Paeoniflorin (purity ≥98%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant IL-1β was purchased from PeproTech (Rocky Hill, NJ, USA). Dulbecco's modified Eagles medium (DMEM), penicillin and streptomycin, fetal bovine serum (FBS), 0.05% trypsin, and collagenase II were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Rat articular chondrocytes for primary culture were obtained from the tibial plateau and femoral condyle of a 4-week-old Sprague-Dawley rat (The Animal Center of Zhejiang University, Hangzhou, China). In brief, cartilage was rinsed in phosphate-buffered saline (PBS) three times and finely cut into pieces of 1–3 mm³, digested with 0.2% pronase for 0.5 h, and then digested with 0.1% collagenase for 4 h at 37°C. Cells were cultured in complete DMEM containing antibiotic-antimycotic solution and 10% FBS at 37°C under a humidified 5% CO₂ atmosphere. The medium was replaced every 2 days. The animal experiments performed in the present study were approved by the University of Zhejiang Institutional Animal Care and Use Committee, Hang Zhou, China.

According to our previous MTT assay, paeoniflorin concentrations ranging from 12.5 to 100 µM did not show any significant toxicity to chondrocytes (Fig. 1A). Therefore, concentrations from 25 to 50 µM were used in subsequent experiments. The lactate dehydrogenase (LDH) cytotoxicity assay was performed using the LDH Cytotoxicity Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China) (12). In brief, chondrocytes were cultured in 96-well plates. Cells were pretreated with 25 or 50 µM paeoniflorin for 3 h and then incubated with IL-1β (10 ng/ml) for 24 h. The release of LDH was measured according to the manufacturer's instructions.

Annexin V-fluorescein isothiocyanate (FITC) antibody immunofluorescence combined with propidium iodide (PI)/DNA binding was used to analyze apoptosis. Chondrocytes were pretreated in growth medium supplemented with paeoniflorin for 3 h and then incubated in the absence or presence of rat recombinant IL-1β (10 ng/ml) for 24 h. Next, according to the instructions with the Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology), 1×10⁵ cells were treated with Annexin V-FITC and PI in the provided binding buffer for 0.5 h in the dark at 4°C. Cells were then subjected to flow cytometry at the emission wavelength of 488 nm.

Caspase-3 activity was determined using a Caspase-3 Cellular Activity Assay Kit (Cell Signaling Technology, Inc., Danvers, MA, USA). During the assay, activated caspase-3 cleaves the fluorogenic substrate (N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin [Ac-DEVDAMC]) between DEVD and AMC. Thus, we can determine highly fluorescent AMC concentrations using a fluorescence reader with excitation at 380 nm. Cells were pre-incubated in growth medium supplemented with different concentrations of paeoniflorin for 3 h, and then incubated with rat recombinant IL-1β (10 ng/ml) for 24 h. According to the manufacturer's protocol, chondrocytes were collected and lysed using cell lysis buffer in the presence or absence of 5 µl DEVD-pNA for 1 h at 37°C. Caspase-3 activity was measured at 405 nm on a microplate reader. Experiments were performed in triplicate.

Chondrocytes were incubated in growth medium supplemented with 25 or 50 µM paeoniflorin for 3 h and then incubated in the absence or presence of rat recombinant IL-1β (10 ng/ml) for 24 h. Total RNA was isolated using TRIzol reagent (Sigma-Aldrich). Briefly, 1 µg of total RNA after genomic DNA deletion by DNase I was reverse transcribed in 10 pmol of random hexanucleotide primers (Promega, Madison, WI, USA), 0.5 mM dNTPs, and 200 U of Moloney murine leukemia virus reverse transcriptase (Promega). Then, the Bcl-2 and Bax mRNA levels were quantified by RT-qPCR, using the iQ™ SYBR-Green SuperMix PCR Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol with 5 ng of template cDNA, 45 cycles: 95°C/15 sec, 60°C/15 sec with the primers listed in Table I. Rat-GAPDH (NM_017008) was used as a parallel amplification to normalize the expression data of the targeted genes. The relative gene expression was calculated using the formula: n = 100 × 2 ^{− (ΔCq targeted gene - ΔCq GAPDH)}.

Rat articular chondrocytes were plated onto 6-well plates at a density of 5×10⁴ cells/cm². Then, the cells were treated using the same settings for RT-qPCR. After rinsing with ice-cold PBS, the cells were lysed using cell lysis buffer and boiled at 100°C for 10 min. Western blotting was carried out following our reported protocol. Targeted protein was probed with primary antibodies against Bax (Cell Signaling Technology, Inc.), Bcl-2, protein kinase B (Akt), and phosphorylated Akt (p-Akt; Abcam, Cambridge, UK). After incubation with horse radish peroxidase (HRP)-labeled secondary antibodies, the blots were detected using enhanced chemiluminescent (ECL) substrate and exposure to Kodak X-Omat film.

All experiments were performed in triplicate. Results are expressed as the mean ± standard deviation (SD) of three experiments. The data were evaluated using one-way ANOVA and followed by Dunnett's analysis. Statistical significance was set at P<0.05. The statistical analyses were performed with SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL, USA).

In our previous study of paeoniflorin and chondrocytes (unpublished data), we found that paeoniflorin concentrations from 12.5 to 100 µM caused no significant toxicity to chondrocytes (Fig. 1A). Therefore, in the present study, we used paeoniflorin concentrations from 25 to 50 µM. According to the LDH release assay, IL-1β significantly increased the levels of LDH release. Paeoniflorin (25–50 µM) suppressed the LDH release induced by IL-1β in a dose-dependent manner and showed a protective effect in vitro (Fig. 1B).

We assessed chondrocyte apoptosis by flow cytometric analysis. The percentage of apoptotic chondrocytes was significantly increased in the IL-1β group compared with the controls. When chondrocytes were pretreated with paeoniflorin for 3 h, a decrease in the percentage of apoptotic chondrocytes was observed compared with the IL-1β alone group (Fig. 2A and B).

After treatment of chondrocytes with IL-1β for 24 h, caspase-3 activity increased significantly. However, chondrocytes treated with paeoniflorin exhibited markedly decreased caspase-3 activity caused by IL-1β stimulation (Fig. 3).

Using RT-qPCR (Fig. 4) and western blot analyses (Fig. 5), IL-1β-stimulation alone significantly increased the level of Bax and decreased the level of Bcl-2. Moreover, the production of Bax in the low- and high-dose paeoniflorin pretreated groups was significantly decreased compared with the IL-1β group (P<0.05). However, the transcript and protein levels of Bcl-2 in the paeoniflorin groups were significantly increased compared with the IL-1β group (P<0.05).

In the present study, we also examined Akt and p-Akt to evaluate the involvement of the Akt pathway. As expected, the western blot assay showed that IL-1β-stimulation alone decreased the phosphorylation level of Akt, which was consistent with another study (24286347). Different concentrations of paeoniflorin increased the IL-1β-induced activation of Akt, as shown in Fig. 6 (*P<0.05 vs. IL-1β cells).