RAW264.7 murine macrophage-like cells were obtained from the American Type Culture Collection (Manassas, VA, USA; ref. no. CRL-1589). The cells were cultured in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) and 1% (v/v) penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used at 37°C in a humidified atmosphere of 5% CO₂. CA and LPS from Escherichia coli (Serotype 0111:B4) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). STAT3 inhibitor (STAT3i) S3I-201 was obtained from Sigma-Aldrich; Merck KGaA and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA). Cells were pretreated with 2 mM CA or 50 µM STAT3i prior to stimulation with 100 ng/ml LPS for 24 h.

RAW264.7 cells were plated on a 96-well plate at a density of 1×10⁴ cells/well and were incubated for 24 h at 37°C prior to treatment with CA. Cells were then treated with various concentrations of CA (0, 0.05, 0.1, 0.2, 0.5, 1, 2 mM) or LPS (50, 100 and 200 ng/ml) for 24 h. Following treatment, cell viability was determined using an EZ-Cytox (WST-1 tetrazolium salt) Colorimetric Cell Viability assay kit (Daeil Lab Service Co., Ltd, Seoul, Republic of Korea) according to the manufacturer's protocols. Following incubation of cells with the WST-1 reagent (100 µl/well) diluted 1:20 for 90 min at 37°C, the absorbance at 450 nm was measured using a microplate reader (Infinite M200; Tecan Trading AG., Männedorf, Switzerland). Each experiment was performed in triplicate wells and repeated at least three times.

RAW264.7 cells were plated on a 12-well plate at a density of 2×10⁵ cells/well, and then treated with 2 mM CA or 100 ng/ml LPS for 0 to 4 or 16 h. Total RNA was isolated from cells by using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The quantity and purity of total RNA was determined using a NanoDrop spectrophotometer (NanoDrop Technologies, LLC; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Total RNA (1 µg) was reverse transcribed to cDNA using the Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA) according to manufacturer's protocols. Semi-qRT-PCR was performed to measure the mRNA level of target genes and reference gene (GAPDH) using gene-specific primers (Table I) and the GoTaq DNA Polymerase (Promega Corporation) according to manufacturer's protocols. The Semi-qRT-PCR was performed by initial incubation at 94°C for 5 min followed by 30 cycles of 94°C for 20 sec, 58°C for 30 sec and 72°C for 30 sec, with a final extension step of 72°C for 5 min. The PCR products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide. The signals were quantified by densitometric analysis using Multi-Gauge software version 3.0 (Fujifilm Holdings Corporation). Experiments were performed in triplicate.

RAW264.7 cells plated on a 6-well plate at a density of 3×10⁵ cells/well and then treated with 2 mM CA or 100 ng/ml LPS. Cells were lysed in radioimmunoprecipitation extraction solution containing 1X Halt^™ Phosphatase inhibitor and 1X Halt^™ Protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) for 15 min in an ice bath. The protein concentration of lysates from treated cells was measured using a bicinchoninic protein assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Equal quantities of protein (20 µg) were separated using SDS-PAGE (10–12%) and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Antibodies against the following proteins were employed: iNOS (cat. no. 2977), cyclooxygenase-2 (COX-2; cat. no. 4842), STAT3 (cat. no. 9139), phosphorylated (p)-STAT3 (cat. no. 8336), JAK2 (cat. no. 3230), p-JAK2 (cat. no. 4406), extracellular signal-regulated kinase1/2 (ERK1/2; cat. no. 4695), p-ERK1/2 (cat. no. 4370), p38 (cat. no. 9212), p-p38 (cat. no. 4511), c-Jun NH2-terminal kinase (JNK; cat. no. 9252), p-JNK (cat. no. 4668), nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα; cat. no. 9242), p-IκBα (cat. no. 9246), p65 (cat. no. 4764), p-p65 (cat. no. 3033) all from Cell Signaling Technology, Inc. (Danvers, MA, USA), and lamin A (cat. no. sc-20680) and GAPDH (cat. no. sc-25778) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The primary antibodies were diluted 1:1,000 and incubated with membranes for 15 h at 4°C. Following a final rinsing with Tris-buffered saline-Tween (TBST) each membrane was incubated with 1:1,000 diluted secondary horseradish peroxidase (HRP)-linked anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) immunoglobulin (Ig)G from Cell Signaling Technology, Inc. for 1 h at room temperature. Protein bands were analyzed using an enhanced chemiluminescence detection system (Amersham; GE Healthcare Life Sciences) and the ImageQuant LAS-4000 luminescent image analyzer (Fujifilm Holdings Corporation, Tokyo, Japan) Immunoblots were quantified using Multi-Gauge software version 3.0 (Fujifilm Holdings Corporation). Experiments were performed in triplicate.

RAW264.7 cells were plated on a 12-well plate at a density of 2×10⁵ cells/well and pretreated with CA (1–2 mM) for 2 h followed by stimulation with 100 ng/ml LPS for 24 h. The cell culture medium was then collected and centrifuged at 15,000 × g at 4°C for 7 min. The levels of IL-6, TNF-α, MIP-2 and IL-1β in the culture medium were determined using Quantikine ELISA mouse MIP-2 (cat. no. MM200) and mouse IL-1b kits (cat. no. MLB00C) from R&D Systems Inc. (Minneapolis, MN, USA), and the OptEIA™ kits for mouse interleukin-6 (IL-6; cat. no. 555240) and mouse TNF (cat. no. 558534) from BD Biosciences (San Jose, CA, USA) according to the manufacturer's protocols. The quantity of IL-6, TNF-α, MIP-2 and IL-1β was determined based on the optical density values (read at 450 nm) obtained using a Bio-Rad Model 550 Microplate Reader (Bio-Rad Laboratories, Inc.) and a standard curve. Experiments were performed in triplicate.

RAW264.7 cells were plated on a 12-well plate at a density of 2×10⁵ cells/well and pretreated with varying concentrations of CA (0 to 2 mM) for 2 h followed by stimulation with 100 ng/ml LPS for 24 h. NO synthesis was determined by analyzing the level of nitrite in cell culture supernatants using Griess Reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in 5% phosphoric acid solution) following incubation at room temperature for 30 min. The absorbance was measured at 540 nm and the nitrite concentration was determined using sodium nitrite as a standard. Three replicates were performed for each of the different treatments.

RAW264.7 cells were plated on a 60 mm dish at a density of 2×10⁶ cells and pretreated with CA (2 mM) for 2 h followed by stimulation with 100 ng/ml LPS for 24 h. The cells were washed with phosphate-buffered saline and lysis the pellet cells by centrifugation at 250 × g for 5 min at 4°C. Nuclear protein extracts were prepared using the ProteoJET^™ Cytoplasmic and Nuclear Protein Extraction kit (Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA) according to the manufacturer's protocols.

The results are presented as the mean ± standard deviation. Differences between groups were determined by Student's t-test. The statistical analysis was performed with the SPSS version 15.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

RAW264.7 cells were treated with CA and LPS at a range of different concentrations (CA, 0 to 2 mM; LPS, 0 to 200 ng/ml) for 24 h. As shown in Fig. 1, CA and LPS treatment did not exhibit cytotoxic effects on RAW264.7 cells.

The present study investigated the effects of CA on the expression of iNOS and COX-2 in response to LPS challenge of RAW264.7 cells. The cells were pretreated with varying concentrations of CA for 2 h, and then stimulated with LPS for 16 or 24 h. LPS-induced mRNA and protein expression of iNOS and COX-2 was measured by Semi-qRT-PCR and western blotting. LPS-induced expression of iNOS mRNA and protein was significantly inhibited in a dose-dependent manner by CA pretreatment, whereas COX-2 expression was not (Fig. 2A and B). In addition, LPS-induced NO production was inhibited in a dose-dependent manner by CA pretreatment (Fig. 2C).

The effect of CA on the production of pro-inflammatory mediators was investigated. Cells were pretreated with CA for 2 h, and were then stimulated with LPS for 0, 1, 4 or 24 h. CA pretreatment significantly inhibited the LPS-induced expression of IL-6, TNF-α, MIP-2 and IL-1β at the mRNA and protein levels (Fig. 3A and B).

The expression of pro-inflammatory mediators is regulated by the activation of the MAPKs, NF-κB, JAK2 and STAT3 signaling pathways (17). Therefore, the present study determined the effect of CA on the LPS-induced MAPKs, NF-κB, JAK2 and STAT3 by western blotting. Cells were pretreated with specific concentrations of CA for 2 h, prior to stimulation with LPS for various lengths of time. CA pretreatment resulted in a decrease in the level of p-JAK2 and p-STAT3 (Fig 4A and B), whereas the levels of ERK1/2, JNK, p38, IκBα and p65 remained unchanged (Fig. 4C).

A STAT3i was used to determine whether the STAT3 signaling pathway may be involved in the anti-inflammatory effects of CA. Cells were pretreated with CA or STAT3i for 2 h, and then stimulated with LPS for 3 h. The level of p-STAT3 was determined by western blotting. CA and STAT3i pretreatments significantly inhibited LPS-induced nuclear translocation of p-STAT3 (Fig. 5).

In order to examine the effect of STAT3 signaling on LPS-induced NO and pro-inflammatory mediator expression, cells were pretreated with CA or STAT3i for 2 h, and then stimulated with LPS for 16 or 24 h. STAT3i pretreatment inhibited the LPS-induced expression of iNOS protein, whereas the LPS-induced mRNA and protein expression of COX-2 remained unchanged (Fig. 6). Pretreatment with CA significantly reduced the LPS-induced expression of iNOS mRNA and protein, whereas no effect on COX-2 mRNA and protein was observed (Fig. 6). In addition, STAT3i pretreatment significantly inhibited the LPS-induced expression of NO and IL-1β when compared to LPS treatment alone (Fig. 7A and B); however, the expression levels of IL-6, TNF-α, and MIP-2 remained unaffected (Fig. 7C-E). Pretreatment with CA significantly inhibited LPS-induced expression of NO and all of the pro-inflammatory mediators investigated (Fig. 7). These results suggest that the inhibitory effect of CA on LPS-induced NO and IL-1β expression may be mediated by inhibiting the STAT3 signaling pathway.

CA inhibited LPS-induced JAK2 and STAT3 phosphorylation, the nuclear translocation of p-STAT3 and the expression of NO and IL-1β in RAW264.7 cells (Fig. 8). These results indicate that CA may suppress LPS-induced NO and IL-1β expression by inhibiting JAK2/STAT3 activation in RAW264.7 cells.