Regulation of NCTD on LPS induced NF-κB activation.

<p>A. Little influence was observed by NCTD on IκBα and MAPK signaling pathway. RAW264.7 cells were treated with PBS (containing 0.1% DMSO) or 1–10 μM NCTD for 24 h and then stimulated with 100 ng/ml LPS for different time. The protein levels of IκBα and MAPK signaling pathway in RAW264.7 cells were determined using western blotting. B. The translocation of p65 to nucleus was increased in NCTD treated macrophages. Cells (2×10⁶ cells/ml) were incubated with PBS (containing 0.1% DMSO) or 1–10 μM NCTD for 24 h and treated with 100 ng/ml LPS for 15 min. Cytoplasmic extracts (CE) and nuclear extract (NE) were prepared and analyzed by western blot analysis using specific antibodies. And the translocation of p65 to nuclear was also quantitatively analyzed. C. LPS induced DNA binding ability of NF-κB was enhanced by NCTD in a dose dependent manner. Cells were treated with PBS (containing 0.1% DMSO) or 1–10 μM NCTD at 37°C for 24 h and then activated with 100 ng/ml LPS for 30 min. Finally, nuclear extracts were prepared and then assayed for NF-κB activation by EMSA as described under Materials and Methods. Three independent experiments were performed. D, Diagram of NCTD facilitates LPS-mediated immune responses by up-regulation of AKT/NF-κB associated signaling pathway.</p

NCTD enhances LPS induced cytokines expression at mRNA level.

<p>A, The cytotoxicity of NCTD is much lower than CTD. RAW264.7 cells were treated with NCTD or CTD for 24 h and cell viability was tested with MTS assay as described under Materials and Methods. B, Cytokines production was enhanced obviously by NCTD in RAW264.7 cells. The RAW264.7 cells were pretreated with PBS (containing 0.1% DMSO) or 1–10 μM NCTD for 24 h before stimulation with 100 ng/ml LPS for 1 h. Total cellular RNA were collected and subjected to Real time -PCR analysis. C, Only LPS induced cytokine production was enhance by NCTD obviously. The RAW264.7 cells were pretreated with 0.1% DMSO, 5 μM NCTD for 24 h before stimulation with LPS (100 ng/ml), PMA (100nM) or LTA (10 µg/ml) for 1 h. Total cellular RNA were collected and subjected to Real time PCR analysis. Columns, mean from three independent experiments with three duplicates; bars, SE (*, P<0.05; **, P<0.01 versus control).</p

NCTD facilitates NF-κB nuclear translocation.

<p>A–B, RAW264.7 cells (A) and peritoneal macrophages (B) were stimulated with 100 ng/ml LPS in absence or presence of 5 µM NCTD that had been added 24 h before. When LPS addition was added for 15 min, subcellular location of NF-κB p65 subunit was detected using immunofluorescence assay. Scale bar, 10 μm. One of three experiments was shown.</p

NCTD protects mice from bacterial-induced peritonitis.

<p>A, Chemical structure of NCTD and CTD. B, The survival of peritonitis mouse was increased by NCTD obviously. The mice were treated with NCTD or PBS at 24 h and 6 h before infection. Both NCTD and PBS (containing 0.1% DMSO) treated mice (n = 8) were intraperitoneal injected with <i>E.coli 0111</i> (8*10⁷) and monitored every 3 h for 72 h. C, Serum concentration of IL-6 and TNF-α in mouse model was enhanced by NCTD. The blood was obtained 6 h after infection and the serum concentration of IL-6 and TNF-α was measured by ELISA assay. (<i>*</i>, P<0.0<i>5</i>) Data are representative of three independent experiments with similar results. D, The quantity of bacterial was reduced in NCTD treated mouse. The mice were treated with 1 ml vehicle control (containing 0.1% DMSO), 100 ug/ml Ampicillin or NCTD (10 mg/kg) respectively followed by an i.p injection with 5×10⁷ CFU of <i>E. coil</i>-GFP in 1 ml PBS for 16 h. Then the bacteria resident in abdomen was visualized by fluorescent stereomicroscope. Also, their peritoneal cavities were lavaged with 3 ml PBS, and bacterial counts were determined by plating on agar plates. n = 4 mice per group. E, NCTD has no effects on the growth of <i>E. coil</i>-GFP. E.coli-GFP were cultured with 100 µg/ml Ampicillin (Amp+) and 5 μM NCTD for 24 h respectively. Then the bacteria number was detected by spectrophotometer. Data are representative of three independent experiments with similar results (mean ± s.d. in B, D).</p