A phospho-specific antibody against NF-κB p65 (Ser-536; cat. no. 93H1) was purchased from Cell Signaling Technology, Inc. Antibodies against β-actin (C-11; cat. no. sc-1615) and NF-κB p65 (C-20; cat. no. sc-372) were obtained from Santa Cruz Biotechnology, Inc. Secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit (cat. no. P0448) or anti-mouse (cat. no. P0260) immunoglobulin G (Dako; Agilent Technologies, Inc.). Negative control small interfering (si)RNA and TLR4-siRNA were purchased from Santa Cruz Biotechnology. Inc. Dectin-1 siRNA was purchased from Thermo Fisher Scientific, Inc. Lipofectamine^® RNAiMAX transfection reagent was obtained from Thermo Fisher Scientific, Inc. Normocin, Zeocin and Quanti-Blue were purchased from InvivoGen. Dexamethasone (Dex), an NF-κB inhibitor, was purchased from Wako Pure Chemical Industries, Ltd. Block Ace for western blotting was obtained from DS Pharma Medical Glycyrrhizae radix was obtained from Tochimoto Tenkaido, Co., Ltd.

RAW-blue cells were cultured in DMEM, High Glucose with L-glutamine, phenol red and sodium pyruvate (Wako Pure Chemical Industries, Ltd.) supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, 50 µg/ml streptomycin and 100 µg/ml normocin at 37˚C in a humidified incubator with 5% CO₂.

Boiled herbal water extracts were prepared by gently boiling 100 g Glycyrrhizae radix in 500 ml water for 50 min at 95˚C and then filtering the decoction. The decoction was centrifuged at 3,000 x g for 5 min at 4˚C (Kubota 6800; Kubota Corporation) and the supernatant was collected and then centrifuged at 20,000 x g for 20 min at 4˚C. The supernatant (60 ml) was collected again and ultra-centrifuged at 140,000 x g for 50 min twice at 4˚C, according to a previously described method for preparation of exosomes (8). After removal of the supernatant, the transparent pellet was dispersed in distilled water (40 ml) and freeze-dried.

Negative staining was performed as follows: Collodion mesh (Nisshin EM Co., Ltd.) was placed on a 20 µl droplet of nanoparticle solution (100 µg/ml) for 30 sec and the solution was absorbed with filter paper. Subsequently, the mesh was placed on a droplet of 2% uranyl acetate for 5 sec, which was absorbed with filter paper, and dried. Nanoparticles which were negatively stained were observed using a JSM-6700F SEM (JEOL, Ltd.) operated using PCSEM software version 1 (JEOL, Ltd.) at a calibrated magnification of x50,000.

A total of 0, 1, 1.5, 5 and 10 µg/ml nanoparticles were added to the cell culture medium of RAW-blue cells and incubated for 20 h, where 0 mg/ml was used as the control. The cell culture supernatant was collected and transferred to QUANTI-Blue medium (InvivoGen) and SEAP expression was then measured after 1 h or 90 min at 620 nm using a spectrophotometer.

Whole cell lysates were extracted using lysis buffer (20 mM HEPES-NaOH, 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.001% Triton X-100, 1 mM DTT, 1 mM sodium orthovanadate, 20 mM β-glycerophosphate disodium salt hydrate, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM PMSF). Protein concentrations in lysates were quantified using a Bradford assay, (Bio-Rad Laboratories, Inc.). The lysates were mixed with an equivalent volume of SDS sample buffer (100 mM Tris-HCl, pH 6.8; 2.0% SDS; 70 mM DTT; 10% glycerol; and 0.10% bromophenol blue) and heated at 95˚C for 5 min. Samples were loaded onto a 9% gel, resolved using SDS-PAGE and subsequently transferred onto an Immobilon-P nylon membrane (EMD Millipore). The membrane was blocked using 4% Block Ace overnight at 4˚C, and probed with primary antibodies (NF-κB p65, rabbit anti-phospho NF-κB p65 and β-actin; all at 1:1,000), for 90 min at room temperature. Primary antibodies were detected using the horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000) and visualized using an ECL system (GE Healthcare). The density of the blots was quantified using ImageJ version 1.8.0_172 (National Institutes of Health). Experiments were repeated at least three times and representative results are shown.

Mouse TLR4-siRNA (cat. no. sc-40261), mouse Dectin-1-siRNA (cat. no. sc-63277) and negative control (cat. no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. RAW-blue cells were transfected with siRNAs at a final concentration of 100 nM using Lipofectamine^® RNAiMAX transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 12 h, the medium was replaced with fresh medium and the cells were cultured in the presence of nanoparticles for a further 24 h. TLR4 siRNA (cat. no. sc-40261) is a pool of 3 different siRNA duplexes. sc-40261A sense, CUAGCCUUCUUCAAUCUUAtt and antisense, UAAGAUUGAAGAAGGCUAGtt; sc-40261B sense, CCGUUGGUGUAUCUUUGAAtt and antisense, UUCAAAGAUACACCAACGGtt; and sc-40261C sense, GAAGGCCCAUAUUUGACUAtt and antisense, UAGUCAAAUAUGGGCCUUCtt. Dectin-1 siRNA sequences were as follows: Dectin-1 sense, GACAACUUCCUAUCAAGAAtt and antisense, UUCUUGAUAGGAAGUUGUCtt. The sequences of the negative control siRNAs used were not disclosed by the manufacturer.

Total RNA from the RAW 264.7 cells was extracted using RNeasy Mini kit (Qiagen, Inc.) according to the manufacturer's protocol. First-strand cDNA synthesis was performed using the RNA as the template (2 µg) using oligo(dT)18 primer and SuperScript III reverse transcriptase (Invitrogen; Thermo Fisher Scientific Inc.). Reverse transcription was performed at 42˚C for 50 min and then at 70˚C for 15 min. qPCR amplification was performed using a FastStart Essential DNA Green Master mix (Roche Diagnostics). The thermocycling conditions were: Denaturation at 94˚C for 5 sec, annealing at 60˚C for 5 sec and extension at 72˚C for 10 sec for 28 cycles qPCR was performed using a Lightcycler nano system (Roche Diagnostics) according to the manufacturer's protocol. β-actin was used as the internal control. The relative quantification of mRNA expression was calculated as a ratio of the target gene to β-actin (12). Primer sequences were as follows: TLR4 forward, 5'-GGACTCTGATCATGGCACTG-3' and reverse, 5'-CTGATCCATGCATTGGTAGGT-3'; Dectin-1 forward, 5'-TTGTGTCGCCAAAATGCTAGG-3' and reverse, 5'-CTGATCCATGCATTGGTAGGT-3'; IL-6 forward, 5'-GCTACCAAACTGGATATATAATCAGGA-3' and reverse 5'-GGTCTGGGCCATAGAACTGA-3'; TNF-α forward 5'-TCTTCTCATTCCTGCTTGTTG-3' and reverse, 5'-GGTCTGGGCCATAGAACTGA-3'; and β-actin forward, 5'-CTAAGGCCAACCGTGAAAAG-3' and reverse, 5'-ACCAGAGGCATACAGGGACA-3'.

Data are presented as the mean ± standard deviation of at least 3 independent experiments. Differences between groups were compared using an ANOVA with a post-hoc Tukey-Kramer test. Statistical analyses were performed using JMP Pro software version 13 (SAS Institute). P<0.05 was considered to indicate a statistically significance difference.

Freeze-dried nanoparticles from boiled Glycyrrhizae radix water extracts were visualized using SEM. The diameter of the nanoparticles were 80-100 nm (Fig. 1).

The effects of the nanoparticles on RAW-blue cells were investigated using a reporter gene assay with NF-κB. NF-κB activation in RAW-blue cells was significantly higher when treated with nanoparticles compared with the control, and NF-κB activation was increased in a dose-dependent manner (Fig. 2A). This activation was suppressed by the NF-κB inhibitor Dex (Fig. 2B). Furthermore, nanoparticles induced the phosphorylation of the NF-κB subunit p65 (Fig. 3A and B) and this phosphorylation was suppressed by Dex in the western blotting analysis (Fig. 3C and D).

Based on the activation of NF-κB, cytokine production induced by nanoparticles in RAW-blue cells was examined. The mRNA expression levels of the inflammatory cytokines IL-6 and TNF-α, which are regulated by NF-κB, were increased in RAW-blue cells exposed to nanoparticles compared with the control cells (Fig. 4).

Based on the activation of NF-κB by nanoparticles, the receptors the nanoparticles bound to, to exert their effect in RAW-blue cells, were determined (Fig. 5A). Activation of NF-κB by nanoparticles was significantly suppressed when cells were transfected with TLR4 siRNA, but not by Dectin-1 siRNA. The reduction in SEAP activity incells transfected with si-TLR4 was ~50% (Fig. 5B).

Together, these results suggest that the signaling pathway by which nanoparticles obtained from boiled Glycyrrhizae radix water extracts at least partially involved TLR4, and signal transmission induced the expression of the inflammatory cytokines IL-6 and TNF-α in RAW-blue cells.