Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F-12 (DMEM/F-12) (cat. no. 11330-032), fetal bovine serum (FBS) (cat. no. 26140-079) and antibiotics (cat. no. 15140122) were obtained from Gibco; Thermo Fisher Scientific, Inc. N-acetylcysteine (NAC) (cat. no. A7250), Bay 11-7082 (cat. no. B5556), Parthenolide (cat. no. P0667) BBR (cat. no. B3412; Purity: 99%), LPS from Escherichia coli (cat. no. L2880; serotype 055:B5) and paraformaldehyde (cat. no. 158127) were purchased from Sigma-Aldrich; Merck KGaA. Tris-HCl (cat. no. #161-0798; #161-0799) and pre-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) marker (cat. no. 1610395) were purchased from Bio-Rad Laboratories, Inc. Sodium dodecyl sulfate (cat. no. 18220) was purchased from Affymetrix; Thermo Fisher Scientific, Inc. Antibodies against iNOS (cat. no. sc-651), COX-2 (cat. No. sc-17454), Inhibitor kappa B alpha (Iκ-Bα) (cat. no. sc-371), NF-κB (cat. no. sc-372), β-actin (cat. no. sc-47778), and horseradish peroxidase-conjugated mouse anti-rabbit IgG (cat. no. sc-2357) and goat anti-mouse IgG (cat. no. sc-2005) were purchased from Santa Cruz Biotechnology. Antibodies against pERK (cat. no. #9101), ERK (cat. no. #9102), pJNK (cat. no. #9251), JNK (cat. no. #9252), pP38 (cat. no. #9211), P38 (cat. no. #9212), and Inhibitory kappa B alpha (Iκ-Bα) (cat. no. #2859) were purchased from Cell Signaling Technology, Inc. Easy-blue™ Total RNA extraction kit (cat. no. 17061) was purchased from iNtRON Biotechnology. Enzyme-linked immunosorbent assay (ELISA) kits for anti-mouse IL-1β (cat. no. MAB401), IL-6 (cat. no. MAB406) and TNF-α (cat. no. MAB425) antibodies, and mouse IL-1β (cat. no. BAF401), IL-6 (cat. no. BAF406) and TNF-α (cat. no. BAF425) biotinylated antibodies were purchased from R&D Systems. Nuclear extraction kit (cat. no. 2900) was purchased from EMD Millipore. Trans AM NF-κB activation assay kit (cat. no. 40096) was purchased from Active Motif.

Mouse inner medullary collecting duct (mIMCD-3) cells, the mouse renal epithelial cell line (cat. no. CRL2123), were purchased from the American Type Culture Collection (ATCC). mIMCD-3 cells were routinely cultured in DMEM/F-12 supplemented with 10% FBS and 1% penicillin/streptomycin, and were maintained in a humidified chamber containing 5% CO₂ at 37°C and were used in passages 3–5.

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to detect the cytotoxicity of BBR on mIMCD-3 cells. The cells were seeded in each well of 24-well plates (5×10⁴ cells/well) and incubated with BBR at a concentration of 0.05, 0.1, 0.5, 1 or 10 µM for 24 h. Thereafter, the medium was changed, and cells were incubated with MTT solution (0.5 mg/ml) for 30 min at 37°C. The medium was removed and the purple colored precipitate of formazan was dissolved in 200 µl of dimethyl sulfoxide. Aliquots of the dissolved precipitate were taken in a 96-well plate, in duplicates, and estimated at 540 nm using a micro-ELISA plate reader.

Total RNAs were isolated with Easy-Blue™, and the purity of the samples was confirmed by RNA calculator (Gene Quant Pro, Biochrom). The total RNA extraction kit was used according to the manufacturer's instructions and reverse transcription of RNA (10 µg) to cDNA was performed using the ABI cDNA synthesis kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) (conditions: 37°C for 1 h, followed by 95°C for 5 min). TaqMan quantitative RT-PCR with an ABI StepOne Plus detection system was performed according to the manufacturer's instructions (Applied Biosystems; Thermo Fisher Scientific, Inc.). All qPCR data were normalized to the expression levels of the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT). The thermocycling conditions used for qPCR were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The commercial forward, reverse, and probe oligonucleotide primers for multiplex real-time TaqMan PCR were purchased from ABI (cat. no. 4304437; Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2^−ΔΔCq method was used to determine the relative mRNA expression level (21).

mIMCD-3 cells treated with BBR (0.1, 0.5 or 1 µM) and LPS (5 µg/ml) for 24 h were harvested with cell scraper and washed with PBS by centrifugation for 5 min. Cells were then incubated with the blocking solution for 30 min at RT. Subsequently, cells were incubated with a PE-conjugated anti-mouse TLR4/MD-2 complex antibody (1:250; cat. no. 117605; BioLegend) for 30 min at 4°C. Data were acquired by BD FACS calibur cell analyser and analysed in CellQuest Pro software (BD Biosciences; Thermo Fisher Scientific, Inc.).

The cells were washed with PBS and lysed with lysis buffer (1% cocktail of protease inhibitor and 1% phosphatase inhibitor in 1X RIPA Buffer). Protein concentration was determined by bicinchoninic acid assay. Subsequently, Total cell proteins (20 µg) were then separated by SDS-PAGE on a 10% gel and transferred to a PVDF membrane (cat. no. 10600023; GE Healthcare Life Sciences). The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) with Tween-20 (PBST) for 2 h at room temperature (RT) and washed PBST for 10 min, three times. Then, incubated with primary antibody (1:1,000) at 4°C overnight: iNOS, COX-2, Ik-Ba, b-actin, pERK, ERK, pJNK, JNK, pP38 and P38. After washing three times with PBST, each blot was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or donkey anti-goat IgG secondary antibody for 1 h at RT. The proteins were visualized using an enhanced chemiluminescence detection system (cat. no. RPN2232; Amersham; GE Healthcare). Captures protein bands and quantitative analysis were performed using Quantity One^® software version 4.6.6 (Bio-Rad Laboratories, Inc.).

ELISAs for IL-1β, IL-6, and TNF-α were carried out in duplicates in 96-well plates coated with anti-mouse IL-1β, IL-6, and TNF-α monoclonal antibodies in PBS (pH 7.4) at 4°C overnight. The plates were washed with PBST and blocked with PBS containing 10% FBS for 2 h at RT. After two times washes, standards and samples were added and incubated for 2 h at RT, and then the wells were washed and biotinylated anti-mouse IL-1β, IL-6, or TNF-α were added and incubated for 1 h at RT. The wells were washed, avidin-peroxidase was added, and plates were incubated for 30 min at RT. The wells washing again and followed by the addition of TMB substrate. Color development was measured at 450 nm using an automated microplate ELISA reader. Standard samples were run on each assay plate by using serial dilutions of recombinant IL-1β, IL-6, and TNF-α.

mIMCD-3 cells were plated in a chamber slide and incubated with LPS (5 µg/ml) for 30 min at 37°C. The cells were treated with BBR (1 µM) for 1 h before LPS treatment. The cells were fixed in 4% paraformaldehyde for 15 min at RT and washed 3 times with PBS. The cells were treated with 0.1% TritonX-100 for 15 min at RT. After washing, non-specific binding sites were blocked with serum (3% BSA) for 1 h at RT, and incubated with NF-κB antibody (1:250) at 4°C overnight. The cells were then washed and incubated with AlexaFluor^®568 goat anti-rabbit IgG (1:2,000; cat. no. A-11011) for NF-κB antibody for 2 h at RT in a darkened room. For nuclear staining, the cells were incubated with DAPI (cat. no. H-1200; Vector Laboratories) at 5 µg/ml for 5 min at RT. The slide was finally washed and mounted for microscopic examination. Stained sections were visualized using a confocal laser microscope (Olympus).

mIMCD-3 cells were plated in 100-mm dishes (1×10⁷ cells/dish). Then, LPS (5 µg/ml) treated for 0, 15, 30 and 60 min with or without pretreatment of BBR (1 µM) for 1 h. Nuclear extraction assay performed according to the manufacturer's instructions. Briefly, cells were harvested with cell scraper and washed with PBS by centrifugation at 250 × g for 5 min. Add 1X Cytoplasmic Lysis Buffer (containing DTT and Protease inhibitor cocktail) to the pellet and incubation on ice for 15 min. Centrifuge at 250 × g for 5 min, discard the supernatant and add 1X Cytoplasmic Lysis Buffer. Five times drawing and ejecting by 27-gauge syringe, then centrifuge at 8,000 × g for 20 min. Discard supernatant, and add Nuclear Extraction Buffer (containing DTT and Protease inhibitor cocktail). Five times drawing and ejecting by 27-gauge syringe, then gently agitate on rotator at 4°C for 60 min. Centrifuge at 16,000 × g for 5 min, then supernatant was used for experiments.

NF-κB activation was determined in the nuclear extracts of mIMCD-3 cells, following the manufacturer's instructions. Briefly, 15 µg nuclear extract was added to a biotinylated oligonucleotide containing the NF-κB consensus site attached to the streptavidin-coated 96-well plates with agitation on rocking platform at 100 rpm for 1 h. Plates were washed with wash buffer to remove all the unbound reagents. NF-κB p65 primary antibody (1:1,000) was added for 1 h at room temperature without agitation, followed by a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:1,000) at room temperature without agitation. Subsequent to an incubation for 1 h at room temperature, 100 µl developing solution was added to all wells for 3 min at room temperature protected from direct light. The blue color development in the sample wells was monitored until it turned medium to dark blue. Subsequently, 100 µl stop solution was added and the blue color turned yellow. Finally, the absorbance value was ascertained using a spectrophotometer at a wavelength of 450 nm. As a positive control for NF-κB p65 activation, nuclear extracts from Jurkat cells, provided with the kit, were used.

Results are expressed as the mean ± standard error of the mean (SEM). Statistical significance of intergroup differences was evaluated using two-way ANOVA, with time and dose as variables, followed by a Duncan's post-hoc test. All statistical analyses were performed using SPSS. P<0.05 was considered to indicate a statistically significant difference. All experiments were carried out in triplicates.

Before studying the biological activity of BBR, we performed the MTT assay to evaluate the cytotoxicity of BBR in mIMCD-3 cells. As shown in Fig. 1A, up to a dose of 1 µM, BBR did not affect cell viability. Thus, we chose BBR concentrations of 0.1, 0.5 and 1 µM for further experiments.

To determine the pro-inflammatory effects of BBR against LPS in mIMCD-3 cells, production of pro-inflammatory molecules such as iNOS and COX-2 after LPS stimulation in mIMCD-3 cells was investigated. In accordance with our previous report (2), production of iNOS and COX-2 was elevated in LPS-exposed mIMCD-3 cells. However, the elevation of iNOS and COX-2 was significantly inhibited by BBR in these cells (Fig. 1B-E).

IL-1β, IL-6, and TNF-α are the representative pro-inflammatory cytokines known to be induced by LPS treatment in mIMCD-3 cells (2). Thus, to measure the effects of BBR on the production of these pro-inflammatory cytokines in LPS-exposed mIMCD-3 cells, we investigated the mRNA and protein expression of IL-1β, IL-6, and TNF-α after LPS stimulation. As reported earlier (2), LPS treatment increased the mRNA and protein levels of IL-1β, IL-6, and TNF-α in mIMCD-3 cells. However, BBR treatment inhibited the induction of IL-1β, IL-6, and TNF-α mRNA and protein levels in a dose-dependent manner (Fig. 2).

Since BBR was effective in inhibiting pro-inflammatory mediators in LPS-treated mIMCD-3 cells, we sought to investigate the mechanistic details of the beneficial activity of BBR. Firstly, to rule out the possibility that BBR directly regulates LPS or TLR4, we examined the effect of BBR on TLR4/MD2 complex expression. The methods of Limulus amebocyte lysate (LAL) assay and RT-qPCR are described in Data S1. As shown in Fig. 3A and Fig. S1, BBR did not directly affect LPS and TLR4 expression in mIMCD-3 cells, which means BBR regulates the down-streams upon TLR4-LPS interaction. Thus, we ought to examine the activation of NF-κB and MAPKs, because activation of both signaling pathways produce pro-einflammatory mediators (22). As shown in Fig. 3B-E, LPS stimulation triggered Iκ-Bα degradation, NF-κB p65 translocation from cytosol to nucleus, elevation of NF-κB p65 binding activity, and MAPK phosphorylation. However, treatment with BBR inhibited Iκ-Bα degradation, NF-κB p65 translocation and NF-κB p65 binding activity elevation but not MAPK phosphorylation.

To evaluate whether NF-κB deactivation could contribute to amelioration of inflammatory responses in LPS-treated mIMCD-3 cells, we used three well known NF-κB inhibitors (NAC, Bay 11-7082, and Parthenolide). As is known, three inhibitors could block NF-κB p65 translocation from nucleus to cytosol against LPS treatment in mIMCD-3 cells (Fig. S2). Next, we hypothesized that NF-κB inhibitors might downregulate the production of pro-inflammatory mediators, similar to BBR. As expected, NF-κB inhibitors treatment reduced the mRNA levels of iNOS, COX-2, IL-1β, IL-6, and TNF-α after LPS treatment in mIMCD-3 cells to a similar degree as in the cells treated with BBR (Fig. 4).