Human OA chondrocytes were obtained from the Department of Anatomy & Neurobiology, Northeast Ohio Medical University (Ohio, USA) and cultured in RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂. Cells were treated with IL-1β (2.0 mg/ml, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and/or hesperidin (2.0 mg/ml), NF-κB inhibitor (NF-κBIR; cat. no. LY-294.002; 2.0 mg/ml, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or PBS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 24 h at 37°C. The groups are as follows: PBS, IL-1β, NF-κBIR, hesperidin and IL-1β+hesperidin.

Total RNA from human OA chondrocytes (1×10⁸ cells) was extracted using an RNAeasy Mini kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA) following the manufacturer's protocol. RNA was reverse transcribed into cDNA at 42°C for 2 h using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Expression levels of mRNAs in cells were measured by qPCR. Forward and reverse primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.; Table I). PCR amplification started with a preliminary denaturation step at 94°C for 2 min, followed by 45 cycles of 95°C for 30 sec, 56°C for 30 sec (annealing) and 72°C for 45 sec. The reaction volume was 20 µl, containing 50 ng genomic cDNA, 200 µM dNTPs, 200 µM primers, 2.5 U Taq DNA polymerase and 2.5 U SYBR-Green (Thermo Fisher Scientific, Inc.). Relative mRNA expression changes were calculated by the 2^−ΔΔCq method (21). Results are presented as the n-fold change compared with β-actin.

Human OA chondrocytes (1×10³/well) were seeded in 96-well plates following treatment with IL-1β (2.0 mg/ml) and/or hesperidin (2.0 mg/ml; IL-1β, IL-1β+hesperidin and hesperidin groups) for 24 h at 37°C, and treated with 10 µl MTT (5 mg/ml, Sigma-Aldrich; Merck KGaA) for 3 h at 37°C. Following incubation, purple formazan crystals were dissolved using isopropanol (15 µl). The absorbance was recorded using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Inc.) at 570 nm. The effects of hesperidin on cell viability were determined by the percent of cell viability, calculated as the ratio between mean absorbance of three samples and mean absorbance of the IL-1β group.

Human OA chondrocytes (1×10⁶/well, untreated cells) were cultured in six-well plates for 12 h at 37°C. The NF-κB overexpression plasmid was synthesized by cloning human NF-κB cDNA into plasmid pcDNA3.1 (pNF-κB; Suzhou GenePharma Co., Ltd., Suzhou, China) and the empty plasmid pcDNA3.1 served as the control (pControl). Cells were transfected pNF-κB (100 pmol) or pControl (100 pmol) using Lipofectamine 3000 reagent (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following 48 h transfection, NF-κB-overexpressed cells were treated with hesperidin (2.0 mg/ml) for 24 h at 37°C further analysis.

After 48 h transfection, NF-kB-overexpressed cells, treated with hesperidin or PBS, were used to detect NO and PGE2 production. Production of NO was assessed by measuring the nitrite content in the culture supernatant was obtained via centrifugation at 4,000 × g at room temperature for 10 min following mixing of the culture with Griess reagent (1% sulphanilamide, 0.1% N−1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid). The absorbance was measured at 540 nm following 10 min of incubation at 37°C. The levels of PGE2 were analyzed using the Prostaglandin E2 Parameter Assay kit (KGE004B, Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions.

After 48 h transfection, NF-kB-overexpressed human OA chondrocytes (1×10⁷) treated with hesperidin or PBS were lysed using radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of proteins (10 µg) were separated using 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Merck KGaA). Following blocking with 5% bovine serum albumin for 2 h at 37°C, membranes were incubated with primary antibodies for 12 h at 4°C: IL-1β (1:1,000; ab200478; Abcam, Cambridge, UK), IL-17A (1:1,000; ab193955; Abcam), IL-6 (1:1,000; ab7737; Abcam), IL-10 (1:1,000; ab19969; Abcam), COX-2 (1:1,000; ab15191; Abcam), PGE-2 (1:1,000; ab181249; Abcam), inducible nitric oxide synthase (iNOS; 1:1,000; ab15323; Abcam), MMP-3 (1:1,000; ab53015; Abcam), MMP-13 (1:1,000; ab39012; Abcam) and β-actin (1:2,000; ab8226; Abcam). Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000; PV-6001; OriGene Technologies, Inc., Beijing, China) was incubated with the membrane for 24 h at 4°C. Membranes were washed with PBS and visualized with an electrochemiluminescence western blotting detection reagent (Amersham; GE Healthcare, Chicago, IL, USA). The band intensities were analyzed by ImageJ software 1.0 (National Institutes of Health, Bethesda, MD, USA).

NF-kB-overexpressed with or without hesperidin human OA chondrocytes (1×10⁶/well) were cultured in six-well plates for 12 h at 37°C. The 3′-untranslated region (3′-UTR) sequence of NF-κB was inserted into the pGL3 control vector (Promega Corporation, Madison, WI, USA). The construct was referred to as NF-κB-3′-UTR. Cells were washed with PBS and transfected with NF-κB-3′-UTR or control (NF-κB-3′-mimic; Promega Corporation) using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were collected using centrifugation in 4,000 × g at room temperature for 15 min after 48 h at 37°C and Renilla luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocols. Results were obtained from three independent experiments performed in duplicate and normalized to Renilla.

Data are expressed as the mean ± standard deviation and statistical analysis was performed with SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Statistically significant differences between groups were analyzed by one-way analysis of variance followed by Tukey's honest significant difference test. P<0.01 was considered to indicate a statistically significant difference.

Anti-inflammatory effects of IL-1β on human OA chondrocytes were investigated in vitro. As illustrated in Fig. 1A, IL-1β significantly decreased the viability of human OA chondrocytes after 24 h compared with the control (P<0.01). IL-1β significantly increased iNOS, COX-2, MMP-3 and MMP-13 gene and protein expression in human OA chondrocytes compared with the PBS-treated cells (P<0.01; Fig. 1B and C). These results indicated that IL-1β stimulated inflammation in human OA chondrocytes.

Effects of hesperidin on inflammation in IL-1β-stimulated human OA chondrocytes were investigated in vitro. Hesperidin significantly increased the viability of human OA chondrocytes compared with the PBS-treated group (P<0.01; Fig. 2A). Hesperidin treatment significantly decreased IL-1β and IL-17A gene and protein expression (P<0.01; Fig. 2B and C) and significantly increased IL-6 and IL-10 gene and protein expression in IL-1β-stimulated human OA chondrocytes compared with the PBS-treated cells (P<0.01; Fig. 2D and E). These results indicated that hesperidin may attenuate inflammation in IL-1β-stimulated human OA chondrocytes.

Hesperidin effects on NO, PGE2, iNOS and COX-2 expression in IL-1β-stimulated human OA chondrocytes were further investigated. It was revealed that hesperidin significantly decreased NO and PGE2 production (P<0.01; Fig. 3A and B) and significantly inhibited iNOS and COX-2 gene and protein expression in IL-1β-stimulated OA chondrocytes compared with the PBS-treated cells (P<0.01; Fig. 3C and D). Hesperidin further significantly inhibited IL-1β-induced MMP-3 and MMP-13 release in human OA chondrocytes compared with the control (PBS-treated cells; P<0.01; Fig. 3E and F). These results suggested that hesperidin may inhibit the IL-1β-induced NO and MMP signaling pathways in human OA chondrocytes.

The potential mechanism mediated by hesperidin was analyzed in human OA chondrocytes. It was observed that hesperidin significantly decreased the activity of NF-κB in IL-1β-stimulated human OA chondrocytes compared with the PBS-treated cells (P<0.01; Fig. 4A). Additionally, NF-κBIR treatment significantly decreased iNOS, COX-2, MMP-3 and MMP-13 gene and protein expression in IL-1β-stimulated human OA chondrocytes compared with the PBS-treated cells (P<0.01; Fig. 4B and C). NF-κB overexpression, induced by transfection with pNF-κB, significantly increased iNOS, COX-2, MMP-3 and MMP-13 gene and protein expression in IL-1β-stimulated human OA chondrocytes compared with the pControl (P<0.01; Fig. 4D and E). The results also demonstrated that NF-κB overexpression reversed hesperidin-inhibited iNOS, COX-2, MMP-3 and MMP-13 gene and protein expression in IL-1β-stimulated human OA chondrocytes compared with the PControl group (Fig. 4F and G). These results indicated that hesperidin inhibited IL-1β-stimulated inflammation by the inhibiting NF-κB activity in human OA chondrocytes.