Following approval from the Institutional Ethics Committee Board of The First Affiliated Hospital of Jinan University (Guangzhou, China) and obtainment of written informed consent, cartilage was obtained from five female patients (64±6 years old) with OA who had undergone total knee joint replacement surgery at the First Affiliated Hospital of Jinan University between May and July 2014. The preparation of chondrocytes from cartilage was performed as previously described (14). The full-thickness articular cartilage was removed from the underlying bone and homogenized. Following enzymatic digestion with 0.25% trypsin for 30 min, the specimens were digested with 0.25 mg/ml collagenase I (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) overnight at 37°C. The articular chondrocytes were cultured in DMEM containing 10% FBS, penicillin (100 units/ml; Sigma-Aldrich; Merck KGaA) and streptomycin (100 µg/ml; Sigma-Aldrich; Merck KGaA) at 37°C in a humidified atmosphere containing 5% CO₂. Chondrocytes were used for further experiments when they had reached 80–90% confluence, and passage 2–4 chondrocytes were used.

Cell viability was evaluated using an MTT assay. Human OA chondrocytes at a density of 1×10⁴ cells/well were pretreated with 10, 50 or 100 µg/ml ISH (Sigma-Aldrich; Merck KGaA) for 2 or 48 h at 37°C. Chondrocytes were subsequently incubated with or without 10 ng/ml IL-1β to stimulate for 24 h. The medium was removed, and 20 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to each well and cultured for an additional 4 h at 37°C. Following removal of the supernatant, 150 µl dimethyl sulfoxide was added to each well. The absorbance of each well was determined at a wavelength of 570 nm using a Dynatech MR5000 plate reader (Dynatech Laboratories, Inc., Chantilly, VA, USA).

Total RNA was isolated from chondrocytes using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A total of 2 µg total RNA was reverse-transcribed using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The qPCR analysis was performed using an ABI PRISM 7700 Sequence Detector System and the SYBR-Green PCR Master Mix kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The sequences of the human-specific primers were: Sense, 5′-TTTCCAAGACACACTTCACCA-3′ and antisense, 5′-ATCTCCTTTGTTACCGCTTCC-3′ iNOS; sense, 5′-GAGAGATGTATCCTCCCACAGTCA-3′ and antisense, 5′-GACCAGGCACCAGACCAAAG-3′ for COX-2; and sense, 5′-ATGACAACTCCCTCAAGAT-3′ and antisense, 5′-GATCCACAACGGATACATT-3′ for GADPH. The PCR cycling program was as follows: 95°C for 3 min, followed by 35 cycles of 95°C for 20 sec, 59°C for 20 sec and 72°C for 20 sec, and a final extension at 72°C for 5 min. The data obtained were analyzed using the 2^−ΔΔCq method (15).

Proteins were extracted from chondrocytes using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and the protein concentration was quantified using the Bradford assay. Equal amounts of protein (30 µg) were separated using SDS-PAGE on a 10% gel and blotted onto a nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, UK). Following blocking with 2% non-fat milk in TBS with 0.1% Tween-20 (TBST) at room temperature for 1 h, the blots were incubated with primary antibodies, including anti-iNOS (SAB4502012; Sigma-Aldrich; Merck KGaA), anti-COX2 (sc-19999), anti-phosphorylated p65 (sc-135769), anti-IκBα (sc-1643) and anti-GAPDH (sc-365062) (all 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. The membranes were subsequently incubated with the horseradish peroxidase-conjugated secondary antibody goat anti-rabbit immunoglobulin G (1:5,000; sc-2004; Santa Cruz Biotechnology, Inc.) or goat-anti-mouse immunoglobulin G (1:5,000; sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (GE Healthcare Life Sciences). Quantification was performed using Gel-Pro Analyzer software (version 4.0; Media Cybernetics, Inc., Rockville, MD, USA).

The concentrations of stromelysin-1 (MMP-3; BMS2014-3), collagenase 3 (MMP-13; EHMMP13) and PGE2 (KHL1701) in the culture medium were measured using ELISA kits (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The nitrite levels in the cell culture supernatant were measured using the Griess reaction, as previously described (16).

Data was analyzed using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Experimental data are presented as the mean ± standard deviation of triplicate independent samples. One-way analysis of variance followed by Dunnett's multiple comparisons post hoc test and Student's unpaired t-tests were used for the statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

Cell viability was evaluated following treatment with ISH using an MTT assay. The results demonstrated that there were no significant differences in absorbance among the treated groups, indicating that ISH exerted no cytotoxic effects on chondrocytes at the assayed concentrations (Fig. 1A).

The protective effect of ISH against IL-1β-induced cytotoxicity was investigated. As presented in Fig. 1B, treatment with IL-1β significantly decreased cell viability, and this effect was reversed by the addition of ISH at concentrations of 10, 50 or 100 µg/ml.

As MMPs serve important roles in the development and progression of OA, the effects of ISH on the production of MMP-13 and MMP-13 in IL-1β-treated human OA chondrocytes were investigated. As presented in Fig. 2, IL-1β significantly promoted the production of MMP-3 and MMP-13 in human OA chondrocytes compared with the control group. However, treatment with ISH significantly inhibited the increased production of MMP-3 and MMP-13 which had been induced by IL-1β.

The effects of ISH on the expression of iNOS and COX-2 in IL-1β-treated human OA chondrocytes were investigated. The results of the RT-qPCR analysis demonstrated that IL-1β induced a significant upregulation of iNOS and COX-2 in IL-1β-stimulated human OA chondrocytes compared with the control group. ISH significantly suppressed the mRNA expression of iNOS and COX-2 in IL-1β-stimulated human OA chondrocytes (Fig. 3A and B). Western blot analysis demonstrated that ISH suppressed the protein expression levels of iNOS and COX-2 in IL-1β-stimulated human OA chondrocytes (Fig. 3C).

The effect of ISH on NO and PGE2 production in human OA chondrocytes induced by IL-1β was analyzed, following 24 h of incubation. The results of the present study demonstrated that treatment with IL-1β induced nitrite accumulation, which was significantly attenuated by ISH (Fig. 4A). Similarly, ISH significantly suppressed the IL-1β-induced production of PGE2 in human OA chondrocytes (Fig. 4B).

In order to further analyze the effect of ISH on the NF-κB signaling pathway, western blotting was performed to analyze the effect of ISH on the expression of p65 and IκBα in IL-1β-stimulated human OA chondrocytes. As presented in Fig. 5, ISH suppressed the activation of p65 and the degradation of IκBα, induced by IL-1β, in human OA chondrocytes.