Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin solution, Trypsin-EDTA (0.05%) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc.). Antibodies specific for β-actin (cat. no. sc-47778) and TLR-4 (cat. no. sc-293072) and secondary antibodies [goat anti-mouse (cat. no. sc-516102) and anti-rabbit (cat. no. sc-2357)] were obtained from Santa Cruz Biotechnology, Inc. The anti-IL-6 (cat. no. ab6672) and Tata-binding protein (TBP; cat. no. ab63766) antibodies were purchased from Abcam, and phosphorylated (p)-Jak2 (cat. no. 3776s), Jak2 (cat. no. 3230s), p-STAT3 (cat. no. 9145) and STAT3 (cat. no. 9139) antibodies were purchased from Cell Signaling Technology, Inc. NTS was donated by Nara Bio Co., Ltd. LPS (cat. no. L4391), and the STAT3 inhibitor 2-hydroxy-4-methylphenyl sulfonyl oxy acetyl amino-benzoic acid (cat. no. S3I-201) and TLR-4 inhibitor (TLR4-C34; cat. no. SML0832) were purchased from Sigma-Aldrich (Merck KGaA).

C2C12 myoblasts (cat. no. CRL-1772; American Type Culture Collection) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in 5% CO₂. For each experiment, cells at 70–80% confluency cells were gently washed twice with phosphate-buffered saline (PBS). Unless otherwise specified, cells were treated with 0.2 µg/ml NTS in dimethyl sulfoxide (DMSO; cat. no. D8418; Sigma-Aldrich; Merck KGaA) for 24 h at 37°C.

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; cat. no. M2128; Sigma-Aldrich; Merck KGaA) assay. Briefly, cells were re-suspended in DMEM 1 day prior to drug treatment at a density of 1×104 cells/well in 24-well culture plates. The next day, the culture medium was replaced with fresh medium containing DMEM (vehicle control) or different concentrations of NTS (0.1–2 µg), followed by incubation for 24 h at 37°C. Following this treatment, MTT (5 mg/ml) was added and the culture plates were incubated at 37°C for 4 h. The resulting formazan product was dissolved in DMSO and absorbances were measured at 560 nm using the Ultra Multifunctional Microplate Reader (Tecan US, Inc.). Cell viability was determined from these readings using the formula % viability = (fluorescence value of MSM/fluorescence value of non-treated control) ×100. All measurements were performed in triplicate and experiments were repeated at least three times. Cell viability was confirmed by WST-1 (Roche Diagnostics GmbH) assay. Briefly, cells were re-suspended in DMEM 1 day prior to drug treatment at a density of 4×103 cells/well in 96-well culture plates. The next day, the culture medium was replaced with fresh medium containing DMEM (vehicle control) or different concentrations of NTS (0.1–2 µg) and cells were incubated for 24 h at 37°C. Following this treatment, 10 µl/well cell proliferation Reagent WST-1 was added and the culture dishes were incubated at 37°C for 4 h. Absorbances were measured at 460 nm using an Ultra Multifunctional Microplate Reader (Tecan US, Inc.). Cell viability was determined from these readings using the formula % viability = (fluorescence value of MSM/fluorescence value of non-treated control) ×100. All measurements were performed in triplicate and experiments were repeated at least three times.

Fluorescein-conjugated Annexin V (Annexin V-FITC) was used to measure the apoptosis in C2C12 cells. The NTS or LPS treated cells were washed with PBS and re-suspended in a binding buffer at a concentration of 1×106 cells. Then, the cells were stained with Annexin V-FITC and PI for 10 min in the dark at room temperature. The percentage of apoptotic cells was measured by flow cytometry using a FACSCalibur flow cytometer (Bio-Rad Laboratories, Inc.) and the analysis was performed using FlowJo software v10 (FlowJo LLC). Apoptotic rate was calculated using live cells vs. dead cells, which included early apoptosis, late apoptosis or necrosis.

Whole cell lysates were prepared from untreated or NTS-treated C2C12 myoblasts by incubating the cells on ice with radioimmunoprecipitation lysis buffer (cat. no. 20-188; EMD Millipore) containing phosphatase and protease inhibitors. Cells were lysed by aspirating through a 23-gauge needle and the lysate was centrifuged at 18,300 × g for 10 min at 4°C to remove cellular debris. Protein concentrations were measured using the Bradford method (Thermo Fisher Scientific, Inc.). Equal amounts of protein (100 µg per lane) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the separated proteins were transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skimmed milk (cat. no. 90002-594; BD Biosciences) in TBS with Tween-20 (TBST) buffer [20 mM Tris-HCl (cat. no. 10708976001; Sigma-Aldrich; Merck KGaA), pH 7.6, 137 mM NaCl (Formedium Ltd.) and 0.1X Tween-20 (cat. no. 0777; Scientific Sales, Inc.)]. Membranes were then probed overnight at 4°C with the indicated primary antibodies (1:1,000; anti-TLR-4, anti-IL-6, anti-p-Jak2, anti-Jak2, anti-p-STAT3, anti-STAT3 and anti-β-actin) diluted in 5% bovine serum albumin (BSA; EMD Millipore) or 5% skimmed milk (Difco™ skim milk; BD Biosciences). Membranes were then washed with TBST and incubated for 1–1.5 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000). Detection was performed using the Enhanced Chemiluminescence Plus detection kit (Cytiva) and LAS-4000 imaging device (Version 1.0; Fujifilm Corporation). Blots were stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Inc.). Densitometry was performed using ImageJ software (version 1.8.0_172; National Institutes of Health).

Total RNA was extracted from the cells using the RNeasy Mini kit (Qiagen GmbH) according to the manufacturer's instructions. RNA was spectrophotometrically quantified at 260 nm. Subsequently, RT-semi-quantitative PCR analyses were performed to detect TLR-4, IL-6 and GAPDH RNA expression. Briefly, cDNA was synthesized from total RNA by incubating the samples at 42°C for 1 h and then at 95°C for 5 min using a First-Strand cDNA Synthesis kit (cat. no. K-2041; Bioneer Corporation) and oligod(T) primers. The RT-PCR Premix kit (cat. no. K-2016; Bioneer Corporation) was used to amplify TLR-4, IL-6 and GAPDH with primers synthesized by Bioneer Corporation. To generate a 359-bp TLR-4 fragment, the following primers were used: TLR-4 sense, 5′-GCTTTCACCTCTGCCTTCAC-3′ and antisense, 5′-CGAGGCTTTTCCATCCAATA-3′. To generate a 396-bp IL-6 fragment, the following primers were used: IL-6 sense, 5′-AGCCCTGAGAAAGGAGACAT-3′ and antisense, 5′-CTGCGCAGAATGAGATGAGT-3′. Then, a 320-bp GAPDH mRNA fragment was generated with the following primers: GAPDH sense, 5′-AAGGCCATCACCATCTTCCA-3′ and antisense, 5′-ACGATGCCAAAGTGGTCATG-3′. The PCR conditions for TLR-4 were as follows: 95°C for 5 min, 32 cycles of 95°C for 60 sec, 58°C for 60 sec and 72°C for 60 sec and 72°C for 10 min. The PCR conditions for IL-6 and GAPDH were as follows: 95°C for 5 min, 32 cycles of 95°C for 60 sec, 60°C for 60 sec and 72°C for 60 sec and 72°C for 10 min. PCR products were resolved via electrophoresis on a 2% agarose gel and visualized with ethidium bromide (cat. no. E7637; Sigma-Aldrich; Merck KGaA) staining.

After cultured cells were washed with cold PBS, cell pellets were isolated by centrifugation at 14,000 × g for 5 min at 4°C and incubated with 10% BSA on ice for 20 min. Then, cells (3×106) were stained with the following antibodies (1:200) on ice for 30 min: PE CD282 (TLR4; cat. no. 312806; BioLegend, Inc.). Stained cells were washed with pre-cold PBS and flow cytometric analysis was performed using the FACSCalibur flow cytometer (BD Bioscience) and analysis was performed using FlowJo software v10 (FlowJo LLC).

The ChIP assay was performed using the Imprint ChIP kit (cat. no. CHP1; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, C2C12 myoblasts were fixed by adding 1% formaldehyde to media containing cells immediately and quenched with 1.25 M glycine at room temperature. Following washing with PBS, the cells were suspended in nuclei preparation buffer and shearing buffer (provided in ChIP kit) and sonicated under optimized conditions (20 sec on and off for 15 min with 25% amplitude on ice). The sheared DNA was then centrifuged at 16,000 × g for 10 min at 4°C and the cleared supernatant was used for protein/DNA immunoprecipitation. The clarified supernatant was diluted with buffer (1:1 ratio) and 5-µl aliquots of the diluted samples were used as internal controls. Then, the diluted supernatant was incubated with anti-STAT3 antibody (1:1,000) in pre-coated wells for 90 min at 4°C. The controls were incubated with normal goat IgG (1:1,000) and then with anti-RNA polymerase II for 90 min at 4°C. The unbound DNA was washed with immunoprecipitation wash buffer and the bound DNA was collected by the cross-link reversal method using a DNA release buffer containing proteinase K. The released DNA and DNA from the internal control were purified using the GenElute Binding Column G (Sigma-Aldrich; Merck KGaA). The DNA was then quantified via RT-qPCR. The primer sequences were: IL-6 sense, 5′-GACTGAGCCTAAGGGTGCAT-3′ and antisense, 5′-ACCACTAGAGGGCCAAGTCA-3′.

Nuclear protein extracts were isolated using a nuclear extract kit (cat. no. AY2002; Panomics, Inc.). Briefly, cells were washed with PBS and buffer A containing DTT, Protease Inhibitor, Phosphates Inhibitor I and Phosphates Inhibitor II were added and incubated in ice on a rocking platform at 200 rpm for 10 min. The samples were transferred to micro centrifuge tubes and the supernatant removed by centrifugation at 14,000 × g for 3 min at 4°C. Then buffer B containing DTT, Protease Inhibitor, Phosphates Inhibitor I and Phosphates Inhibitor II were added and incubated on ice for 1 h. The nuclear extract was collected by taking the supernatant by centrifugation at 14,000 × g for 5 min at 4°C and western blotting was performed as described above for further analysis.

ELISA was performed for the quantitative detection of IL-6 using a mouse IL-6 ELISA kit (cat. no. ab100713; Abcam). C2C12 cells were treated with NTS for 24 h and with 100 ng/ml LPS for 4 h and the spent media were used for the assay. The samples were added to anti-mouse IL-6-coated microwells along with sample diluent and a biotin-conjugate solution. Following incubation, streptavidin-HRP was added and the plates were further incubated for 1 h in a shaker at room temperature. Then, 3,3′,5,5′-tetramethylbenzidine solution was added after washing. Finally, a stop solution was added once the high-concentrated standard turned a dark blue color. The absorbance was read with a microplate reader at 450 nm and calculations were performed according to the assay protocol.

All experiments were performed at least three times. Results are expressed as the mean ± standard error of the mean. Statistical analyses were conducted using the one-way analysis of variance (ANOVA) or unpaired Student's t-test. One-way ANOVA was followed by Tukey's post hoc test. Analyses were performed using the SAS 9.3 program (SAS Institute, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To examine whether NTS exhibits a protective effect on cell viability, the MTT assay (Fig. 1A) and WST-1 assay (Fig. 1B) were performed. It was found that LPS induced ~20% of cell death during a treatment period of 24 h. To determine the optimum concentration of NTS, cells were treated with 0.1, 0.2, 0.5, 1 and 2 µg/ml NTS in the presence of 100 ng/ml LPS. A non-significant increase in cell numbers with 0.1, 0.2 and 0.5 µg/ml NTS was observed, whereas 1 and 2 µg/ml NTS decreased cell viability. In order to confirm the cyto protective effect of NTS, flow cytometry analysis was conducted in C2C12 myoblasts (Fig. 1C). The results confirmed an increase in cell proliferation compared with LPS-treated cells (Fig. 1D). The pro-apoptotic factor BAX protein was also analyzed and this confirmed the cyto protective effect of NTS against LPS-induced cell death (Fig. S1). From these results, 0.2 and 0.5 µg/ml NTS were selected for further studies.

From the cell viability assay, the optimal concentrations of NTS were determined to be 0.2 and 0.5 µg/ml. Subsequently, it was determined whether NTS can inhibit LPS-induced expression of the inflammatory receptor TLR-4 and pro-inflammatory cytokine IL-6. C2C12 cells were treated with LPS or NTS and then mRNA was isolated from these cells to analyze the expression of TLR-4 and IL-6 via RT-semi-quantitative PCR (Fig. 2A). The obtained results suggested an increase in the expression of TLR-4 and IL-6 with LPS treatment, which was inhibited by NTS treatment (Fig. 2B). These results suggested the anti-inflammatory effect of NTS.

It was observed that NTS inhibited LPS-induced mRNA expression of TLR-4 and IL-6 mRNA. Therefore, the protein expression of TLR-4 and JAK2/STAT3/IL-6 was measured with western blotting. The obtained results demonstrated an increase in the expression of TLR-4 and JAK2/STAT3/IL-6 in LPS-treated cells, which was downregulated by 0.5 µg/ml NTS (Fig. 3A) without affecting the expression of total JAK2 and STAT3. To determine the role of TLR-4 and STAT3 in the anti-inflammatory effect of NTS, cells were treated with a TLR-4 inhibitor (TLR4-C34) or STAT3 inhibitor S3I-201 (100 µM). It was observed that the TLR-4 inhibitor demonstrated a similar significant pattern to NTS; by contrast, the STAT3 inhibitor inhibited the expression of p-STAT3 and IL-6. These results provided evidence of the role of TLR-4 and STAT3 in the anti-inflammatory activity of NTS (Fig. 3B). The ratio of phosphorylated proteins to their total form also demonstrated similar result to the phosphorylated proteins compared with β-actin (Fig. 3C). In addition, the role of TLR-4 in inflammation was confirmed by examining LPS-induced expression of TLR-4 and its inhibition by NTS treatment via flow cytometry analysis (Fig. 3D).

The present study demonstrated that NTS inhibited LPS-induced expression of TLR-4, JAK2, STAT3 and IL-6. It was hypothesized that STAT3 would translocate from the cytoplasm to the nucleus to bind to the promoters of pro-inflammatory cytokines in LPS-induced inflammation. Therefore, C2C12 cells were treated with or without LPS or NTS for 24 h and nuclear proteins were isolated to analyze the expression of STAT3 via western blotting. The expression of p-STAT3 increased in LPS-treated cells, whereas total STAT3 levels remained unchanged in non-treated control cells (Fig. 4A). NTS inhibited the expression of p-STAT3 without affecting the expression levels of STAT3, indicating the possible inhibition of the nuclear translocation of STAT3 by NTS (Fig. 4B). The ratio of nuclear p-STAT3 protein to total nuclear STAT3 protein also demonstrated a similar result to the p-STAT3 expression compared with TBP (Fig. 4C). It was hypothesized that nuclear p-STAT3 may bind to the IL-6 promoter. To confirm this, the expression of the STAT3-IL-6 complex was analyzed by ChIP assay (Fig. 4D). The result demonstrated a significant increase in LPS-induced expression of the STAT3-IL-6 complex, which was downregulated by 0.5 µg/ml NTS (Fig. 4E).

The inhibition of the STAT3-IL-6 complex by NTS was demonstrated in LPS-induced inflammation. The present study assessed whether NTS could inhibit IL-6 release into the media. C2C12 cells were treated with or without LPS or NTS and the media was collected to conduct a mouse IL-6 ELISA. The result demonstrated a significant increase in LPS-induced IL-6 expression, whereas NTS decreased IL-6 expression (Fig. 5). A similar pattern was observed in the STAT3 inhibitor-treated group. These results demonstrated that NTS exhibited an anti-inflammatory effect by regulating STAT3. Taken together, NTS inhibited LPS-induced inflammation by inhibiting LPS-induced expression of TLR-4, JAK2, STAT3 and IL-6. It also inhibited the expression of IL-6 mRNA to induce its anti-inflammatory effects (Fig. 6).