Inflammatory responses elicit a host defense for outer stimuli, including bacterial components and invasion of pathogens trigger sequential innate immune responses. The main immune cells that act in innate immune responses include neutrophils, dendritic cells and macrophages, whose inflammatory properties involve phagocytic action, antigen presentation and inflammatory mediator production (
The ethnopharmacological anti-inflammatory effects of
A methanol extract (cat. no. KRIB0043997) of the
RAW 264.7 macrophages (ATCC, Manassas, VA, USA), a mouse monocytic cell line, were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (both GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in humidified air containing 5% CO2. Rabbit anti-inhibitor of κBα (IκBα; sc-371) and anti-GAPDH (sc-25778) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit anti-inducible NO synthase (iNOS; 2982), anti-cyclooxygenase (COX)-2 (4842), anti-p-IκBα (Ser32/36; 9246), anti-p38 (9212), anti-p-p38 (Thr180/Tyr182; 9211), anti-extracellular signal-regulated kinase (ERK; 9102), anti-c-Jun N-terminal kinase (JNK; 9252), anti-p-JNK (Thr183/Tyr185; 9251), anti-transforming growth factor beta-activated kinase 1 (TAK1; 4505), anti-p-TAK1 (Thr184/187; 4508) antibodies, and mouse anti-p-ERK (Thr202/Tyr204; 9106) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Goat anti-rabbit IgG (LF-SA8002) and goat anti-mouse IgG (LF-SA8001) were purchased from AbFrontier Co., Ltd. (Seoul, Korea). DMSO was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Ez-cytox solution was purchased from Daeil Lab Service Co., Ltd. (Seoul, Korea). Ready-SET-Go! ELISA kits for the detection of IL-6 (88–7064) and tumor necrosis factor (TNF)-α (88–7324) were from eBioscience (San Diego, CA, USA). The PGE2 ELISA kit (510410) was from Cayman Chemical Company (Ann Arbor, MI, USA). Accuzol reagent was from Bioneer Corporation (Daejeon, Korea) and TOPscript cDNA synthesis kit was from Enzynomics Co., Ltd. (Daejeon, Korea). The iTaq Universal SYBR-Green Supermix was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).
RAW 264.7 macrophages were pretreated with MSA (50, 100, 200, 300 and 400 µg/ml) for 2 h and further incubated for 24 h at 37°C in the absence or presence of LPS (1 µg/ml). Following incubation, Ez-cytox solution (1/10 dilution of culture medium) was added to each well and incubated for 1 h. Supernatants were transferred to new 96-well plates and the absorbance was measured at 450 nm using Synergy H1 Microplate reader (BioTek Instruments, Inc., Winnoski, VT, USA).
Cells were seeded at 96-well plates (4.0×104 cells/well) and incubated at 37°C overnight. Cells were pretreated with various concentrations of MSA (50, 100, 200 and 300 µg/ml) for 2 h prior to LPS treatment. Following stimulation with LPS (1 µg/ml) for 24 h, the supernatants (100 µl) were transferred to new 96-well plate and 100 µl Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid) was added to each well. NaNO2 solution (2.5, 5, 10, 25, 50 and 100 M) was used to generate standard curve for calculating the quantity of NO in supernatants. The absorbance was measured at 540 nm using Synergy H1 Microplate reader (BioTek Instruments, Winooski, VT, USA).
Cells were seeded at 96-well plates (4.0×104 cells/well) and incubated at 37°C overnight. Cells were pretreated with various concentrations of MSA (50, 100, 200 and 300 µg/ml) for 2 h prior to LPS treatment. Following stimulation with LPS (1 µg/ml) for 24 h, the supernatants were collected and diluted according to predetermined dilution rate for each proinflammatory cytokines. The production of proinflammatory cytokines, including IL-6 and TNF-α, was measured using Ready-SET-Go! ELISA kits for each cytokines according to manufacturer's protocol. Briefly, the 96-well plate was coated with coating solution for overnight at 4°C, washed with 1X phosphate-buffered saline/0.05% Tween 20 (PBST) for 3 times, and treated with 1X Assay Diluent (from the ELISA kit) for 1 h at room temperature. Following the emptying of the wells, diluted supernatants and standard solutions were added to each well. At 2 h after treatment at RT, the plate was washed with 1X PBST for 3 times and detection Ab solution (also from the ELISA kit) diluted in 1X Assay Diluent was added to plate. The plate was washed following a 1 h treatment, horseradish peroxidase-streptavidin solution was added for 30 min, and washed with 1X PBST 5 times. A solution of 3,3′,5,5′-Tetramethylbenzidine was added to the plate and incubated for 10 min at the dark. An additional 1 N H3PO4 was added to the plate to stop the reaction and absorbance of each well was measured using Synergy H1 Microplate reader at 450 nm.
The production of PGE2 was measured using PGE2 ELISA kit according to manufacturer's protocol. Briefly, 96-well plate pre-coated with goat anti-mouse IgG was incubated with tracer, antibody and either standards or samples for 16 h. Then, the plate was washed with supplied washing buffer 5 times to remove unbound reagents and developed with Ellman's reagent for 1 h. The absorbance of each well was measured at 405 nm and the obtained values were analyzed by performing 4-parameter logistic fit.
RAW 264.7 macrophages were seeded in a 12-well plate (8×105 cells/well) and incubated at 37°C overnight. Cells were pretreated with MSA (50, 100, 200 and 300 µg/ml) for 2 h and additionally stimulated with LPS (1 µg/ml) for 3 h. Total RNA was prepared from the cells using Accuzol (Bioneer Corporation) and reverse-transcribed into cDNA using a TOPscript cDNA synthesis kit, according to the manufacturer's protocol.
PCR amplification of the cDNA was performed using iTaq Universal SYBR-Green Supermix according to the manufacturer's protocol. The PCR was run for 40 cycles of denaturation at 94°C for 5 sec and annealing/extension at 60°C for 30 sec using a CFX Connect real-time thermal cycler (Bio-Rad Laboratories, Inc.). Based on the 2−ΔΔCq method (
PCR primers used in these experiments were listed in a previous report of the authors (
RAW 264.7 macrophages pretreated with MSA were further stimulated with LPS (1 µg/ml) for the optimized time for the detection of target proteins (IκBα and TAK1 for 3 min; MAPKs for 15 min; iNOS and COX-2 for 24 h). Following stimulation for the indicated times, cells were washed 3 times with ice-cold PBS. Lysis buffer, containing 0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF and 1 mM Na3VO4, was added to the washed wells and then collected in each microtube after 10 min. Following centrifugation at 15,814 × g for 30 min at 4°C, the supernatants were prepared in new microtubes.
Protein concentration was measured using the Bradford reagent (Bio-Rad Laboratories, Inc.). Briefly, optical density was measured at 595 nm and the concentration of each lysate was calculated by applying the values to the bovine serum albumin standard plot. After boiling the mixture of lysates and sample buffers, aliquots of the samples (20 µg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes with transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH 8.8), and 20% methanol (v/v)]. Following blocking with 5% non-fat dried milk, each membrane was incubated overnight at 4°C with primary antibodies (1:1,000 dilution for all primary antibodies). Each membrane was incubated for an additional 1 h with secondary peroxidase-conjugated IgG (1:5,000) at room temperature. After washing 5 times with 1X PBST, the target proteins were detected using Pierce ECL Western Blotting Substrate for enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). Protein levels were quantified by scanning the immunoblots and analyzing them with LabWorks software version 4.6 (UVP, LLC; Analytik Jena AG, Upland, CA, USA).
The data are represented as means ± standard error of the mean. Comparisons between multiple experimental groups were performed using one-way analysis of variance followed by Dunnett's post-hoc test using GraphPad Prism (version 3.0; GraphPad Software, Inc., La Jolla, CA, USA) and P<0.01 was considered statistically significant. The data from nine replicates were analyzed, including three independent experiments with three replicates in each.
Since the inhibitory effect of an anti-inflammatory reagent should be assessed under non-cytotoxic concentrations, the authors determined the maximal effective and non-cytotoxic concentration with a cell viability assay. MSA did not induce cytotoxicity in concentrations up to 300 µg/ml. However, clear cytotoxicity was observed at 400 µg/ml for both groups with LPS (1 µg/ml) and without LPS, when compared to their respective 0 µg/ml MSA groups (P<0.05;
Following this, the effect of MSA was measured on the mRNA and protein expression levels of iNOS, an NO-synthesizing enzyme, and COX-2, a responsible enzyme for the production of PGE2, to investigate the transcriptional regulation of proinflammatory mediators. As presented in
Since excessive production of inflammatory mediators, including IL-1β, IL-6, and TNF-α as well as NO and PGE2, in macrophages is accompanied by severe inflammation (
The major regulatory signaling pathways for the production of inflammatory mediators are NF-κB and MAPK. To assess whether MSA regulates NF-κB activation, MSA-mediated IκBα phosphorylation levels and total IκBα levels were measured in LPS-treated RAW 264.7 cells. The p-IκBα levels were reduced by MSA in a dose-dependent manner and IκBα levels was increased by the MSA treatment (
Further investigations were conducted to elucidate the action point of MSA in LPS-treated RAW 264.7 cells. Since MSA inhibits both NF-κB and MAPKs, the authors investigated the regulation of TAK1 phosphorylation level by MSA treatment in LPS-stimulated RAW 264.7 cells. As demonstrated in
The production of all of inflammatory mediators measured in the current study was inhibited by MSA treatment whereas TNF-α was not significant. Therefore, the authors assumed that the major inflammatory signaling pathways, including NF-κB and MAPKs, are involved in the MSA-mediated regulation of the production of inflammatory mediators, although each inflammatory mediator has different regulatory factors for those activation. LPS-induced NF-κB activation in macrophages is primarily mediated by IκBα phosphorylation at Ser-32/36, followed by IκBα degradation and the translocation of released cytoplasmic p50 and p65 complex to the nucleus (
LPS binding to toll-like receptor 4 leads to the recruitment of accessory molecules including myeloid differentiation primary response 88, interleukin-1 receptor-associated kinase 1 and TNF receptor associated factor 6, and this complex formation induces phosphorylation and activation of transforming growth factor β-activated kinase 1 (TAK1) (
Regulatory mechanisms of many natural extracts for their anti-inflammatory effects are primarily focused on the regulation of NF-κB and MAPK signaling pathways. However, the targeted inflammatory mediators and action points of each extract are relatively selective when compared to the inhibitory properties of single compounds. Based on many studies for the anti-inflammatory properties by natural extracts, the selective inhibitory effects are due to multiple components that show anti-inflammatory effects. A recent report has revealed that mulberry fruit extract inhibits acute colitis by selective inhibition of the NF-κB and ERK pathways (
In conclusion, MSA exhibits anti-inflammatory properties by inhibiting the production of various inflammatory mediators through the regulation of both NF-κB and MAPK signaling pathways. Although more studies are required to elucidate concise action mechanism for the anti-inflammatory effects of MSA, these results suggested that MSA may be a valuable candidate as an alternative medicine for the treatment of severe inflammation states.
The present research was supported by the National Research Foundation of Korea grant funded by the Ministry of Science, ICT & Future Planning (grant no. NRF-2015R1A2A2A11001446) and by a Chung-Ang University Research Scholarship grant.
Effects of MSA on cell viability and the production of NO. (A) MSA-pretreated (50, 100, 200, 300 and 400 µg/ml) RAW 264.7 macrophages were incubated for 24 h in the presence or absence of LPS. Cell viability of each group was compared with that of the LPS-treated or untreated control group. Data were represented as mean ± standard error of the mean and analyzed using one-way analysis of variance. *P<0.01 vs. LPS-untreated or-treated control groups. (B) RAW 264.7 macrophages were pretreated with MSA (50, 100, 200 and 300 µg/ml) and then incubated with LPS (1 µg/ml). Following 24 h stimulation, NO and PGE2 levels in the supernatants were measured. Relative production of NO and PGE2 levels to LPS-treated group (100%) by MSA was represented as bar graphs. Data were represented as mean ± standard error of the mean and analyzed using one-way analysis of variance. *P<0.0001 vs. LPS-untreated control groups. aP<0.01, bP<0.001 and cP<0.0001 vs. LPS-treated groups. MSA,
Inhibitory effects of MSA on the production of mRNA and protein expression levels of iNOS and COX-2. RAW 264.7 macrophages were pretreated with MSA (50, 100, 200 and 300 µg/ml) and then incubated with LPS (1 µg/ml) for the indicated times. (A)
Inhibitory effect of MSA on the production of proinflammatory cytokines. RAW 264.7 macrophages were pretreated with MSA (50, 100, 200 and 300 µg/ml) and then incubated with LPS (1 µg/ml) for the indicated times. Following 24 h stimulation, an ELISA was used to measure levels of (A) TNF-α and (B) IL-6. The production of each cytokine was determined using a standard curve. Data were represented as mean ± standard error of the mean and analyzed using one-way analysis of variance. *P<0.0001 vs. LPS-untreated control groups. aP<0.01 and cP<0.0001 vs. LPS-treated groups. (C and D) At 3 h following stimulation, total RNA was extracted and reverse transcribed to cDNA. (C)
Inhibitory effects of MSA on NF-κB and MAPK. RAW 264.7 macrophages were pretreated with various concentrations of MSA (50, 100, 200 and 300 µg/ml) for 2 h and then incubated with LPS for 3 min (for detection of IκBα and TAK1) or 15 min (MAPKs). Total cell lysates were prepared and subjected to immunoblot analyses. The expression levels of (A) p-IκBα, IκBα, (B) p-JNK, JNK, p-ERK, ERK, p-p38, p38, (C) p-TAK1 and TAK1 were detected using specific antibodies. Relative expression levels of IκBα and p-IκBα were normalized to GAPDH levels. Levels of phosphorylated MAPKs and TAK1 were normalized to the corresponding MAPK and TAK1 levels. Quantitative analyses of phosphorylation and protein levels are shown as bar graphs following normalization. Data are represented as mean ± standard error of the mean and analyzed using one-way analysis of variance. *P<0.0001 vs. LPS-untreated control groups. aP<0.01 and bP<0.001 vs. LPS-treated groups. MSA,