Rat primary chondrocytes (cat. no. CP-R087) were obtained from Procell Life Science & Technology Co., Ltd. The cells were suspended in rat chondrocyte complete medium (cat. no. CM-R087; Procell Life Science & Technology Co., Ltd.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-100 µg/ml streptomycin (penicillin-streptomycin liquid; Beijing Solarbio Science & Technology Co., Ltd.), and were incubated at 37°C in 5% CO₂. The cells were divided into control, low-concentration (25 nM) TBTC (MilliporeSigma), medium-concentration (50 nM) TBTC and high-concentration (100 nM) TBTC groups at 37°C for 24 h. The experiments were repeated three times for each group.

The cells were resuspended and seeded in 96-well plates at a density of 1×10⁴ cells/well. When the cells reached 50–60% confluence, the medium was discarded, and TBTC was added at final concentrations of 0, 25, 50 and 100 nM for 24 h at 37°C. The cells were then incubated with CCK-8 reagent (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at 37°C and the absorbance values were determined at 450 nm.

The cells were lysed with RIPA buffer (high; Beijing Solarbio Science & Technology Co., Ltd.) and the lysate was centrifuged at 11,000 × g for 15 min at 4°C, after which the supernatant was collected. The protein concentration was quantified using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.), and proteins (20 µg) were separated by SDS-PAGE on 10% gels. The proteins were then transferred to PVDF membranes, which were incubated with 5% nonfat milk for 1 h at 37°C. Subsequently, primary antibodies against β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.), NLRP3 (1:1,000; cat. no. ab263899; Abcam), interleukin (IL)-1β (1:1,000; cat. no. ab315084; Abcam), IL-18 (1:1,000; cat. no. ab223293; Abcam), matrix metalloproteinase (MMP)-1 (1:500; cat. no. 70R-50066; AmyJet Scientific, Inc.), MMP-13 (1:1,000; cat. no. ab219620; Abcam), caspase-1 (1:1,000; cat. nos. ab286125 for rat or ab138483 for mouse; Abcam), PYD and CARD domain containing (ASC; 1:1,000; cat. no. ab307560; Abcam) and gasdermin D (GSDMD; 1:1,000; cat. no. ab219800; Abcam) were incubated with the PVDF membranes overnight at 4°C. The membranes were then incubated with an HRP-labeled goat anti-rabbit IgG (H+L) secondary antibody (1:3,000; cat. no. A0208 Beyotime Institute of Biotechnology) at room temperature for 1 h. After washing the membranes three times with TBS-Tween (0.1%), the protein bands were detected using an ECL western blotting substrate (Beijing Solarbio Science & Technology Co., Ltd.). The intensities of the protein bands were analyzed using ImageJ 1.8.0 software (National Institutes of Health), and the relative protein expression levels were calculated using β-actin as a reference.

Rat primary chondrocytes (1×10⁶ cells/well in a 6-well plate) were divided into control, low-concentration (25 nM) TBTC, medium-concentration (50 nM) TBTC and high-concentration (100 nM) TBTC groups and were treated at 37°C for 24 h. Cytotoxicity was evaluated by determining the LDH leakage rate in cell supernatants using a cytotoxicity LDH assay kit (MedChemExpress) according to the manufacturer's instructions.

Rat primary chondrocytes (1×10⁶ cells/well in a 6-well plate) were divided into control, low-concentration (25 nM) TBTC, medium-concentration (50 nM) TBTC and high-concentration (100 nM) TBTC groups and were treated at 37°C for 24 h. The chondrocytes were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 20 min and then blocked with 5% donkey serum at room temperature for 2 h (Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, the cells were incubated with a primary antibody against NLRP3 (1:50; cat. no. ab263899; Abcam) or GSDMD (1:50; cat. no. ab219800; Abcam) at 4°C overnight. After being washed three times with PBS, the slides were incubated with Alexa Fluor 350-labeled goat anti-rabbit IgG (H+L) (1:200; cat. no. A0408; Beyotime Institute of Biotechnology) at room temperature for 1 h, followed by incubation with antifade mounting medium containing DAPI (Beyotime Institute of Biotechnology). Images were observed under a fluorescence microscope (magnification, ×20; Olympus Corporation).

Rat primary chondrocytes (1×10⁶ cells/well in a 6-well plate) were divided into control, low-concentration (25 nM) TBTC, medium-concentration (50 nM) TBTC and high-concentration (100 nM) TBTC groups and were treated at 37°C for 24 h. The cell cycle assay was performed using the DNA Content Quantitation Assay (Cell Cycle) (cat. no. CA1510; Beijing Solarbio Science & Technology Co., Ltd.). Briefly, cells were collected and fixed with 70% precooled ethanol (500 µl) at 4°C overnight. The cells were then centrifuged at 800 × g at 4°C for 15 min, 100 µl RNase A solution was added to the cell precipitate and the resuspended cells were incubated in a water bath at 37°C for 30 min. Subsequently, 400 µl PI staining solution was added to the cell suspension, which was incubated at 4°C for 30 min in the dark. Finally, the cells were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences) and data analysis was performed using FlowJo 10 software (FlowJo, LLC).

Rat primary chondrocytes (1×10⁶ cells/well in a 6-well plate) were divided into control, low-concentration (25 nM) TBTC, medium-concentration (50 nM) TBTC and high-concentration (100 nM) TBTC groups and were treated at 37°C for 24 h. EdU staining was performed using an EdU cell proliferation assay kit (Shanghai Recordbio Biological Technology). Rat primary chondrocytes (1×10⁶ cells/well in a 6-well plate) were incubated with 50 µM EdU (20 µl total volume) for 2 h at 37°C. After three washes with PBS, the cells were incubated with 4% paraformaldehyde for 15 min at room temperature and then with 0.5% Triton X-100 for 15 min at room temperature. Subsequently, the samples were blocked with antifade mounting medium containing DAPI (Beyotime Institute of Biotechnology) and were observed under a fluorescence microscope (Olympus Corporation). Cell proliferation rate was calculated as follows: Cell proliferation rate (%)=EdU-positive cells/DAPI-positive cells ×100.

The present study used rat primary chondrocytes in vitro and a mouse model in vivo. The present study aimed to verify that TBTC damaged articular chondrocytes in different species and, compared with rat models, mouse models are easier to generate and are less costly. C57BL/6J male mice [n=20; Shulaibao (Wuhan) Biotechnology Co., Ltd.] were acclimated for 1 week at 22–24°C and 45% humidity. All mice were given free access to food and water under a 12-h light/dark cycle. All mice were randomly divided into the following four groups: Control, OA, TBTC and TBTC + OA groups (n=5 mice/group). The mouse OA model was induced by injection of monosodium iodoacetate (MIA; cat. no. I2512; MilliporeSigma) as previously described (16). Specifically, MIA was dissolved in a 0.9% NaCl solution to a final concentration of 60 mg/ml, and 50 µl MIA solution was injected into the right knee joint cavity of the mice in the OA groups. The control mice were injected with 50 µl 0.9% NaCl solution. After 2 weeks of modeling, the OA model mice were further divided into two groups: In the TBTC group, the mice were injected with 50 µl 0.9% NaCl solution and were also administered TBTC daily at a dose of 10 µg/kg for 2 weeks, and in the other groups, the mice were administered an equal volume of saline for a total of 2 weeks. All of the mice were euthanized at the termination of the experiment, ensuring minimal suffering. No mice died naturally during the study. All mice were monitored twice daily for signs of discomfort, illness or distress by trained personnel. To minimize suffering and distress, isoflurane was utilized at an induction concentration of 5% and a maintenance concentration of 2% prior to cervical dislocation. Humane endpoints were established based on the guidelines from the Institutional Animal Care and Use Committee of Yangzhou University, and included >20% weight loss or gain, inability to eat or drink, persistent lack of mobility, signs of severe and chronic pain, and any conditions notably interfering with daily activities. When the mice were fully unresponsive to nociceptive stimuli indicating that they were fully anesthetized, cervical dislocation was used for euthanasia. Death was verified by the absence of respiration, heartbeat and reflexes. The knee joint tissues were subsequently collected.

Cartilage sections (1.0×1.0×0.5 cm) were cut from the mouse subchondral bone and fixed in 4% formaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) for 3 days at 4°C. Subsequently, the sections were decalcified in a 30% formic acid solution for 14 days at room temperature, dehydrated in an increasing ethanol gradient, embedded in paraffin and cut into 5-µm sections. The sections were stained using the modified Safranin-O and Fast Green stain kit (for bone) (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Finally, the sections were blocked with neutral gum (Beijing Solarbio Science & Technology Co., Ltd.). Normal cartilage was red in color, and the background was green, and staining was observed under a light microscope (Olympus Corporation).

Cartilage samples were cut into 5-µm sections as aforementioned and were incubated using a H&E stain kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Briefly, the sections were incubated with hematoxylin at room temperature for 10 min, rinsed in tap water, and then stained with eosin at room temperature for 30 sec. After rinsing in tap water, and following dehydration, clearing and sealing, the sections were observed under a light microscope (Olympus Corporation).

Cartilage samples were cut into 5-µm sections as aforementioned and were deparaffinized and stained with a 1% toluidine blue O solution (Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at 55°C, according to the manufacturer's instructions. After being rinsed with distilled water, the sections were dehydrated with 95% ethanol, cleared with xylene and blocked with resin. Subsequently, the tissues were observed under a light microscope (Olympus Corporation).

Rat primary chondrocytes (1×10⁶ cells/well in a 6-well plate) were divided into control, low-concentration (25 nM) TBTC, medium-concentration (50 nM) TBTC and high-concentration (100 nM) TBTC groups and were treated at 37°C for 24 h. Total RNA was extracted from chondrocytes using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was reverse transcribed into cDNA using PrimeScript^™ RT master mix (cat. no. RR036A; Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was performed on an ABI StepOnePlus real-time fluorescence qPCR instrument (Thermo Fisher Scientific, Inc.) using the SYBR^® Select master mix (Beijing Think-Far Technology Co., Ltd.). The thermocycling conditions were as follows: Denaturation at 95°C for 30 sec, followed by 35 cycles of annealing at 60°C for 1 min and extension at 95°C for 5 sec. The sequences of the primers used were as follows: NLRP3, forward 5′-ATAGCGTCATACGGGGGAAG-3′, reverse 5′-CACCCACGGGCAAGATGAAA-3′; GAPDH, forward 5′-TGAGGAGTCCCCATCCCAAC-3′ and reverse 5′-ATGGTATTCGAGAGAAGGGAGG-3′. Relative gene expression was calculated using the 2^−∆∆Cq method (17) with GAPDH as an internal control.

The siRNA targeting NLRP3 (sense 5′-AAUUUAAAUACAUUUCACCCA-3′; antisense 5′-TGGGTGAAATGTATTTAAATT-3′) and the negative control (NC) siRNA (sense 5′-UUCUCCGAACGUGUCACGUUU-3′; antisense 5′-AAACGUGACACGUUCGGAGAA-3′) were purchased from Shanghai GenePharma Co., Ltd. Briefly, primary rat chondrocytes (1×10⁶ cells/well in a 6-well plate) were transfected with si-NLRP3 or NC at a final concentration of 20 nM using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h, according to the manufacturer's instructions before TBTC treatment. Subsequently, the cells were collected immediately for subsequent experiments.

SPSS 18.0 statistical software (SPSS, Inc.) was used for data analysis and all experiments were repeated at least three times. The data are presented as the mean ± standard deviation. Differences among more than two groups were examined by one-way ANOVA, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

First, the effects of TBTC on the cell cycle of rat chondrocytes were explored. Compared with in the control group, TBTC increased the number of cells in G₁ phase in a concentration-dependent manner and decreased the number of cells in the G₂ and S phases (Fig. 1A-C). EdU staining also showed that the proliferation of rat chondrocytes was significantly inhibited in response to an increasing concentration of TBTC (Fig. 1D). These results preliminarily suggested that TBTC was detrimental to the proliferation of rat chondrocytes.

The CCK-8 assay results showed that TBTC reduced rat chondrocyte viability in a concentration-dependent manner (Fig. 2A). LDH leakage is the main indicator of pyroptosis, and LDH leakage was significantly greater in the TBTC groups than that in the control group (Fig. 2B). Furthermore, the mRNA expression levels of NLRP3 in rat chondrocytes in the TBTC group were significantly greater than those in the control group (Fig. 2C). After incubation with TBTC, the protein expression levels of NRLP3, IL-1β, IL-18, MMP-1 and MMP-13 in rat chondrocytes were significantly greater than those in the control group (Fig. 2D). These results suggested that TBTC serves a critical role in the destruction of chondrocytes by activating the NLRP3 inflammasome.

IF experiments revealed that the expression of NLRP3 and GSDMD was significantly greater in rat chondrocytes in the TBTC groups than that in the control group (Fig. 3A). Moreover, western blot analysis revealed that the expression levels of caspase-1, ASC and GSDMD were significantly elevated in the TBTC groups compared with those in the control group (Fig. 3B), indicating the induction of pyroptosis by TBTC in rat chondrocytes.

To further verify whether TBTC induces rat chondrocyte pyroptosis via NLRP3, cells were transfected with a siRNA specifically targeting NLRP3. The siRNA targeting NLRP3 effectively inhibited NLRP3 expression compared with NC group (Fig. 4A). Compared with that in the TBTC group, the LDH leakage rate was significantly decreased in the si-NLRP3 + TBTC group (Fig. 4B). Moreover, the expression levels of pyroptosis-related proteins, including caspase-1, ASC and GSDMD, were significantly reduced in the si-NLRP3 + TBTC group than those in the TBTC group (Fig. 4C). These results suggested that TBTC induced rat chondrocyte pyroptosis by activating NLRP3 signaling.

H&E staining showed that in the cartilage of the control group, the cell nuclei were dark blue, the cytoplasm and cartilage matrix were pink, and the coloring was homogeneous; the surface of the cartilage was smooth and flat, and was arranged parallel to the surface of the joint (Fig. 5A). In the OA group, the surface of the cartilage was rough, the integrity of the cartilage was disrupted, and the surface cartilage exhibited fibrotic degeneration and defects. In the TBTC-treated group, the cartilage surface also showed some defects. Moreover, the integrity of the cartilage was markedly disrupted, and the cartilage defects were more pronounced in the TBTC + OA group (Fig. 5A). The results of Safranin O-Fast Green staining showed that the cartilage matrix was intact in the control group, as evidenced by proteoglycans stained in red, and by fibers, the subchondral bone cortex and trabeculae stained in green (Fig. 5B). In the OA and TBTC groups, the cartilage matrix exhibited some degree of disruption, as evidenced by reduced proteoglycan coloration. In the TBTC + OA group, the reduction in proteoglycan coloring was further exacerbated (Fig. 5B). Toluidine blue staining also revealed greater articular cartilage degeneration in the mice from the OA and TBTC groups than in those from the control group (Fig. 5C). This cartilage degeneration was more pronounced in the mice from the TBTC + OA group (Fig. 5C). These results suggested that TBTC enhanced articular cartilage degeneration in a mouse model of OA.

The present study then examined the effects of TBTC on cartilage tissue damage in mice. The results revealed that the protein expression levels of NLRP3, IL-1β, IL-18, MMP-1 and MMP-13 were significantly greater in the OA and TBTC groups than those in the control group (Fig. 6A). Similarly, the proteins were also upregulated in the TBTC + OA group (Fig. 6A). The expression levels of pyroptosis-related proteins were also detected. Notably, the expression levels of the pyroptosis-related proteins caspase-1, ASC and GSDMD were significantly greater in the OA and TBTC groups than those in the control group (Fig. 6B). These proteins were also increased in the TBTC + OA group (Fig. 6B). These results confirmed that TBTC could increase the NLRP3-mediated inflammatory response and pyroptosis, thereby promoting osteoarticular injury in mice.