The human chondrocyte cell line C28/I2 purchased from the National Cell Bank of Pasteur Institute, Iran, Tehran, was cultivated in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA) containing 10 % fetal bovine serum (FBS) (Anacell, Tehran, Iran) and 1 % penicillin and streptomycin (Bioideaco, Tehran, Iran). The culture was maintained at 37 °C in a humidified environment with 5 % CO₂. Every two days, a new medium was used. Four groups were investigated: A: Control (cells with no treatment), B: IL-1β (cells stimulated with IL-1β), C: IL-1β+Si-TRPV3 (cells stimulated with IL-1β and transfected with TRPV3-specific siRNA), and D: IL-1β+Si-NC (cells stimulated with IL-1β and transfected with siRNA negative control).

Metabion, Germany, generated the small interfering RNA (siRNA) and controls against TRPV3. The sequences were as follows: si-TRPV3 sense, 5´-GGGCGAACAUGCUCUACUAUATT-3´, and antisense, 5´-UAUAGUAGAGCAUGUUCGCCCTT-3´, and Si-NC sense, 5´-AACAGCUUCGGGUACAAGUCUTT-3´, and Si-NC antisense, 5´-AGACUUGUACCCGAAGCUGUUTT-3´. For cell transfection, 2.5×10⁵ C28/I2 cells per well were seeded into 6-well plates and allowed to grow to a cell density of 70 %. Next, 20 nM oligonucleotides were transfected using lipofectamine®2000 (Invitrogen; Thermo Fisher Scientific, USA). After six hours, the medium was changed to a new one, and the culture was allowed to continue for a full twenty-four hours. Following transfection, cells were treated for 24 hours with 10 ng/ml IL-1β ((Purity: >95 %) (Sino Biological, Beijing, China)) (Liu et al., 2023[21]) before being utilized in the following tests.

Human C28/I2 chondrocytes in the logarithmic phase were taken, seeded into 96-well plates (1 × 10⁴ cells per well), and placed in an incubator. Subsequently, three repetitions of each set of cells treated with various substances were added (a blank control group received an equivalent amount of PBS). Following a 24-hour culture period, 50 μL MTT (Sigma, St. Louis, MO, USA) (5 g/L) was added, and the cells were then cultivated for an additional 4 hours at 37 °C. Afterward, we removed the supernatant, filled each well with 150 μL of DMSO (Sigma, St. Louis, MO, USA), and gave it a good shake using a plate shaker. Following the crystals' dissolution, the OD value of each well was measured at 490 nm using a microplate reader.

The cells were separated from the plates using 0.25 % EDTA-trypsin (Sigma, St. Louis, MO, USA). As directed by the manufacturer, total RNA was extracted using column RNA extraction (DENAzistAsia, Mashhad, Iran). The RNA samples were quantified and qualified with a NanoDrop spectrophotometer and 2 % agarose gel electrophoresis. The cDNA was synthesized using the Parstous kit following the kit instructions and used in qRT-PCR. The housekeeping gene U6 was used to normalize the expression data. The qRT-PCR protocol involved 15 min at 95 °C for primary denaturation, which was contained with 40 cycles at 95 °C for 15 sec, annealing temperature for 30 sec, and at 72 °C for 30 sec, and finished with a final extension at 72 °C for 5 min. The 2-∆∆Ct method was used to determine the fold change of each mRNA expression. Table 1(Tab. 1) listed the primer sequences and their corresponding amplification products.

To study the impacts of TRPV3 knockdown on apoptosis, we utilized the Annexin V-FITC/PI apoptosis kit (ImmunoStep, Salamanca, Spain). After cultivating 2.5×10⁵ C28/I2 cells in 6-well plates, all four groups underwent a 24-hour treatment, were re-suspended in 197 μL binding buffer, and were stained with 3 μL Annexin V-FITC and 1 µL PI in a dark, room-temperature environment for 15 minutes. The cells were then examined by flow cytometry (Becton Dickinson, San Jose, CA, USA). Using the FlowJo analytic software version 10, the percentages of early and late apoptotic cells were calculated.

After plating 2.5×10⁵ C28/I2 cells per well per 2 mL on a six-well culture plate, the cells were incubated for 24 hours at 37 °C in a CO₂ incubator. Trypsin was used in this instance to separate the cells after 24 hours, and they were then washed with cold phosphate-buffered saline (PBS) and preserved in 90 % ethanol at -20 °C for the night. After centrifuging the ethanol-fixed cells and washing them with PBS, they were stained with a PI and RNase A (Simbiolab, Mashhad, Iran) solution at final concentrations of 50 mg/mL and 20 mg/mL, respectively. The BD FACSCalibur cytometer (Becton Dickinson, San Jose, CA, USA) was used for assessing the cell cycle phases.

The cells were lysed using a lysis buffer composed of 0.1 % SDS, 1 % Triton X-100, 1.5 M NaCl, 1 mM EDTA, and 20 mM Tris HCl (pH 7.4), supplemented with 1 % phenylmethanesulfonyl fluoride. Total protein was purified by centrifugation at 16,000 g for 10 minutes at 4 °C. The protein concentration was determined using the DC protein assay from BioRad. A constant voltage of 80 mV was applied for 30 minutes, followed by 120 mV for 120 minutes, to separate 30 µg of protein from each group via electrophoresis. The proteins were then transferred to PVDF membranes using the Bio-Rad TransBlot system at 100 mV for 120 minutes (Bio Rad Laboratories, Inc.). A solution of 5 % non-fat dried milk in 10 mM Tris HCl, 0.2 % Tween-20 (TBST), and 0.15 M NaCl was utilized to block the membrane for 2 hours at room temperature. The membrane underwent incubation with primary antibodies targeting TRPV3 (mouse; cat. no. sc-45407), Bax (mouse; cat. no. sc-7480), Bcl-2 (rabbit; cat. no. sc-492), caspase-3 (mouse; cat. no. sc7272), iNOS (mouse; cat. no. sc-7271), COX-2 (mouse; cat. no. sc-514489), and β-Actin (mouse; cat. no. sc-47778) overnight at 4 °C. The membrane underwent incubation with mIgG κBP-HRP (mouse; cat. no. sc-516102) and mouse anti-rabbit IgG HRP (mouse; cat. no. sc-2357) secondary antibodies at room temperature for 2 hours, following three rinses with TBST for 5 minutes each. The membrane underwent treatment with an ECL chemiluminescence kit and was subsequently analyzed using the Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad). Densitometry quantification of the images was performed using ImageJ v1.8.0 software from the National Institutes of Health.

Data analysis was conducted using GraphPad Prism (San Diego, CA, USA) version 9.0 and SPSS version 26. Data normalization was performed as necessary. A two-tailed t-test was conducted to compare the two groups, with the data presented as the mean ± SD. FlowJo v10.8 was employed to evaluate the data obtained from flow cytometry assays. Image processing and quantification of the western blotting data were done using ImageJ v1.8.0 software. Statistical significance was defined as P < 0.05.

To investigate the role of TRPV3 in OA, we assessed its expression in the IL-1β-treated C28/I2 chondrocyte cells by qRT-PCR and western blot. As shown in Figure 2(Fig. 2), IL-1β-treated C28/I2 chondrocyte cells upregulated the expression of TRPV3 in both mRNA and protein levels, implying that IL-1β enhances TRPV3 expression in chondrocyte cells. We applied TRPV3-specific siRNA (si-TRPV3) to knockdown its expression. As Figure 2(Fig. 2) shows, transfection of the IL-1β-treated C28/I2 cells with si-TRPV3 effectively repressed TRPV3 expression in the mRNA and protein levels.

Chondrocyte cell viability was assessed after treatments of the cells with IL-1β, Si-TRPV3+IL-1β, and siRNA negative control +IL-1β (Si-NC+IL-1β). The cell viability was significantly decreased in response to the IL-1β treatment compared to the control (Figure 3F(Fig. 3)). To verify whether the cytotoxicity induced by IL-1β occurred via apoptosis, we employed flow cytometry to measure the apoptosis of the C28/I2 cells treated with IL-1β. Figure 3A and B(Fig. 3) demonstrate that IL-1β significantly induced apoptosis in C28/I2 cells, with 9.65 % early and 25.8 % late apoptosis compared to the control group. To understand whether TRPV3 knockdown could affect chondrocyte cell viability, C28/I2 cells were pretreated with Si-TRPV3 or Si-NC for 6 hours before treatment with IL-1β. As Figure 3F(Fig. 3) shows the cell viability was significantly increased in response to the knockdown of TRPV3. Besides, the findings indicated a notable decrease in the percentage of apoptotic chondrocytes in the Si-TRPV3+IL-1β treated cells (early apoptosis 9.79 % and late apoptosis 15.6 %) in comparison to the IL-1β-treated cells (early apoptosis 9.65 % and late apoptosis 25.8 %) (Figure 3C(Fig. 3)). However, treatment of the cells with Si-NC +IL-1β resulted in non-significant differences compared to the IL-1β-treated cells (Figure 3D(Fig. 3)). Figure 3E(Fig. 3) presents the results derived from three independent experimental replicates.

Flow cytometry assessed the effect of Si-TRPV3+IL-1β, IL-1β, and Si-NC +IL-1β interventions on the cell cycle progression of C28/I2 chondrocyte cells. The results indicated that treatment with IL-1β considerably elevated the sub-G₁ cell phase population to 33.4 % compared to the untreated control cells, which exhibited 4.55 %. Conversely, the sub-G₁ cell phase population of the Si-NC +IL-1β group exhibited an insignificant change (33.3 %) in comparison to the IL-1β treatment group (33.4 %) (Figure 4B and D(Fig. 4)). The Si-TRPV3+IL-1β treatment group exhibited a reduction in the sub-G₁ cell phase population (23.1 %) in comparison to the IL-1β treated cells (33.4 %) (Figure 4B and C(Fig. 4)). Figure 4E(Fig. 4) delineates the findings derived from three independent experiments.

Quantitative Real-time PCR and western blotting were used to assess the expression of Bax, Caspase-3, and Bcl-2. Figure 5(Fig. 5) illustrates how IL-1β treatment significantly increased the mRNA and protein levels of pro-apoptotic Bax and Caspase-3 genes and significantly decreased the mRNA and protein levels of anti-apoptotic Bcl-2. Si-TRPV3 treatment notably elevated Bcl-2 expression (anti-apoptotic) and reduced the expression of Bax and Caspase-3 (pro-apoptotic) genes. Western blotting validated these effects at the protein level (Figure 5A-H(Fig. 5)).

Measurements of iNOS and COX-2, mRNA and protein levels, were also conducted using qRT-PCR and western blotting, respectively, to ascertain the impact of TRPV3 knockdown on osteoarthritis inflammation. Our findings indicated that IL-1β treatment elevated both mRNA and protein levels of iNOS and COX-2 expression; however, the treatment of chondrocyte cells with Si-TRPV3 considerably inhibited the expression of these genes (Figure 6A-E(Fig. 6)).

See also the supplementary data.

The authors appreciate the research deputy of the University of Tabriz for general support of the study.

Not applicable.

Data are available upon reasonable request.

The authors declare no competing interest.

No funds, grants, or other support were received.

SG, AM, and MKK, designed the study. SG collected data and wrote the manuscript. MKK supervised, directed, and managed the study. SG, AM, and MKK approved the version to be published.