Preparation of compoundsBer was solubilized in dimethyl sulfoxide to achieve a 10 mM concentration and subsequently stored at − 80 °C for preservation. This compound was sourced from MedChemExpress (New Jersey, USA).Network pharmacological analysisWe obtained the molecular structure of Ber from PubChem. The potential binding protein targets of Ber were then identified using PharmMapper and BATMAN-TCM online tools. Moreover, we used the DisGENET database (http://www.disgenet.org), GeneCards database (http://www.genecards.org), DrugBank (https://go.drugbank.com), TTD database (http://db.idrblab.net/ttd/) and PharmGKB (https://www.pharmgkb.org/) to retrieve target genes related to OA. Subsequently, we employed the R language to obtain the main osteoarthritis-related targets potentially targeted by Ber and visualized the protein interactions (PPIs) using Cytoscape software. Key functions and pathways were identified by KEGG and GO analyses, and the main mechanisms by which Ber acts on OA were analysed by Metoscape.Molecular dockingWe removed the ligands, solvent molecules, etc., of the predicted target proteins via Chimera software. At the same time, we extracted the drug structure from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and pre-processed the structure of Ber following the same steps as those described above. Subsequently, we calculated the center and size of Gasteiger and docking stations using AutoDock Tools. Finally, we used AutoDock Vina to perform molecular docking and calculate the binding affinity. The results were displayed by Chimera software.Cell viability assayChondrocyte viability was meticulously evaluated utilizing the Cell Counting Kit-8 (CCK-8) assay in strict accordance with the manufacturer’s comprehensive guidelines. Initially, chondrocytes were seeded onto 96-well plates at a density of 70,000 cells per cm2 and allowed to adhere for 24 h. Subsequently, the chondrocytes were treated with varying concentrations of Ber (0, 2.5, 5, 10, 20, 40, or 80 μM) for 24, 48, 72, or 96 h. Following the incubation period, 10 μl of CCK-8 solution was added to each well, and the plates were incubated in a humidified environment with 5% CO2 at 37 °C. After a 2-h incubation, the optical density (OD) values were measured at 450 nm using a microplate reader (Bio-Tek Instruments, USA). Furthermore, the cytotoxic effects of Ber on bone marrow macrophages (BMMs) were evaluated employing the same CCK-8 assay methodology. To ensure the reliability and reproducibility of the results, all experiments were conducted in triplicate.High-density culture and toluidine blue stainingTo accurately assess the ECM levels in mouse cartilage, we implemented a precise experimental protocol as follows. Initially, approximately 12 × 106 primary chondrocytes were enzymatically dissociated and resuspended in trypsin. Subsequently, 10 μl of the cell suspension was seeded into each well and allowed to adhere at 37 °C for approximately 1 h to facilitate cellular attachment to the substrate. Next, 500 ml of DMEM supplemented with 10% fetal bovine serum (FBS) was added to each well, and the plates were incubated at 37 °C for 24 h. Concurrently, during the incubation phase, interleukin-1 beta (IL-1β) and varying concentrations of Ber (0, 10, and 20 μM) were added to the culture wells. Ultimately, between days 7–9 of the culture period, the cells were fixed with 4% formaldehyde for approximately 30 min and subsequently stained with toluidine blue. The staining intensity was quantified using ImageJ software to determine the ECM levels in the cartilage samples.RNA extraction and quantitative RT‒PCR analysisIn the presence or absence of Ber at concentrations of 10 and 20 μM, 2 × 105 chondrocytes per well were seeded into 6-well plates and subjected to IL-1β treatment for 48 h. BMMs at a density of 1 × 105 cells per well were seeded into 6-well plates and stimulated with RANKL (50 ng/ml) and M-CSF (30 ng/ml) for 5‒7 days. Total RNA was extracted from chondrocytes using TRIzol (Takara Bio, Otsu, Japan) according to the manufacturer’s protocol. RNA purity and quality were assessed by measuring the A260/A280 ratio. Subsequently, 1000 ng of total RNA was reverse transcribed into cDNA using reverse transcriptase (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. Real-time PCR was conducted using the LightCycler®480 system (Roche, Germany) and FastStart Universal SYBR Green Master Mix (Roche, Germany) with the following cycling conditions: initial denaturation at 94 °C for 5 s, followed by extension at 60 °C for 30 s, followed by 40 cycles. The expression levels of the target mRNAs were normalized to the GAPDH levels and compared to those in the control group. The specific primer sequences utilized are detailed below (Table1):Table 1 Sequences used for quantitative real-time PCR.Western blot analysisTotal protein was extracted using RIPA lysis buffer, followed by centrifugation, and the protein concentrations were determined using a BCA protein assay kit following the manufacturer’s protocol. Equal amounts of protein (40 ng) were separated by 10% SDS‒PAGE and then transferred to PVDF membranes (0.45 μm, Millipore, Bedford, MA, USA). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4 °C (details of the primary antibodies used in this study can be seen in the Table 2 below). Subsequently, the cells were washed three times with TBST and incubated with the appropriate secondary antibody (1:500) at room temperature for 2 h. Finally, protein bands on the membrane were visualized using an enhanced chemiluminescence (ECL) kit and analysed for quantification using ImageJ software (National Institutes of Health, Bethesda, MD) (Supplementary Information).Table 2 The Company and Catalogue number of all primary antibodies.Immunofluorescence stainingChondrocytes were seeded at a density of 3 × 104 cells per well in 24-well plates and cultured for 48 h. Subsequently, the chondrocytes were fixed for 30 min at room temperature and treated with 0.1% Triton X-100 (Sigma Aldrich, Germany) for 10 min. Next, the cells were blocked with 1% BSA (Sigma Aldrich, Germany) for 30 min, followed by overnight incubation at 4 °C with primary antibodies targeting MMP13 and COL2A1. Finally, the chondrocytes were visualized and imaged using a Leica fluorescence microscope.TUNEL stainingApoptotic cells in the cartilage cell population were identified using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining according to the manufacturer’s instructions. The number of TUNEL-positive cells was quantified.Flow cytometry analysisChondrocytes were seeded on 6-well plates at a density of 3.0 × 105 cells per well and exposed to varying concentrations of Ber (0, 10, or 20 µM) for 24 h. Following treatment, the cells were harvested and subjected to staining with Annexin V-PE and 7-AAD using assay kits according to the manufacturer’s protocol.TRAP staining assayBMMs were cultured in a 96-well plate at a density of 8 × 103 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 30 ng/mL macrophage colony-stimulating factor (M-CSF), 50 ng/mL receptor activator of nuclear factor kappa-Β ligand (RANKL), and varying concentrations of Ber (0, 10 and 20 μM) for 5–7 days. The culture medium was refreshed every three days. Upon the appearance of a substantial population of mature osteoclasts in the control well, these cells were specifically stained utilizing a tartrate-resistant acid phosphatase (TRAP) staining kit. Cells exhibiting TRAP positivity with three or more nuclei were counted, and their respective areas were quantitatively assessed.F-actin ring formation evaluationBMMs were cultured on glass coverslips and treated with 50 ng/mL RANKL and Ber at concentrations of 0, 10, and 20 μM. After 5–7 days of cell culture, mature osteoclasts fixed in 4% formaldehyde for 20 min were subjected to permeabilization with 0.1% (v/v) Triton X-100 (Sigma‒Aldrich) for 5 min and rinsed three times with phosphate-buffered saline (PBS). Nuclei were stained using 4ʹ,6-diamidino-2-phenylindole (DAPI), while F-actin was visualized with phalloidin-tetramethylrhodamine isothiocyanate. The arrangement of F-actin rings was examined using an LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany) and analysed with Zeiss ZEN software.DMM-induced OA mice modelC57BL/6 mice (10 weeks old, male, n = 18) were anaesthetised by intraperitoneal injection of 10% chloral hydrate before knees were prepared for aseptic surgery. The connection between the medial meniscus and the tibial plateau (medial meniscus ligament) of the right knee of the mice was subsequently severed using a microsurgical scalpel. The surgical approach is described as follows22: We first dissected the fat pad on the cranial horn of the medial meniscus using Jewelers forceps, then took care to identify and avoid the lateral meniscus ligament (LMTL), which is located posteriorly and has fibres oriented in a similar direction to the lateral meniscus ligament. The medial meniscus ligament is then incised with microsurgical scissors, a microsurgical scalpel, or an 11-gauge blade with the blade facing proximally, thereby destabilising the medial meniscus (DMM). After destabilisation of the medial meniscus, the medial meniscus undergoes a medial shift and weight bearing is concentrated in a smaller area, resulting in increased local mechanical stress. The animals were randomly divided into three groups: non-DMM group, DMM group, and DMM + 25 mg/kg Ber group. In the non-DMM group, only the right knee joint was incised, and the tibiofibular ligament of the medial meniscus was not removed. 25 mg/kg was injected intra-articularly into the joint cavity of the DMM plus 25 mg/kg Ber group twice a week for 8 weeks. The non-DMM and DMM groups received the same dose of PBS. All mice were euthanised and knee tissue samples were collected for further analysis at 8 weeks postoperatively. All animal experiments comply with the ARRIVE guidelines and be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. And we confirmed that all animal experiments were reviewed and approved by the Animal Ethics Committee of the First Affiliated Hospital of Nanchang University (protocol number: CDYFY-IACUC-202310QR044).Histological assessmentKnee joints of mice in each group were fixed in 4% formaldehyde for 24 h, then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for four weeks and embedded in paraffin. Five micron thick mouse knee joints in paraffin blocks were cut into different planes and stained with hematoxylin and eosin (H&E) and safranin O-fast green.Statistical analysisAll the data are expressed as the means ± SDs (standard deviations). Each experiment was conducted independently at least three times. Statistical analyses were performed utilizing Student’s t test or analysis of variance (ANOVA) with GraphPad Prism software version 8.0 (GraphPad Software, San Diego, CA, USA). P values less than 0.05 were considered to indicate statistical significance.
