CAI was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) as a 40 mM stock solution. Cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.) and the COX inhibitor screening assay kit, sc-560 and DuP697 were purchased from Cayman Chemical Company. Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. LPS (Escherichia coli 055:B5) was obtained from Sigma-Aldrich (Merck KGaA). TRIzol^® reagent and SYBRGreen dye were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The NO assay kit (cat. no. A013-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute. TNF-α (cat. no. EM008), IL-1β (cat. no. EM001) and IL-6 (cat. no. EM004) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai ExCell Biology, Inc.. The TransScript II First-Strand cDNA Synthesis SuperMIX was purchased from Transgen Biotech Co., Ltd. The TransAM NF-κB p65 transcription factor assay kit (cat. no. 40096) and nuclear extract kit (cat. no. 40010) were purchased from Active Motif Inc.. Primary antibodies against inducible nitric oxide synthase (iNOS, cat. no. 13120), phosphorylated (p)-MAPKs (p-MAPK family antibody sampler kit; cat. no. 9910), MAPKs (MAPK family antibody sampler kit; cat. no. 9926), p-IκB (cat. no. 2859), IκB (cat. no. 4814), p-p65 (cat. no. 3033) and p65 (cat. no. 8242) were purchased from Cell Signaling Technology Inc., while anti-β-actin primary antibody (cat. no. sc-47778) and horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. nos. sc-2004 and sc-2005) were purchased from Santa Cruz Biotechnology, Inc.. The chemiluminescent reagent was purchased from EMD Millipore. The immunofluorescence staining kit (cat. no. P0176), containing goat anti-rabbit immunoglobulin G (IgG) conjugated with Alexa Fluor 488 was purchased from Beyotime Institute of Biotechnology. Other chemical reagents including Tris, NaCl, glycerol, Triton X-100, egtazic acid, sodium orthovanadate and sodium fluoride were of analytical grade.

The COX inhibitor screening kit, which can directly detect prostaglandin F_2α (PGF_2α) produced by tin chloride reduction of COX-derived prostaglandin H₂ generated in the COX reaction, was used to evaluate the effects of CAI on COX-1 and COX-2 activity. The assay was performed according to the manufacturer's instructions. Heme and the reaction buffer (Tris-HCl buffer containing 5 mM EDTA and 2 mM phenol, pH 8.0) were added to test tubes. Following addition of CAI and COX-1 or COX-2, the tubes were incubated at 37˚C for 10 min. Subsequently, the substrate (arachidonic acid) was added and the tubes were incubated at 37˚C for a further 2 min. The amount of PGF_2α produced was determined by utilizing ELISA contained within the aforementioned kit. The absorbance was quantified using spectrophotometry at a wavelength of 405 nm and PGF_2α content was determined from a standard curve. CAI was dissolved in DMSO at final concentrations of 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 µM. DMSO alone was placed in the control tubes. Sc-560 and DuP697, COX-1 and COX-2 specific inhibitors, respectively, were used at final concentrations of 20 nM as positive controls for 10 min at 37˚C. All measurements were performed in triplicate.

The RAW264.7 mouse macrophage cell line was provided by the Chinese National Infrastructure of Cell Line Resource. Cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM L-glutamine and 10% fetal bovine serum at 37˚C in a humidified incubator with 5% CO₂.

The CCK-8 assay was used to measure cell viability. In brief, RAW264.7 cells were plated at a density of 2x10⁴ cells/well in a 96-well plate, and either untreated (control), or pretreated with vehicle (0.1% DMSO) or various concentrations of CAI (10, 20 and 40 µM) for 2 h in a 37˚C incubator, and subsequently stimulated for 22 h with 1 µg/ml LPS at 37˚C. CCK-8 solution (20 µl/well) was then added to the cells and incubated for an additional 2 h at 37˚C. Subsequently, the absorbance was determined at a wavelength of 450 nm to measure cell viability.

RAW264.7 cells were cultured at a density of 2x10⁵ cells/well in 24-well plates, and either untreated (control), or pretreated with vehicle (0.1% DMSO) or various concentrations of CAI (10, 20 and 40 µM) for 2 h in a 37˚C incubator, and subsequently stimulated with 1 µg/ml LPS at 37˚C. The culture supernatants were collected after 6 h of LPS stimulation (for detection of cytokine levels) or 24 h (for detection of NO levels) (21). The levels of TNF-α, IL-1β and IL-6 in the supernatants were determined using corresponding ELISA kits, and NO levels were measured using an NO assay kit according to the manufacturer's instructions.

Following either untreated (control), pretreatment with vehicle (0.1% DMSO) or various concentrations of CAI (10, 20 and 40 µM) for 2 h in a 37˚C incubator, RAW264.7 cells were stimulated for 6 h with 1 µg/ml LPS at 37˚C. Total mRNA was extracted from the cells using TRIzol^® reagent and RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, total RNA from each sample was reverse transcribed into cDNA using the TransScript II First-Strand cDNA Synthesis SuperMIX according to the manufacturer's instructions. RT-qPCR was performed using the CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.) and SYBRGreen dye. The sequences of the primers used are shown in Table I. The following thermocycling conditions were used for the qPCR: Initial denaturation at 94˚C for 3 min; 40 cycles of 94˚C for 30 sec, 60˚C for 40 sec, and a final extension at 72˚C for 1 min. mRNA levels were quantified using the 2^-ΔΔCq method (22). β-actin was used as the endogenous control.

Western blot analysis was used to analyze the protein expression levels of corresponding proteins. For the detection of iNOS, cells were either untreated (control), or preincubated with vehicle (0.1% DMSO) or various concentrations of CAI (10, 20 and 40 µM) for 2 h in a 37˚C incubator and subsequently stimulated for 6 h with 1 µg/ml LPS at 37˚C. Since inflammatory pathways, including NF-κB and MAPKs, are activated quickly following LPS stimulation, the time of LPS incubation for analysis of the two pathways was set to 1 h following CAI pretreatment, as previously described (23,24). Briefly, RAW264.7 cells were washed twice with phosphate-buffered saline (PBS) and lysed on ice for 30 min with ice-cold lysis buffer (10 mM Tris, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM egtazic acid, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 1% protease inhibitor mix; pH 7.5), followed by centrifugation for 30 min at 12,000 x g (4˚C). The supernatants were collected, and protein concentrations were determined using a Bradford assay. Equivalent amounts of protein (60 µg/lane) from the supernatant were transferred onto polyvinylidene difluoride membranes following separation on 10% SDS-PAGE. The membranes were subsequently blocked with Tris-buffered saline solution containing 0.1% Tween-20 (TBS-T), supplemented with 5% skimmed milk for 2 h at room temperature. Membranes were then probed overnight (4˚C) with primary antibodies against iNOS (1:500), IκB (1:1,000), p-IκB (1:1,000), p65 (1:1,000); p-p65 (1:1,000), β-actin (1:1,000), p-p38 (1:500), p38 (1:500), p-JNK (1:500), JNK (1:800), p-ERK (1:1,000) and ERK (1:1,000). Following washing with TBS-T three times, the membranes were incubated for 2 h at room temperature with HRP-labeled secondary antibodies at a dilution of 1:5,000. Protein bands were visualized using an enhanced chemiluminescent reagent and the band density was quantified using Kodak 1D software (version 3.5; Kodak). β-actin served as the internal control and total levels of the proteins were used in the evaluation of the phosphorylated forms of the proteins.

RAW264.7 cells were either untreated (control), or preincubated with vehicle (0.1% DMSO) or various concentrations of CAI (10, 20 and 40 µM) for 2 h in a 37˚C incubator and then stimulated for 1 h with 1 µg/ml LPS at 37˚C. Cellular nuclear protein was extracted using a nuclear extraction kit and analyzed using the NF-κB p65 transcription factor assay kit, which uses an ELISA-based assay to determine NF-κB p65 activity. Briefly, the kit contained a 96-well microtiter plate to which an oligonucleotide containing the NF-κB consensus binding site (5'-GGGACTTTCC-3') was immobilized. The nuclear extracts were added to the 96-well plate and incubated for 1 h at room temperature, allowing the active form of p65 contained in the extracts to bind to the oligonucleotide. Following sufficient washing with the wash buffer contained in the kit, anti-NF-κB p65 primary antibody (1:1,000) was added and incubated for 1 h at room temperature. The plate was washed extensively, and subsequently incubated with HRP-conjugated secondary antibody (1:1,000). Finally, tetramethyl benzidine substrate was added for color development and the absorbance was quantified using spectrophotometry at a wavelength of 450 nm.

RAW264.7 cells were cultured on coverslips and either untreated (control), or preincubated with vehicle (0.1% DMSO) or various concentrations of CAI (10, 20 and 40 µM) for 2 h in a 37˚C incubator followed by stimulation for 1 h with 1 µg/ml LPS at 37˚C. Following washing with PBS, cells were fixed for 20 min with 4% paraformaldehyde at room temperature and permeabilized for 10 min using 0.1% Triton X-100 on ice. Subsequently, the cells were treated for 1 h with blocking solution (1% bovine serum albumin) and incubated overnight with a primary antibody against p65 (1:400) at 4˚C. Subsequently, Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (1:1,000) was added to the cells for 30 min at 37˚C. The nuclei were stained with DAPI at room temperature for 5 min. Images were acquired using a Zeiss LSM 780 laser scanning confocal microscope (Zeiss AG) and a Leica DM4000 upright fluorescence microscope (Leica Microsystems GmbH) under x400 magnification. The pixel intensities of p65 in the nuclear area were measured as the percent area of immunoreactivity using ImageJ software (version 1.52u; National Institutes of Health) (25,26).

SPSS software (version 20.0; IBM Corp.) was used for statistical analysis. Data are presented as mean ± SD and were analyzed using one-way ANOVA followed by Bonferroni's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the main drugs used for inflammation-related diseases, which have been known to possess anti-inflammatory action via inhibiting COX activity and numerous arachidonic acid-related pro-inflammatory factors, such as prostaglandins and leukotrienes (27). By contrast, CAI did not affect the enzyme activities of both COX-1 and COX-2, at concentrations ranging from 1.56 to 100 µM. Furthermore, sc-560 (COX-1-specific inhibitor) and DuP697 (COX-2-specific inhibitor), significantly suppressed COX-1 and COX-2 activity, respectively, compared with control cells (Fig. 1). These results suggested that CAI exerted its anti-inflammatory action via a different mechanism to COX-inhibiting NSAIDs.

The anti-inflammatory effects and mechanism of CAI on RAW264.7 macrophages was investigated. To rule out the possibility that decreased cellular inflammatory responses were caused by direct toxicity of CAI to the cells, the potential cytotoxicity of CAI was evaluated using a CCK-8 assay. As shown in Fig. 2, CAI did not affect cell viability at all the concentrations tested compared with controls, which was subsequently found to inhibit LPS-induced inflammatory responses. Therefore, the effect of CAI on RAW264.7 macrophages was not due to cytotoxicity.

The anti-inflammatory action of CAI in macrophages was firstly investigated by determining the levels of NO production following stimulation of RAW 264.7 cells with LPS. NO production significantly increased following LPS stimulation compared with controls. However, cells pretreated with CAI (20 and 40 µM) showed a significant downregulation in NO levels compared with the LPS + DMSO group (Fig. 3A).

Since iNOS is an important NO-generating enzyme (4), western blot analysis was performed to further analyze the protein expression levels of iNOS. iNOS expression was almost undetectable in the absence of LPS but was significantly increased following LPS stimulation compared with controls. However, CAI pretreatment (20 and 40 µM) significantly suppressed the increased protein expression of iNOS in LPS-induced RAW264.7 cells compared with the LPS + DMSO group (Fig. 3B). In addition, LPS-induced mRNA expression levels of iNOS were also significantly inhibited by pretreatment with CAI (20 and 40 µM) compared with the LPS + DMSO group, as shown from the RT-qPCR analysis (Fig. 3C).

The production of pro-inflammatory cytokines was measured using ELISA. Pretreating the cells with LPS alone significantly upregulated the levels of TNF-α, IL-1β and IL-6 in the supernatant compared with controls, while CAI (20 and 40 µM) prior to LPS treatment inhibited the elevated secretion of these cytokines compared with the LPS + DMSO group (Fig. 4). RT-qPCR analysis further confirmed that treatment with LPS increased the mRNA expression levels of the aforementioned cytokines, which were attenuated by CAI at 20 and 40 µM (Fig. 5).

The transcription factor NF-κB is known to be implicated in the regulation of numerous genes that code for the mediators of inflammatory and immune responses (28). To explore the potential mechanism involved in the anti-inflammatory action of CAI in the macrophages, a DNA-binding assay was performed to determine the effects of CAI on LPS-induced NF-κB activation. The DNA-binding activity of NF-κB p65 in nuclear extracts was significantly increased in response to LPS treatment compared with controls, and this increase was significantly reduced by pretreatment with CAI (20 and 40 µM) compared with the LPS + DMSO group (Fig. 6A).

Since the activation of NF-κB is associated with the sequential transduction cascade, including IκBα phosphorylation and degradation, and translocation of activated NF-κB, which is facilitated by the phosphorylation of the p65 subunit (28), protein extracts of RAW264.7 cells were probed for IκBα, p-IκBα, p-65 and p-p65. The results showed that the phosphorylation of IκBα and p65 in RAW264.7 cells increased after LPS stimulation compared with controls, but was significantly inhibited by CAI pretreatment (10, 20 and 40 µM) (Fig. 6B).

NF-κB nuclear translocation is considered as a hallmark of activation, therefore it was subsequently visualized using immunofluorescence of p65. The expression level of p65 was low in unstimulated cells. Following LPS stimulation, the overlapping of p65-Alexa Fluor 488 green fluorescence with DAPI blue staining was increased, suggesting the enhanced nuclear expression of p65. However, p65 staining was weakened in the nucleus and mainly located in the cytoplasm by pretreatment with CAI (10, 20 and 40 µM), which indicated the inhibitory nuclear translocation of p65 (Figs. 7 and S1, S2). These findings indicated that CAI could inhibit NF-κB activation in LPS-stimulated RAW264.7 cells.

MAPKs play critical roles in the induction of pro-inflammatory gene expression (29). Moreover, the activation of NF-κB is regulated by p38, JNK and ERK (8,30). To further characterize the mechanism underlying the effects of CAI, the MAPK signaling pathway was examined. The phosphorylation of p38, JNK and ERK was significantly increased in RAW264.7 cells stimulated with LPS compared with controls; however, CAI treatment significantly reduced protein levels compared with the LPS + DMSO groups (Fig. 8). However, the total protein expression levels of p38, JNK and ERK were not altered following LPS stimulation or treatment with CAI and LPS. The aforementioned results suggested that CAI inactivated the MAPK signaling pathway to exert its inhibition on the inflammatory responses in macrophages stimulated by LPS.