The present study was approved by the Institutional Care and Use Committee, Experimental Animal Centre of Jinzhou Medical University (Jinzhou, China). A total of 200 male C57BL/6J mice (6–8 weeks old; 20–25 g weight) were obtained from our animal center and housed in standard Plexiglas cages under a controlled temperature (21–25°C) and 50% humidity with food and water available ad libitum under a 12 h light/dark cycle with lights on at 6:00 a.m. Food supply was restricted 3 days prior to the experiments to achieve a target weight of 85% their expected weight under conditions of unrestricted food access.

To isolate and purify mouse peritoneal macrophages, the mice were euthanized and 70% ethanol was subsequently sprayed onto the abdomen. The outer layer of the peritoneum was incised using scissors, and ice-cold RPMI-1640 (cat. no. SH30809.01; HyClone; Thermo Fisher Scientific, Inc., Logan, UT, USA) was subsequently injected into the peritoneal cavity using a 5 ml syringe. Subsequent to gently massaging the peritoneum to dislodge any attached cells into the RPMI-1640 medium, the fluid from the peritoneum was collected into a tube using a 5 ml syringe, kept on ice, and centrifuged at 250 × g at 4°C for 5 min. The supernatant was discarded prior to resuspension of cells in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; cat. no. FSP500; ExCell Bio, Shanghai, China), 100 U/ml penicillin and 3.7 g/l NaHCO₃, and counted using a hemocytometer. Cells were then added into 6-well tissue culture plates at a density of 1×10⁶ cells/well and cultured for 2 h at 37°C to ensure their adherence to the substrate; non-adherent cells were removed by gently washing with warm PBS three times. In total, 90% pure macrophages were collected for the experiments following a previous study (25).

Macrophages were seeded in 6-well plates at a density of 5×10⁵ cells/well or 12-well plates at a density of 2×10⁵ cells/well, cultured overnight and subsequently exposed to different conditions of external stimulations and cultured for 24 h at 37°C in a cell culture incubator with 95% air and 5% CO₂: i) Control group (normal peritoneal macrophages without any treatment); ii) LPS group (normal peritoneal macrophages treated with 500 ng/ml LPS); iii) 8MXF/LPS group (normal peritoneal macrophages treated with 8 µg/ml MXF and 500 ng/ml LPS for 2 h); iv) 16MXF/LPS group (normal peritoneal macrophages treated with 16 µg/ml MXF and 500 ng/ml LPS for 2 h); v) 32MXF/LPS group (normal peritoneal macrophages treated with 32 µg/ml MXF and 500 ng/ml LPS for 2 h; and vi) 64MXF/LPS group (normal peritoneal macrophages treated with 64 µg/ml MXF and 500 ng/ml LPS for 2 h). Subsequently, total cellular RNA and protein were extracted from these treated macrophages for RT-qPCR and western blotting, or cells were fixed in 4% formaldehyde at the room temperature for 20 min prior to immunostaining.

Macrophages were seeded in 96-well culture plates at a density of 5×10³ cells/well and cultured at 37°C for 24 h, prior to the addition of various doses of MXF (0, 50, 100, 200, 400, 800 and 1,600 µg/ml) for 24 h. MTT solution was added to each well to reach a final concentration of 5 mg/ml. After a 2-h incubation at 37°C, the culture medium was replaced with 200 µl dimethyl sulfoxide to solubilize the formazan crystals produced by MTT. The absorbance was measured at 490 nm with a spectrophotometer and the percentage of viable cells was calculated. The experiment was set to five replicates and repeated at least three times. The growth inhibition was calculated by the equation: % cell viability=(ODc-ODt)/(ODc-OD_blank) ×100; where ODt and ODc are the optical densities in treated cultures and control cultures, respectively.

Total RNA was isolated from the treated macrophages using an RNeasy Mini kit (BioTeke Corporation, Beijing, China) and reverse transcribed into cDNA with the M-MuLV Reverse Transcriptase kit (BioTeke Corporation) and incubated at 42°C for 50 min according to the manufacturer's protocols. qPCR was performed in an Exicycler^™ 96 Detection system (Bioneer Corporation, Daejeon, Korea) with 10 µl reaction mixture, containing 5 µl SYBR green Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.5 µl (10 µM) of each forward and reverse primer, and 4 µl cDNA template with the FastStart SYBR Green Reagents kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocol. The qPCR conditions were 95°C for 10 min and 40 cycles of 95°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec and held at 4°C. The primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and the sequences were as follows: SPHK1 forward, 5′-ACGAGCAGGTGACTAATGAAGA-3′ and reverse, 5′-GTGCCCACTGTGAAACGAA-3′; NF-κB forward, 5′-AGCATTAACCTCCTGGAGACG-3′ and reverse, 5′-TTGGGAGCACTGCTTTGGAT-3′; TLR4 forward, 5′-GTAGAGATGAATACCTCCTTAGTGT-3′ and reverse, 5′-TTTTACAGCGACCAATAAGTATCAG-3′; β-actin forward, 5′-CTGTGCCCATCTACGAGGGCTAT-3′ and reverse, 5′-TTTGATGTCACGCACGATTTCC-3′. The relative expression level of mRNA was analyzed using the 2^−∆∆Cq method, where ΔΔCq=ΔCq_treated-ΔCq_control according to a previous study (26).

Treated peritoneal macrophages were washed with ice-cold PBS twice, lysed in ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 2 mM ethylenediaminetetraacetic acid, 50 mM NaF, 10 µg/ml leupeptin, 2 mM Na₃VO₄, 15 µg/ml aprotinin and 1 mM phenylmethane sulfonyl fluoride) on ice for 30 min, homogenized using a vortex and centrifuged 13,000 × g at 4°C for 15 min. The supernatant was transferred into a fresh tube and kept on ice, and the protein concentration was determined with a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein samples (30 µg/lane) were loaded and separated by SDS-PAGE (10% gels), and electrically transferred onto polyvinylidene fluoride membranes at 30 V for 1 h. Following a rinse in tap water, the membranes were blocked in 5% (w/v) fat-free milk at room temperature for 1 h and incubated overnight with primary antibodies against TLR4 (19811-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:500, SPHK1 (10670-1-AP; Proteintech) at a dilution of 1:1,000 and β-actin (CAB340MI22; Cloud-Clone Corp, Atlanta, GA, USA) at a dilution of 1:500, NF-κB (10745-1-AP, Proteintech) at a dilution of 1:500, or PKA (55388-1-AP, Proteintech) at a dilution of 1:500, at 4°C overnight. The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (ZB-2305; OriGene Technologies, Inc., Beijing, China) at a dilution of 1:2,000 at room temperature for 90 min. Following three washes with Tris-based saline-0.1%Tween 20 (T8220; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the blots were visualized with enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and the images were captured using the Kodak Image Station 4000R scanner (Kodak, Rochester, NY, USA). The band intensity of the target proteins was quantified using ImageJ software version 1.5.0 (National Institutes of Health, Bethesda, MD, USA).

Cultured macrophages were fixed in 4% paraformaldehyde at room temperature for 30 min and subsequently incubated with 1% Triton X-100 for 15 min followed by 1 h of blocking in 5% goat serum (Beyotime Biotechnology, Shanghai, China) at the room temperature. Subsequently, the macrophages from different treatment groups were incubated with specific primary antibodies, including mouse anti-TLR4 (1:100; 19811-1-AP; Proteintech), anti-SPHK1 (1:1,000; 10670-1-AP; Proteintech) and anti-NF-κB p65 (1:1,000; 10745-1-AP; Proteintech) at 4°C overnight. Subsequently, macrophages were incubated with Cy3-conjugated secondary anti-mouse antibody (1:2,000; SA00009-1; Proteintech) at them room temperature for 1 h and the macrophages were mounted onto glass slides with mounting medium containing DAPI. Images were captured at 400× magnifications using a Nikon epifluorescence microscope (Nikon Eclipse E800; Nikon Corporation, Tokyo, Japan). Analysis was performed for 30–50 cells from each sample using the Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and a total of 150–500 cells per treatment group were statistically analyzed.

Macrophages were seeded into 24-well culture plates at a density of 1×10⁵ cells/well and subsequently treated as detailed above. The supernatant was collected through centrifugation at 1,000 × g at 4°C for 10 min to assess the expression levels of IL-6 (at a dilution of 1:4, cat. no. SEA079Mu) and TNF-α (cat. no. SEA133Mu) using these ELISA kits according to the manufacturer's protocols (OriGene Technologies, Inc.).

The data were expressed as the mean ± standard error of three repeated experiments and analyzed using one-way analysis of variance followed by Tukey's post-hoc test. All statistical analyses were performed by using Graphpad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

In the present study, a cell viability MTT assay was initially performed to determine cell viability (cytotoxicity) following macrophage treatment with different concentrations of MXF in the presence or absence of 500 ng/ml LPS for 24 h. Higher MXF doses decreased the cell viability and increased the cell inhibition rate (Table I). The IC₅₀ of MXF was 294.8 µg/ml, whereas, IC₅₀ of MXF plus LSP was 281.82 µg/ml (Tables I and II).

To assess the effects of MXF on the inflammatory response of macrophages, LPS was used to induce the inflammatory response. It was observed that LPS significantly induced the expression of TLR4 (P<0.05; Fig. 1A), SPHK1 (P<0.05; Fig. 1B) and NF-κB (P<0.05; Fig. 1C) mRNA, compared with the control group. At doses of 8 and 16 µg/ml, MXF significantly decreased the expression levels of TLR4, SPHK1 and NF-κB mRNA compared with the LPS group (P<0.05), suggesting that MXF had an inhibitory effect on the inflammatory reaction at lower doses. In contrast, the higher MXF doses (32 and 64 µg/ml) increased the expression levels of TLR4 (32 µg/ml, P<0.05; 64 µg/ml, P<0.05), SPHK1 (64 µg/ml; P<0.05) and NF-κB (32 µg/ml, P<0.05; 64 µg/ml, P<0.05) mRNA compared with LPS treatment alone, suggesting that high doses of MXF promoted the inflammatory response, although 32 µg/ml MXF had no significant effect on SPHK1 mRNA expression (P>0.05).

Western blotting (Fig. 2) and immunofluorescence analysis (Fig. 3) was performed to detect alterations in TLR4 (Figs. 2A, 3A and D), SPHK1 (Figs. 2B, 3B and E) and NF-κB p65 (Figs. 2C, 3C and F) following treatment with LPS. Western blotting demonstrated that the protein expression levels of TLR4, SPHK1 and NF-κB p65 increased following treatment with LPS, compared with the control group (P<0.05). MXF treatment at 8 and 16 µg/ml downregulated the expression of TLR4 (P<0.05), SPHK1 (P<0.05) and NF-κB p65 (P<0.05), compared with the LPS alone group. These results suggested that low MXF doses prevented the effects of LPS on the expression of these proteins in intestinal macrophages. However, at doses of 32 µg/ml, MXF had no significant effect on SPHK1 (P>0.05) and NF-κB p65 (P>0.05) protein expression; however, increased TLR4 expression (P<0.05) compared with the LPS alone group. At 64 µg/ml, MXF increased NF-κB p65 (P<0.05) expression; however, had no effect on TLR4 (P>0.05) and SPHK1 (P>0.05) expression, compared with the LPS alone group. These results suggested that MXF promoted the inflammatory response at higher doses, whereas MXF suppressed the inflammatory response at lower doses.

The immunofluorescence experiments demonstrated that the expression of TLR4, SPHK1 and NF-κB p65 was significantly increased by treatment with LPS compared with the control group (P<0.05; Fig. 3). Treatment with MXF at doses of 8 and 16 µg/ml decreased the expression of TLR4 (P<0.05), SPHK1 (P<0.05) and NF-κB p65 (P<0.05) compared with the LPS alone group. However, at doses of 32 and 64 µg/ml, MXF had no significant effects on the expression of TLR4 (P>0.05), SPHK1 (P>0.05) or NF-κB p65 (P>0.05). These results suggested that lower MXF doses inhibited the effects of LPS on the expression of these proteins in intestinal macrophages.

After 24 h of treatment with LPS, the ELISA data demonstrated treatment with LPS resulted in a significant increase of IL-6 and TNF-α expression (P<0.05; Fig. 4) compared with the control group. At doses of 8 and 16 µg/ml, MXF decreased the expression of IL-6 (both P<0.05) and TNF-α (P<0.05 at 8 µg/ml and P<0.05 at 16 µg/ml), compared with the LPS alone group. MXF at 32 µg/ml did not affect the production of IL-6 and TNF-α (P>0.05), whereas, MXF at 64 µg/ml significantly increased the expression levels of IL-6 and TNF-α in macrophages, compared with the LPS alone group (P<0.05). These results supported the effects of MXF on the production of IL-6 and TNF-α by LPS-stimulated intestinal macrophages.

As PKA may mediate the effect of MXF on the regulation of synthesis and secretion of these cytokines (27), PKA protein expression in LPS and MXF-treated macrophages was determined (Fig. 5). It was demonstrated that LPS induced PKA expression, whereas low MXF doses prevented the effects of LPS on PKA expression. Higher MXF doses did not exert inhibitory effects, suggesting that the effects of MXF on the synthesis and secretion of the investigated cytokines may be through PKA suppression in macrophages.