Human chondrocytes, CHON-001 (cat. no. ATCC^®CRL-2846TM), used in the present study were from American Type Culture Collection. Cells were cultured at a concentration of 5×10⁵ cells in 25-cm² flasks with DMEM/F12 (cat. no. 1662222, Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (cat. no. 10099-141, Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (10% penicillin/streptomycin solution) in an incubator at 37°C with 5% CO₂. Chondrocytes, which were identified using collagen II immunohistochemical detection, were inoculated in a culture dishes pre-coated with a coverslip and collected after 4 h, then washed with PBS. The cells were fixed using 4% paraformaldehyde at room temperature for 20 min, embedded with neutral resin at room temperature for 20 min and sectioned at 18 µm. After being washed three times with PBS for 5 min each time and incubated with 0.5% Triton X-100 at room temperature for 20 min, chondrocytes were treated with 3% H₂O₂ at room temperature for 15 min and blocked in 5% BSA (cat. no. mu30432, Bio-Swamp) at room temperature for 20 min after dehydration. Subsequently, chondrocytes were incubated with anti-collagen II antibodies (rabbit; 1:100; cat. no. AF0135; Affinity Biosciences) at 4°C overnight. The following day, sections were washed with PBS and incubated with the secondary antibody [goat anti-rabbit horseradish peroxidase (HRP); 1:50; cat. no. A0208; Beyotime Institute of Biotechnology) for 50 min at 4°C. The sections were then washed three times with PBS, with each wash lasting for 5 min, and were stained with 100 µl 3,3-diaminobenzidine (cat. no. P0203; Beyotime Institute of Biotechnology) at room temperature for 8 min, then counter-stained with hematoxylin (cat. no. H9627, Sigma-Aldrich; Merck KGaA) at room temperature for 25 sec. Images were captured from each section at magnification ×100 and ×400, using an IX51 light microscope (Olympus Corporation).

Human chondrocytes were divided into normal chondrocytes and those from the OA cell model. The OA cell model was induced using IL-1β (cat. no. SRP6169; Sigma-Aldrich; Merck KGaA) at the recommended concentration of 10 ng/ml (14). Chondrocytes were treated with IL-1β at room temperature for 24 h.

Human chondrocytes in the present study were divided into six groups as follows: i) Group A, normal chondrocytes; ii) group B, OA cell model (chondrocytes + IL-1β); iii) group C, OA cell model (chondrocytes + IL-1β) + genistein (cat. no. 1288816, Sigma-Aldrich; Merck KGaA) (25 µg/ml); iv) group D, OA cell model (chondrocytes + IL-1β) + genistein (50 µg/ml); v) group E, OA cell model (chondrocytes + IL-1β) + genistein (100 µg/ml); vi) and group F, OA cell model (chondrocytes + IL-1β) + estradiol valerate (cat. no. 1252003, Sigma-Aldrich; Merck KGaA) (100 µg/ml). After treatment with IL-1β at room temperature for 24 h, chondrocytes in groups B, C, D, E and F were incubated with different concentrations of genistein and estradiol valerate as aforementioned at room temperature for 72 h.

Chondrocyte apoptosis was assessed using flow cytometry (FACSCalibur, BD Biosciences). The apoptotic rate was calculated as the percentage of early + late apoptotic cells. A total of 2×10⁵ cells were seeded in 6-well plates. Briefly, a cell apoptosis detection kit (cat. no. KGA108, Nanjing KeyGen Biotech Co. Ltd.) was used: 500 µl binding buffer mixed with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) was added to each well. Cells were then incubated at 25°C in darkness for 15 min. In the flow cytometry assay, green fluorescence indicated Annexin V-FITC and red fluorescence indicated PI. The excitation wavelength of FITC was 488 nm and was detected in the FL1 channel. Red fluorescence was detected in the FL2 channel. The staining profile was analyzed using Cell Quest Pro software, version 5.1 (BD Biosciences). In the two-color dot plot, the x-axis represented FL1 and y-axis represented FL2. In total, 10,000 events were collected for each sample.

Protein from chondrocytes was extracted using a lysis buffer [containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% TritonX-100, 1.5 M NaCl, 0.1% SDS and 1% phenylmethanesulfonyl fluoride; cat. no. ST505; Beyotime Institute of Biotechnology] and was preserved at −80°C after centrifugation at 16,000 × g for 10 min at 4°C. The protein concentration was determined using the Bio-Rad DC protein assay(ChemiDoc XRS + system, Bio-Rad Laboratories, Inc.). A total of 30 µg protein from each group was analyzed using electrophoresis on 10% SDS-PAGE with a constant voltage initially set at 80 V for 30 min, and then 120 V for 120 min. The proteins were transferred onto PVDF membranes in the Bio-Rad TransBlot apparatus (Bio-Rad Laboratories, Inc.) at 100 V for 120 min. Subsequently, the membrane was blocked with 5% non-fat dry milk, which was dissolved in 0.15 M NaCl containing Tris-buffered saline-0.2% Tween-20 (TBST) and 10 mM Tris-HCl, for 2 h at room temperature. The membrane was incubated overnight at 4°C with the following primary antibodies: Aggrecan (rabbit; 1:1,000; cat. no. DF7561; Affinity Biosciences), caspase 3 (rabbit; 1:1,000; cat. no. AF6311; Affinity Biosciences), collagen II (rabbit; 1:1,000; cat. no. AF0135; Affinity Biosciences), estrogen receptor α (ERα; rabbit; 1:500; cat. no. AF6058; Affinity Biosciences) and GAPDH (rabbit; 1:1,000; cat. no. AP0063; Bioworld Technology, Inc.). The following day, the membrane was washed with TBST three times for 5 min each time at room temperature and was then incubated with secondary antibodies (goat anti-rabbit HRP; 1:50; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 2 h. Subsequently, the membrane was incubated with ECL chemiluminescence reagents (cat. no. NCI5079, Thermo Fisher Scientific, Inc.) and exposed to Kodak X-OMAT films for detection with ImageJ v1.8.0 (National Institutes of Health).

To analyze the expression levels of TNF-α in chondrocytes of each group, a TNF-α ELISA kit (cat. no. EHJ-10039; Xiamen Huijia Biotechnology Co., Ltd.) was used according to the manufacturer's protocol. Optical density values were determined using a microplate reader at 450 nm and the concentration of TNF-α was calculated using the linear regression equation (y=0.004×-0.089, R2=0.9874).

All 40 pathogen-free male juvenile Sprague-Dawley rats (age, 4 weeks; weight, 180–200 g) were provided by the Shanghai Experimental Animal Center of the Chinese Academy of Science [animal certificate no. SCXK (Shanghai 2007-0005)] and housed at a constant temperature of 23±1°C, humidity of 40–70% and under a 12-h light-dark cycle, with free access to water and food. All animal studies, including the mice euthanasia procedure, were performed in compliance with the regulations and guidelines of the Zhejiang Chinese Medical University Institutional Animal Care and Conducted according to the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) (15) and the Institutional Animal Care and Use Committee (IACUC) (16) guidelines. The present study was approved by Zhejiang Chinese Medical University Institutional Animal Care.

The OA rat model used in this study was performed based on a previous report (17). Rats were anesthetized with 2.25% pentobarbital sodium (45 mg/kg, intraperitoneally) and the medial knee joint of one of the limbs of each rat was incised to expose an area of skin ~1×2 cm, thus opening the knee joint cavity. Then, the medial and anterior crucial ligaments were exposed and resected. Subsequently, the medial meniscus was removed and sutured. On days 1–3 post-surgery, rats were treated with an intramuscular injection of 1 ml penicillin (40,000 U/ml). All 40 rats were randomly divided into a normal group, OA group, genistein group and estradiol valerate group (n=10 for each group). Rats in the OA, genistein and estradiol valerate groups underwent OA model surgery as aforementioned and were intragastrically administrated normal saline (2 ml), genistein (20 mg/kg) (18) or estradiol valerate (0.8 mg/kg) (19), respectively, each day for 6 weeks. Rats in the normal group were intragastrically administrated normal saline (2 ml) each day for 6 weeks and housed under the same conditions.

After 6 weeks post-surgery, all the rats were sacrificed by dislocation of the neck and the cartilage of rats in each group was removed to create paraffin-cut sections. Cartilage tissues were fixed with formaldehyde-acetic acid-ethanol fixative (90 ml 70% ethanol, 5 ml acetic acid and 5 ml formaldehyde) at room temperature for 48 h, embedded in paraffin, and sectioned coronally to 18 µm. Sections were dewaxed using xylene (cat. no. 10023418; Sinopharm Chemical Reagent Co., Ltd.) after drying and placed in 100, 95, 85 and 75% ethanol respectively, each for 5 min at room temperature, and finally soaked in distilled water. After staining with Masson trichrome (cat. no. DC0032; Beijing Leagene Biotech Co., Ltd.) and toluidine blue (cat. no. G1032; Servicebio, Inc.) at room temperature for 20 min, the histopathological changes of cartilage were viewed under an optical microscope at ×400 magnification.

Levels of TNF-α and IL-1β in synovial liquid analyzed using ELISA. Synovial liquid (1 ml) was collected from each rat and the concentration of TNF-α and IL-1β in the synovial liquid was calculated using ELISA kits (cat. no. EHJ-10039 and EHJ-10293, respectively; Xiamen Huijia Biotechnology Co., Ltd.) according to the manufacturer's protocol.

The cartilage of rats in each group was removed with a small scalpel and scissors for western blot analysis. Protein from the articular cartilage was extracted using SDS lysis buffer (cat. no. 10014118, Sinopharm Chemical Reagent Co., Ltd.), and the protein expression levels of collagen II, aggrecan, caspase 3 and ERα were detected using the aforementioned protocol.

All data were analyzed using SPSS 19.0 software (IBM Corp.). Each experiment was repeated ≥3 times. Comparisons between experimental groups were performed using one-way ANOVA and Tukey's test. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

The collagen II immunohistochemical identification of human chondrocytes is presented in Fig. 1. Collagen II is specifically secreted by chondrocytes and can be accurately used to localize chondrocytes (20).

After being counterstained with hematoxylin, collagen II was labelled brown and the cell nucleus was stained blue-purple. Collagen II immunohistochemical staining identified the heterochromatism of cultured chondrocytes, which were confirmed to be human chondrocytes (Fig. 1).

The protein expression levels of collagen II, aggrecan, caspase 3 and ERα of chondrocytes in the OA model treated with varying concentrations of genistein and estradiol valerate were detected using western blotting (Fig. 2A). Compared with normal chondrocytes in group A, the expression levels of collagen II (Fig. 2B), aggrecan (Fig. 2C) and ERα (Fig. 2E) decreased in group B (all P<0.01), while those of caspase 3 increased (Fig. 2D; P<0.01). Compared with group B, the expression levels of collagen II and aggrecan increased in groups D and E (P<0.01 and P<0.05), while the expression of caspase 3 decreased in a dose-dependent manner (both P<0.01). Furthermore, compared with group B, the expression of ERα increased in group D (P<0.01). Compared with group E, the expression levels of collagen II and aggrecan decreased in groups C and D (P<0.01 and P<0.05), while the expression of caspase 3 increased in groups C and D (P<0.01), and the expression of ERα increased in group D (P<0.01).

The extent of chondrocytes apoptosis was detected using flow cytometry (Fig. 3). Compared with group A, the rate of apoptosis in groups B, C, D, E and F was significantly increased (P<0.01 and P<0.05). In addition, compared with group B, there was no significant difference in the apoptotic rate of group C with 25 µg/ml genistein (P>0.05); while in groups D, E and F, the rate of apoptosis was significantly decreased (P<0.01). Thus, the results indicated that IL-1β promoted chondrocyte apoptosis, but genistein reversed this effect in a dose-dependent manner.

TNF-α plays an important role in the development of OA (21). The levels of TNF-α in chondrocytes of each group were measured using ELISA. Compared with group A, the levels of TNF-α were higher in groups B, C, D and F (P<0.01; Fig. 4). However, the levels of TNF-α in groups C, D, E and F, with different concentrations of genistein, were significantly decreased compared with group B (P<0.01). Compared with group C, the levels of TNF-α significantly decreased in group D and E (P<0.01).

The Masson staining of the articular cartilage in each group of the OA model rats is presented in Fig. 5. Collagenous fibers were closely arranged and were stained blue in the normal group. Compared with the normal group, collagenous fibers in the OA group were disordered and locally broken, while the articular cartilage was degraded with a decreased collagen content. Furthermore, compared with the OA group, collagenous fibers in the genistein group were more closely arranged and had an increased collagen content, which reduced the cartilage degradation. In the estradiol valerate group, the staining results were similar to the genistein group, although the collagenous fibers were more closely arranged and collagen content was increased compared with the OA group.

The toluidine blue staining of articular cartilage from each group of the OA model rats is presented in Fig. 6. Toluidine blue is a basic dye, which turns blue after combining with acid glycosaminoglycan (22). Acid glycosaminoglycans were abundantly identified in the cartilage matrix of the normal group rats, demonstrating metachromasia. Furthermore, the acid glycosaminoglycan content in the cartilage matrix was markedly decreased in the OA group but notably increased in the genistein group. The acid glycosaminoglycan content in the estradiol valerate group rats increased compared with the OA group but was lower compared with the normal group.

The levels of TNF-α and IL-1β in the synovial fluid of each group were detected using ELISA (Fig. 7). Compared with the normal group, levels of TNF-α and IL-1β in the OA group were significantly increased (both P<0.01). However, in the genistein and estradiol valerate treated groups, TNF-α and IL-1β levels were significantly decreased compared with the OA group (all P<0.01).

The expression levels of collagen II, aggrecan, caspase 3 and ERα from the articular cartilage in the OA model rats treated with genistein and estradiol valerate were measured using western blotting (Fig. 8A). Compared with the normal group, protein expression levels of collagen II (Fig. 8B; P<0.01), aggrecan (Fig. 8C; P<0.01) and ERα (Fig. 8E; P<0.05) were decreased in the OA group, while caspase 3 expression was increased (Fig. 8D; P<0.01). Moreover, compared with the OA group, the expression levels of collagen II (P<0.01), aggrecan (P<0.01) and ERα (P<0.05) were increased in the genistein and estradiol valerate groups, but the expression of caspase 3 was decreased (P<0.05).