2.1 Regents
Hydroxysafflower yellow A was purchased from Chengdu must Biotechnology (CAS. 78281-02-4; Chengdu, China). We bought the Cell Counting Kit-8 (CCK-8) kit (Nanjing, China) from Vazyme Corporation. IL-1β was obtained from Novoprotein Scientific Inc. (Shanghai, China). RIPA lysate was recruited from Beyotime (Shanghai, China). Primary antibodies against Col-II (Abcam, ab34712), SOX9 (Abcam, ab185230), MMP13 (Abcam, ab39012), CH25H (Abcam, ab133933), CYP7B1 (Abcam, ab138497), ABCA1 (Abcam, ab18180), PI3K (Abcam, ab140307), phospho-Akt (Abcam ab1926623), phospho-mTOR (Abcam, ab109268), NF-κB p65 (Abcam, ab16502), phospho-IκBα (Abcam, ab133462), second antibody goat anti-rabbit (Abcam, ab6721), and second antibody goat anti-mouse (Abcam, ab6789) were purchased from Abcam. β-actin (Abclonal, AC026) was from Abclonal.
2.2 Cell Culture and Treatment
SW1353 cells (Chinese academy of sciences cell bank, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) supplemented with 10% FBS (Gibco, Australia), 100 units/ml penicillin and 100μg/ml streptomycin and incubated at 37 °C with 5% CO2. To assess the effects of HSYA on cell viability, SW1353 cells were cultured in 96 wells overnight and then treated with increasing amounts of HSYA (0, 20, 40, 60, 80, 100, 200μmol/L) for 48h. Next, 10 μl of CCK-8 reagent was added to each well for further 2 h incubation. IL-1β was used to induce the OA cell model in vitro. After the cells were cultured in 96 wells overnight, we treated the cells with different doses of IL-1β (0, 2, 4, 6, 8, 10 μmol/L) to choose the optimum concentration. For the protective effect of HSYA, IL-1β (6 μmol/L) was added to SW1353 cells for 48h, and before this, 20, 40, and 60μmol/L of HSYA was pre-treated for 2h. And then treated with CCK-8 reagent incubating for 2 h. The absorbance value was assessed by a microplate reader (BioTek Epoch, USA) at 450nm. The test was repeated three times.
2.3 Flow cytometry
We measured the effects of HSYA on cell apoptosis by Flow cytometry. We divided the cells into control group, IL-1β group, HSYA (40μmol/L) group, and IL-1β+HSYA group. The cells were pre-treated as part of the protective effect of HSYA, and then cells were digested with trypsin. After centrifuging and resuspending in the buffer, we stained the cells with AnnexinV-FITC/PI kit (Invitrogen, USA). The data were collected by flow cytometer (BECKMAN, USA).
2.4 ELISA for conditioned medium
The levels of CRP, TNF-α, and IL-6 in the conditioned medium (collected from cultural supernatant) were measured with CRP ELISA kits (ZC-31853, ZCi Bio, China), TNF-α ELISA kits (ZC-35733, ZCi Bio, China), and IL-6 ELISA kits (ZC-32446, ZCi Bio, China). Experimental procedures followed the manufacturer's instructions.
2.5 Western blot analysis
RIPA buffer containing 0.1% protease inhibitor was used to homogenize cell samples, and the lysates from cells were centrifuged at 12,000 rpm for 15 min at 4 °C, and then collected supernatants for protein detection. Measuring the total protein concentrations with a BCA kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein (20μg per lane for cell samples) were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto PVDF membranes. Then blocking the membranes in TBS-T (20mM Tris-HCl pH 7.6, 150mM NaCl, 0.1% Tween-20) at room temperature for 2 h containing 5% skimmed milk or 5% BSA. After washing in TBS-T three times, incubating the immunoblots with the following primary antibodies (3% BSA dilution)including Col-II, SOX9, MMP13, CH25H, CYP7B1, ABCA1, APOA-1, PI3K, p-Akt, p-mTOR, NF-κB p65, p-IκBα (Details of dilution were in the regents part) overnight at 4°C, respectively. Then using secondary antibodies (1% BSA dilution) to incubate the membranes for 2 h at room temperature. The membranes were washed with TBST again and then detected with ECL. Quantifying the Band sizes by Scion Image 4.0 software (Scion Corporation, Frederick, MD, USA). Sample loading was normalized relative to β-actin as a reference standard. The final results are shown as "fold changes" in comparison with the control group.
2.6 Statistical analyses
We used GraphPad Prism v.6.0 (GraphPad Software, Inc.) to analyze the data and generate the charts in this experiment. All data are presented as the mean ± standard deviation, and all tests were performed three times. Unpaired, two-tailed Student's t-test statistically analyzed differences between the two groups. Differences among the three groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's post‑hoc test. For all experiments, P < 0.05 was considered statistically significant.