = 5). To avoid the randomness of the experiment, five duplicate samples were conducted simultaneously. The model group was induced by incubating the cells with 10 ng/mL IL-1β for 24 h. For the EGCG treatment group, cells were first subjected to IL-1β induction as in the model group and subsequently treated with 10 mg/mL EGCG for an additional 24 h. Both IL-1β and EGCG were diluted in culture medium. At the conclusion of the treatment period, cells were harvested and processed for RNA sequencing and further analysis.

Chondrocytes were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and cultured overnight. Subsequently, the medium was replaced, and the cells were subjected to experimental treatments. After 24 h, a 10 μM working solution of DCFH-DA prepared in serum-free medium was added and incubated with cells for 20 min at 37°C. The cells were then washed three times with serum-free medium to thoroughly remove any extracellular DCFH-DA. Finally, the intracellular ROS levels were visualized and imaged using a laser confocal microscope with an excitation wavelength of 488 nm, and the average fluorescence intensity was quantified using ImageJ software.

To assess the effects of EGCG on the expression of IL-1β, MMP13, and TNF-α in chondrocytes, the cell culture supernatants were collected by centrifugation at 300 g for 10 min to remove any cell debris and stored at −80°C for analysis. ELISA was performed on cell culture supernatants using specific ELISA kits (Ruixin biotech, Quanzhou, Fujian) according to the manufacturer’s protocols.

Cells form each group were collected and washed twice with cold 1×PBS. According to the Illumina TruSeq PE Cluster Kit protocol, total RNA was extracted, and mRNA was purified, qualifying RNA was subjected to PCR amplification for the construction of a cDNA library, and then sequenced on the Illumina NovaSeq platform to generate 150 bp paired terminal reads. Low-quality sequences and adaptor reads were removed using the Hisat2 v2.0.5. Then, according to the length of each gene and the reads count of the gene, the number of fragments sequenced per thousand base pairs (FPKM) of each gene was calculated. Gene expression was annoteted using Cufflinks software, with, default parameters applied. Gene function enrichment analysis was performed using Metscape (https://metascape.org) and KEGG function enrichment webtools.

To identify key targets of epigallocatechin 3-gallate (EGCG), the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) was searched using the term “epigallocatechin 3-gallate” to retrieve its SMILES molecular formula. This molecular formula was then imported into Swiss Target Prediction (http://swisstargetprediction.ch/) with the species restricted to “Rattus sapiens” to obtain predicted targets. These predicted targets were subsequently matched with differentially expressed genes identified through transcriptomic sequencing analysis. The intersection of these mapped targets was considered as the target genes for EGCG therapy in osteoarthritis. These target genes were further analyzed using gene set enrichment analysis, including Gene Ontology (GO) analysis and KEGG pathway enrichment analysis. Finally, the potential key targets were visualized and constructed into a network using Cytoscape 3.9.0.

Thirty Sprague-dawley were divided into three experimental groups: (A) control group (Control group) (n = 10), (B) osteoarthritis model group (OA group) (n = 10), and (C) EGCG treatment group (EGCG group) (n = 10). Following a 7-day acclimatization period, OA group and EGCG group were received a single intra-articular injection of Monoiodoacetic acid (MIA) (2 mg/50 μL, dissolved in normal saline) to induce osteoarthritis. After 2 weeks, the EGCG group received intra-articular administration of 100 μL EGCG (10 μg/mL, dissolved in normal saline), while both the normal control (NC group) and OA group received intra-articular injections of normal saline.

One week post-treatment, the animals were anesthetized with an intraperitoneal injection of isoflurane, and blood samples were collected from the veins. Serum was separated and stored at −80°C for subsequent biochemical analysis. The knee joints were harvested and fixed in 4% paraformaldehyde for further histological examination. All experimental protocols were approved by the Ethics Committee of the Hospital of the Chengdu Office of the People’s Government of the Tibetan Autonomous Region (Approval No. KY2022-86).

Histological analysis of the knee joint tissues was performed using Hematoxylin and Eosin (H&E) staining. After fixation in 4% paraformaldehyde, the knee joint tissues were decalcified in a 10% EDTA solution for 4 weeks, with the solution being refreshed weekly. Following decalcification, tissue samples were dehydrated through a series of graded ethanol solutions, cleared in xylene, and embedded in paraffin. Serial sections of 3 μm thickness were cut using a microtome and mounted on glass slides.

The tissue sections were then deparaffinized with xylene and rehydrated through a descending ethanol series. Sections were stained with hematoxylin for 5 min to highlight cell nuclei, followed by rinsing in running tap water. Slides were then dipped in acid alcohol and rinsed again before staining with eosin for 2 min to stain the cytoplasm and extracellular matrix. After thorough washing in water, the sections were dehydrated through an ascending ethanol series, cleared in xylene, and mounted with coverslips using resinous mounting medium. Histological changes in the articular cartilage and subchondral bone were observed and photographed under a panoramic slice scanner (3DHISTECH, Hungary). The severity of osteoarthritic lesions was evaluated based on established histopathological scoring systems.

To assess the extent of fibrosis and collagen deposition in the knee joint tissues, Masson’s Trichrome staining was performed. After fixation and decalcification as described, serial sections of 3 μm thickness were prepared and mounted on glass slides. The sections were deparaffinized in xylene and rehydrated through descending concentrations of ethanol. The slides were then stained using Masson’s Trichrome staining kit. After staining, the sections were rinsed in distilled water, differentiated in 1% acetic acid solution for 2–5 min, dehydrated through an ascending ethanol series, cleared in xylene, and mounted with coverslips using a resinous medium. The stained sections were examined under a panoramic slice scanner (3DHISTECH, Hungary), and images were captured to evaluate the extent of collagen deposition and fibrosis in the joint tissues. The severity of osteoarthritic lesions was evaluated based on established Masson scoring systems.

Immunohistochemical staining was performed to detect the expression of Pla2g2a in knee joint tissues. Following fixation, decalcification, and paraffin embedding, 5 μm tissue sections were prepared and mounted on glass slides. The sections were deparaffinized in xylene, rehydrated through a graded series of ethanol solutions, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0) at 95°C–100°C for 20 min. To quench endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxide, and non-specific binding was blocked using 5% bovine serum albumin in PBS for 30 min. Subsequently, the sections were incubated overnight at 4°C with a rabbit anti-Pla2g2a antibody (1:200, Thermo fisher, United States). The following day, the sections were incubated with a biotinylated secondary antibody (anti-rabbit IgG, Thermo fisher, United States) for 30 min at room temperature. Signal development was carried out using an avidin-biotin complex (ABC) kit followed by DAB substrate. Finally, sections were counterstained with hematoxylin, dehydrated through an ascending ethanol series, cleared in xylene, and mounted with coverslips. Stained sections were examined under a panoramic slice scanner (3DHISTECH, Hungary), and images were captured for analysis. The expression of Pla2g2a was quantified using ImageJ software by calculating the percentage of positive-stained area relative to the total tissue area.

Primary chondrocytes were isolated from knee joints of healthy SD rats to preserve their inherent characteristics (Figure 1A). Cells were identified by collagen II immunofluorescence and toluidine blue staining. Immunofluorescence confirmed collagen II expression in the cytoplasm and membrane (Figure 1B). Toluidine blue staining revealed blue cytoplasm, blue-violet/dark blue nuclei with distinct nucleoli, and perinuclear metachromatic granules (Figure 1B). Cells displayed short spindle/long fusiform morphology with pseudopodia, typical of fibroblast-like chondrocytes. These results confirmed successful chondrocyte isolation, supporting subsequent experiments.

After successful isolation and identification of primary chondrocytes, we first determined the optimal EGCG concentration by assessing its dose-dependent effects on chondrocyte viability. The data revealed that 10 μg/mL EGCG enhanced cellular viability, whereas higher concentrations (>50 μg/mL) demonstrated cytotoxicity Supplementary Figure S2; therefore, 10 μg/mL was selected for therapeutic intervention. Subsequently, to investigate EGCG’s effects on chondrocytes under osteoarthritis (OA)-mimicking conditions, cells were stimulated with IL-1β (10 ng/mL) for 24 h to establish the disease model group (Model), followed by treatment with 10 μg/mL EGCG for an additional 24 h (EGCG group). Normally cultured cells without IL-1β stimulation served as the control group (Control). As illustrated in Figure 1C, IL-1β stimulation induced significant morphological abnormalities in chondrocytes, which were partially restored upon EGCG treatment. Consistently, the cell viability of chondrocytes decreased to below 75% following IL-1β stimulation but significantly improved after EGCG treatment (Figure 1D), suggesting that EGCG exhibits reparative potential in mitigating OA-induced chondrocyte dysfunction.

Given the critical role of reactive oxygen species (ROS) in OA progression, and EGCG’s ability to scavenge intracellular ROS, we further assessed ROS levels in chondrocytes. After IL-1β stimulation, the ROS levels in chondrocytes significantly increased to approximately 1.8 times those of the control group, whereas EGCG treatment notably reduced ROS levels to near-control levels (Figures 1E,F). This finding suggests that EGCG can significantly reduce the high ROS levels induced by OA in chondrocytes.

EGCG also can inhibit the progression of inflammation by suppressing key inflammatory signaling pathways. Thus, we collected the cell culture supernatants from each group and measured the expression levels of inflammatory factors IL-1β, MMP-13, and TNF-α. As shown in Figure 1G, IL-1β stimulation significantly upregulated the expression of these inflammatory factors, while EGCG treatment notably reduced their levels. Collectively, these results demonstrate that EGCG plays a crucial role in mitigating OA-induced chondrocyte damage by reducing ROS production and suppressing inflammatory factors Expression.

Following the morphological and functional assessments of the impact of EGCG on chondrocytes under inflammatory conditions, we further explored the molecular mechanisms underlying these effects using transcriptome sequencing of mRNA from three distinct groups: Control, Model (IL-1β treated), and EGCG (EGCG-treated, 10 μg/mL). The Differentially expressed genes (DEGs) were defined using a dual-threshold criterion:|log₂(fold change)| ≥ 1 (equivalent to 2-fold change),Adjusted p-value <0.05 (Benjamini–Hochberg FDR correction).The transcriptome analysis revealed 1526 differentially expressed genes (DEGs) between the Model and Control groups, with 919 DEGs upregulated and 607 DEGs downregulated. Comparatively, the EGCG and Control groups exhibited 1171 DEGs, including 421 upregulated and 750 downregulated genes (Figure 2A). When comparing the EGCG and Model groups, a total of 1638 DEGs were identified, of which 697 were upregulated and 941 were downregulated (Figure 2A). A Venn diagram was employed to visualize the overlap of DEGs among the three groups, highlighting 119 shared DEGs (Figure 2B).

To expedite the identification of potential EGCG targets for mitigating the effects of IL-1β on chondrocytes, we utilized network pharmacology to identify 95 EGCG-associated targets relevant to OA (Supplementary Table S1). These targets were then correlated with the 119 differentially expressed genes identified in Figure 3B, leading to the selection of 18 potential EGCG targets for alleviating IL-1β-induced chondrocytes (Figure 2C). Detailed gene information is provided in Supplementary Table S2. Functional annotation analysis of these 18 genes revealed that Gene Ontology (GO) annotations were primarily linked to biological regulation, cellular processes, cell components, and binding (Figure 2D). Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations highlighted signaling molecules and interactions, as well as signal transduction pathways (Figure 2E). Further functional enrichment analysis indicated that GO enrichment was mainly related to multicellular organismal processes (Figure 2F), while KEGG enrichment pathways focused on neuroactive ligand-receptor interactions (Figure 2G). Subsequently, KEGG pathway information was further used to construct a “component-pathway-target” network with Cytoscape 3.7.2, illustrating the complex interactions of EGCG in counteracting IL-1β effects. The network comprises 32 nodes and 138 connections (Figure 2H). By Integrating protein-protein interaction (PPI) network analysis with “target-pathway” network of KEGG, CytoNCA was utilized to calculate the “Degree” score, identifying Mmp2, Hsd11b1, and Pla2g2a as core targets with a Degree greater than 4 (Figure 2I).

To determine the key targets of EGCG in OA therapy, we used the SERING database to identify 9 central genes with a connectivity index greater than 3. These genes, including Pla2g2a, Alox5, Aldh2, Aoc1, Maob, Maoa, Htr2a, and DRD2, were markedly modulated by EGCG in the context of OA (refer to Supplementary Figure S1). A subsequent comparative analysis parallels these 9 pivotal osteoarthritis-associated genes with 3 previously identified chondrocyte-specific targets—namely, Mmp2, Hsd11b1, and Pla2g2a—revealed substantial overlap with Pla2g2a. Such convergence highlights the pivotal role of Pla2g2a, affirming it as a critical molecular target in the alleviation of chondrocyte inflammation via EGCG in OA therapy.

Additionally, we assessed the binding affinity of EGCG for its target gene, Pla2g2a, through molecular docking analysis. We utilized AutoDock Vina v.1.2.2 to calculate the binding posture and interaction dynamics between EGCG and Pla2g2a, including the calculation of binding energy. The findings revealed that EGCG interacts with Pla2g2a predominantly via stable hydrogen bonds and substantial electrostatic interactions. As shown in Figure 2J, the conformational energy of the EGCG-target protein complex was relatively low (−6.97 kcal/mol), signifying a strong binding affinity between EGCG and Pla2g2a.

Based on these findings, our preliminary investigation into the regulatory effect of EGCG on Pla2g2a expression in chondrocytes within an OA model revealed that EGCG upregulates Pla2g2a levels (Supplementary Figure S3), supporting its therapeutic potential for OA intervention. Subsequently, we further assessed the impact of EGCG on cartilage repair using an MIA-induced osteoarthritis model in the knee joints of SD rats. Initially, OA was induced in the OA group via intra-articular injection of MIA, followed by the treatment of EGCG. A control group received an equivalent volume of saline without MIA induction. Cartilage repair was evaluated on day 7 using multiple methodologies (Figure 3A). Micro-CT analysis highlighted significant bone defects post-MIA induction, which were markedly repaired after EGCG treatment (Figure 3B). Further analyses of percent bone volume and trabecular number demonstrated substantial improvements with EGCG treatment, consistent with the Micro-CT findings (Figure 3C).

Subsequent assessments focused on cartilage tissue cells and matrix damage. Hematoxylin and eosin (H&E) staining revealed disorganization and uneven cell distribution, lighter matrix staining, and reduced cell density in the OA group, with notable improvements following EGCG treatment, indicating effective cartilage repair (Figure 3D). H&E staining scores further confirmed the significant role of EGCG in promoting cartilage tissue repair (Figure 3E). Masson staining presented extensive collagen fiber structure damage in the OA group, which was substantially repaired by EGCG, restoring orderly collagen fiber alignment and clear tissue structure without abnormal deposits (Figure 3F). This was supported by Masson staining scores, underscoring the efficacy of EGCG in collagen fiber repair (Figure 3G). Additionally, analysis of Pla2g2a expression revealed a significant increase after EGCG treatment compared to its significant reduction in OA group (Figures 3H,I), indicating the therapeutic potential of EGCG in OA treatment through Pla2g2a mediation in cartilage cells.