Chlorogenic acid (CGA), which is a natural compound found in various plants, has been reported to exert notable anti-inflammatory activities. The present study investigated the effects and underlying mechanism of CGA on interleukin (IL)-1β-induced osteoarthritis (OA) chondrocytes. An
Osteoarthritis (OA) is a common disease of the elderly worldwide, which is characterized by articular cartilage destruction and local inflammation, resulting in pain, disability and a significantly reduced quality of life for the affected individuals (
Previous research has also indicated that IL-1β, or other certain stimuli including tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS), may activate nuclear factor (NF)-κB (
Chlorogenic acid (CGA;
Human SW-1353 chondrosarcoma cells stimulated with IL-1β possess similar properties to the articular chondrocytes of OA (
CGA (purity >98%) was purchased from Beijing Dewei Sodium Biotechnology Co., Ltd. (Beijing, China) and confirmed by high-performance liquid chromatography by the Research and Engineering Center for Natural Medicine, Xi'an Jiaotong University (Xi'an, China). L-15 medium was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum was purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). Trypsin was purchased from Amresco, LLC (Solon, OH, USA). IL-1β was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Ethylenediamine tetra-acetic acid (EDTA) and 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Griess reagent was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kit for human IL-6 (cat. no. D6050) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Antibodies against COX-2 (cat. no. 12375-1-AP), MMP-13 (cat. no. 18165-1-AP), collagen II (cat. no. 15943-1-AP), NF-κB (cat. no. 10745-1-AP) and IκB-α (cat. no. 10268-1-AP) were purchased from ProteinTech, Inc. (Rosemont, IL, USA) Antibodies against phosphorylated (p)-NF-κB (cat. no. 3033) and p-IκB-α (cat. no. 2859) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). PGE2 rabbit polyclonal antibody (cat. no. ab2318) and iNOS rabbit monoclonal antibody (cat. no. ab178945) were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-GAPDH (cat. no. PA1-987), bicinchoninic acid (BCA) protein assay reagent kit and Pierce enhanced chemiluminescent (ECL) Plus Substrate were purchased from Pierce (Thermo Fisher Scientific, Inc.).
Human SW-1353 chondrocytes were purchased from the Shanghai Institute of Cell Biology, the Chinese Academy of Sciences (Shanghai, China) and cultured in L-15 supplemented with 10% fetal bovine serum, 100 U/ml penicillin and, 100 µg/ml streptomycin. Cells were incubated in a humidified incubator in an atmosphere containing 5% CO2 at 37°C. The culture medium was replaced every 3 days.
To evaluate the cytotoxic effects of CGA, cell viability was assessed using the MTT assay. Exponentially growing SW-1353 cells were plated into 96-well plates (2×106 cells/ml) and cultivated overnight. The following day, CGA (31.25, 62.5, 125, 250, 500 or 1,000 µg/ml), or various concentrations of CGA along with IL-1β (10 ng/ml), was added to the cells, which were incubated for 48 h at 37°C. The control cells received an equal amount of dimethyl sulfoxide (DMSO). Following removal of the medium, 180 µl serum-free medium and 20 µl MTT (5 mg/ml) was added to each well, and the plates were incubated at 37°C for 4 h. The supernatants were replaced with 150 µl DMSO to dissolve the crystals, the plates were agitated for 10 min, and the absorbance was measured at 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Survival was calculated according to the formula: Survival=ODtest /ODcontrol, where OD indicates optical density.
NO production was assessed spectrophotometrically via the determination of nitrite concentrations in the cell culture supernatant using Griess reagent. SW-1353 chondrocytes (2×106 cell/ml) were pretreated with various concentrations of CGA (50, 100, 200 or 500 µg/ml) for 24 h, and were subsequently exposed to IL-1β (10 ng/ml) for 24 h at 37°C. After 24 h the supernatants were collected. Each supernatant was mixed with an equal volume of Griess reagent (150 µl) and incubated for 5 min at room temperature. Absorbance was subsequently determined at 540 nm. Sodium nitrite was used to prepare the standard curve.
SW-1353 chondrocytes (2×106 cell/ml) were pretreated with various concentrations of CGA (50, 100, 200 or 500 µg/ml) for 24 h, after which the cells were stimulated with IL-1β (10 ng/ml) for 24 h at 37°C. The amount of IL-6 secreted into the culture medium was measured using a commercially available IL-6-specific ELISA kit according to the manufacturer's protocol.
SW-1353 chondrocytes (2×106 cells/ml) were seeded and cultured in 6-well plates. The cells were pretreated with CGA (50 or 200 µg/ml) for 24 h at 37°C, after which the cells were exposed to IL-1β (10 ng/m) for 30 min. Total proteins were isolated using lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 0.5% NP-40, 1% Triton, 1% protease inhibitor and phosphatase inhibitor cocktail]. Concentrations were determined using a BCA Protein Quantification kit. The proteins (30 µg/sample) were then separated on different percentages of SDS-polyacrylamide gel (6–10%) and were transferred onto polyvinylidene difluoride membranes. Nonspecific sites were blocked with Tris-buffered saline 0.1% Tween-20 (TBST) plus 5% nonfat dry milk for 2 h at room temperature, and the membranes were then incubated with primary antibodies (1:1,000 dilution) in 5% skim milk overnight at 4°C. The blots were subsequently washed three times with TBST, and incubated with horseradish peroxidase-linked secondary antibodies (1:15,000 dilution; cat. no. 31463; Pierce; Thermo Fisher Scientific, Inc.) for 2 h at room temperature, and washed three further times with TBST. Protein bands were detected using a Pierce ECL Plus Substrate. GAPDH band intensity was used as internal control to normalize protein expression levels. The optical density was measured using Quantity One 1-D Analysis software (version 4.4; Bio-Rad Laboratories, Inc.).
All results were presented as the mean ± standard error of the mean of ≥3 independent experiments for each test. Differences among the various groups were assessed by one-way analysis of variance followed by Dunnett's test. Statistical analyses were performed using SPSS v. 12.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
An MTT assay was used to determine the effects of CGA on the cell viability of IL-1β-induced human SW-1353 chondrocytes. The results indicated that CGA was cytotoxic to human SW-1353 chondrocytes at a concentration of 1,000 µg/ml; however, cell viability in cells treated with up to 500 µg/ml CGA was not significantly different from the control cells, with or without IL-1β stimulation (
NO serves a pivotal role in OA progression. The results of the viability assay indicated that treatment with 500 µg/ml CGA for 48 h was not cytotoxic; therefore, the inhibitory effects of CGA (50, 100, 200 and 500 µg/ml) on NO release in stimulated SW-1353 chondrocytes were examined. NO production was significantly enhanced following IL-1β activation for 24 h, and pretreatment with CGA significantly diminished the IL-1β-induced NO release in a concentration-dependent manner (
To evaluate the effects of CGA on IL-6 production in human SW-1353 chondrocytes, IL-6 secretion was examined by ELISA. Human SW-1353 chondrocytes were pretreated with CGA for 24 h and then stimulated with IL-1β for 24 h. IL-6 secretion from human SW-1353 chondrocytes was stimulated by IL-1β treatment, and this was reduced by treatment with CGA (
To evaluate whether CGA affected the levels of COX-2 and PGE2 in IL-1β-induced SW-1353 chondrocytes, the protein expression levels of COX-2 and PGE2 were investigated by western blot analysis (
Protein expression levels of collagen II and MMP-13 were examined using western blot analysis (
The NF-κB signal transduction pathway serves vital roles in cartilage degradation and the inflammatory response. To investigate the effects of CGA-mediated inflammatory response inhibition on IL-1β-stimulated chondrocytes, NF-κB and its inhibitory protein IκBα were detected. Western blot analysis was used to detect the levels of NF-κB p65 phosphorylation and IκB degradation. IL-1β stimulation resulted in the phosphorylation of IκBα and NF-κB p65, whereas CGA reversed this effect (
OA is the most prevalent joint disease, and there are currently no effective treatments to limit disease progression. Inflammation has been recognized as an important process in the pathogenesis of OA. Proinflammatory cytokines, including TNF-α and IL-1, become activated during OA progression, and this induces inflammatory responses and cartilage destruction (
IL-1β is an important proinflammatory cytokine released by activated synoviocytes or chondrocytes, which serves an important role in the progression of OA (
NO is produced from the amino acid L-arginine by the enzymatic action of iNOS, which is induced by inflammatory stimuli, indicating that this proinflammatory mediator could contribute to OA disease (
PGE2 is another key inflammatory mediator; PGE2 is synthesized from arachidonic acid via the stimulation of COX-2 during the inflammatory response. COX-2 has been recognized as an inducible or pathological enzyme that participates in the inflammatory response by facilitating PGE2 generation, and promotes the inflammatory cytokine-induced metabolic imbalance of cartilage proteoglycans, thereby advancing arthritis (
Cartilage destruction is a fundamental disease-progression process that occurs in OA. Therefore, the role CGA serves in abrogating this process was further explored. Chondrocytes are the solitary cellular components of cartilage; under normal conditions, these cells maintain the ECM, and balance anabolic and catabolic metabolism (
NF-κB is a cytokine-induced transcription factor that serves an important role in regulating the expression of various genes, including numerous proinflammatory cytokines, adhesion molecules and proteases in arthritis, such as MMPs, PGE2, NO and IL-6 (
In conclusion, the present study demonstrated the
The present study was supported by the Research Foundation of Xi'an Hong-Hui Hospital (grant no. YJ2014016).
Chemical structure of chlorogenic acid.
Effects of CGA and/or IL-1β on the viability of human SW-1353 chondrocytes. (A) Chondrocytes were treated with CGA (31.25, 62.5, 125, 250, 500, or 1,000 µg/ml) alone for 48 h. (B) Chondrocytes were treated with CGA (31.25, 62.5, 125, 250, 500 or 1,000 µg/ml) and IL-1β (10 ng/ml) for 48 h. Values are expressed as the mean ± standard error of the mean (n=3). **P<0.01; ***P<0.001 vs. the control group. CGA, chlorogenic acid; IL, interleukin.
Effects of CGA on IL-1β-induced NO production in human SW-1353 chondrocytes. Chondrocytes were pretreated with CGA (50, 100, 200 or 500 µg/ml) for 24 h followed by exposure to IL-1β (10 ng/ml) for 24 h. NO production was assessed by the Griess reaction. Values are presented as the mean ± standard error of the mean. ***P<0.05 vs. the control group; ###P<0.001 vs. the IL-1β alone group. CGA, chlorogenic acid; IL, interleukin; NO, nitric oxide.
Effects of CGA on IL-1β-induced protein expression of iNOS in human SW-1353 chondrocytes. Chondrocytes were pretreated with CGA (50 or 200 µg/ml) for 24 h, followed by incubation with IL-1β (10 ng/ml) for 30 min. The expression of iNOS was determined by western blot analysis. The data represents band intensity compared with untreated samples, GAPDH was used for normalization. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the control group; #P<0.05 vs. the IL-1β alone group. CGA, chlorogenic acid; IL, interleukin; iNOS, inducible nitric oxide synthase.
Effects of CGA on the levels of IL-6 from IL-1β-stimulated SW-1353 chondrocytes. Cells were incubated with CGA (50, 100, 200 or 500 µg/ml) for 24 h prior to exposure to IL-1β (10 ng/ml) for 24 h. The levels of IL-6 were measured by ELISA. Data are presented as the mean ± standard error of the mean. ***P<0.05 vs. the control group; ###P<0.001 vs. the IL-1β alone group. CGA, chlorogenic acid; IL, interleukin.
Effects of CGA on the expression of COX-2 and PGE2 from human SW-1353 chondrocytes. Chondrocytes (2×106 cells/ml) were treated with CGA (50 or 200 µg/ml) for 24 h followed by IL-1β (10 ng/ml) treatment for 30 min. The expression levels of (A) COX-2 and (B) PGE2 were determined by western blot analysis. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the control group; #P<0.05 vs. the IL-1β alone group. CGA, chlorogenic acid; IL, interleukin; COX-2, cyclooxygenase 2; PGE2, prostaglandin E2.
Effects of CGA on the expression of collagen II and MMP-13 from IL-1β-stimulated SW-1353 chondrocytes. Chondrocytes (2×106 cells/ml) were treated with CGA (50 or 200 µg/ml) for 24 h, and IL-1β (10 ng/ml) was added for 30 min. The expression levels of (A) collagen II and (B) MMP-13 were analyzed by western blot analysis. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the control group; #P<0.05 vs. the IL-1β alone group. CGA, chlorogenic acid; IL, interleukin; MMP, matrix metalloproteinase.
Effects of CGA on the IκBα/NF-κB signaling pathway from IL-1β-stimulated SW-1353 chondrocytes. Chondrocytes (2×106 cells/ml) were pretreated with CGA (50 or 200 µg/ml) for 24 h, followed by incubation with IL-1β (10 ng/ml) for 30 min. Protein levels of (A) NF-κB p65 and NF-κB p-p65 and (B) IκBα and p-IκBα were examined by western blot analysis. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the control group; #P<0.05 vs. the IL-1β alone group. CGA, chlorogenic acid; IL, interleukin; NF-κB, nuclear factor κB; IκBα, inhibitor-κBα; p, phosphorylated.