Journal: The Journal of infectious diseases

Article Title: Stimulation of the α7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation.

doi: 10.1093/infdis/jit669

Figure Lengend Snippet: Figure 1. Stimulation of α7 nicotinic acetylcholine receptor by nicotine reduces cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced mortality and organ injury. Sham-operated animals were subjected to laparotomy and intestinal manipulation; however, the cecum was neither ligated nor punctured. A and B, Survival rate. Mice were given intraperitoneal nicotine (400 μg/mg) or vehicle (saline) 0, 24, 48, and 72 hours after CLP (A, n = 10 per group) and 6, 30, and 54 hours after LPS treatment (B, n = 10 per group). Mice were treated with intraperitoneal methyllycaconitine (MLA; 5 mg/kg) 5 minutes prior to nicotine treatment. All animals were monitored for 10 days after CLP surgery and LPS administration. P < .01 (log-rank test; significant difference from CLP or LPS group). yP < .05 (log-rank test; significant difference from CLP + nicotine or LPS + nicotine group). Histological micrographs of liver (C) and lung (D). Mice were given intraperitoneal nicotine (400 μg/mg) or vehicle 0 hour after CLP. Mice were treated with intraperitoneal MLA (5 mg/ kg) 5 minutes prior to nicotine treatment. Twenty-four hours after CLP, liver and lung tissues were obtained. Histological micrographs of liver and lung tissues stained with hematoxylin and eosin are shown (magnification ×200; scale bar = 100 μm). Representative images were chosen from each of the experimental groups.

Article Snippet: Methyllycaconitine (MLA, 5 mg/kg; Tocris Bioscience), a selective α7nAChR antagonist, was dissolved in saline and intraperitoneally administered 5 minutes prior to nicotine treatment.

Techniques: Ligation, Saline, Staining

Journal: The Journal of infectious diseases

Article Title: Stimulation of the α7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation.

doi: 10.1093/infdis/jit669

Figure Lengend Snippet: Figure 2. Stimulation of α7 nicotinic acetylcholine receptor by nicotine downregulates Toll-like receptor 4 (TLR4) protein and mRNA expression and PU.1 expression during sepsis. Mice were given intraperitoneal nicotine (400 μg/mg) or vehicle (saline) 0 hour after cecal ligation and puncture (CLP). Mice were treated with intraperitoneal methyllycaconitine (MLA; 5 mg/kg) 5 minutes prior to nicotine treatment. Liver tissues were obtained 6 hours after CLP. Sham-operated animals were subjected to laparotomy and intestinal manipulation; however, the cecum was neither ligated nor punctured. A, TLR4 protein expression in liver. B, TLR4 mRNA expression in liver. C, Nucleus PU.1 protein expression in liver. Densitometry was performed and fold changes in protein expression are shown below the representative bands. Results are expressed as the mean ± standard error of mean of 6–8 animals per group. P < .05, P < .01 (Bonferroni test; significant differences from sham group). yyP < .01 (Bonferroni test; significant differences from CLP group). xP < .05, xxP < .01 (Bonferroni test; significant differences from CLP + nicotine group).

Article Snippet: Methyllycaconitine (MLA, 5 mg/kg; Tocris Bioscience), a selective α7nAChR antagonist, was dissolved in saline and intraperitoneally administered 5 minutes prior to nicotine treatment.

Techniques: Expressing, Saline, Ligation

Journal: The Journal of infectious diseases

Article Title: Stimulation of the α7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation.

doi: 10.1093/infdis/jit669

Figure Lengend Snippet: Figure 3. Nicotine suppresses Toll-like receptor 4 (TLR4) protein expression and PU.1 activity through α7 nicotinic acetylcholine receptor (α7nAChR) activa- tion in lipopolysaccharide (LPS)-treated RAW264.7 cells. A, Cytotoxicity of nicotine on RAW264.7 cells. The cells were treated with various concentrations of nicotine for 24 hours for 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay. B, α7nAChR protein expression. Forty-eight hours after α7nAChR siRNA transfection, α7nAChR protein expression was estimated using Western blot analysis. C, TLR4 protein expression. After siRNA transfection, the cells were treated with nicotine (30 μM) for 1 hour followed by LPS 1 μg/mL treatment for 6 hours. D and E, Translocation of PU.1 to nucleus. The cells were pre- treated with methyllycaconitine (MLA; 30 μM) or wortmannin (20 nM) for 30 minutes, then treated with nicotine (30 μM) for 1 hour, and finally LPS (1 μg/ mL) for 1 hour. Densitometry was performed, and fold changes in protein expression are shown below the representative bands. Results are expressed as the mean ± standard error of mean of 3 independent experiments. P < .01 (Bonferroni test; significant difference from control siRNA or control group). yyP < .01 (Bonferroni test; significant difference from LPS group). xxP < .01 (Bonferroni test; significant difference from LPS + nicotine group). F, DNA binding of PU.1. The cells were pretreated with MLA (30 μM) or wortmannin (20 nM) for 30 minutes, then treated with nicotine (30 μM) for 1 hour, and finally LPS (1 μg/mL) for 1 hour. The cold probe was in 66-fold excess of unlabeled PU.1 oligonucleotide. The autoradiogram is representative of 3 independent experiments.

Article Snippet: Methyllycaconitine (MLA, 5 mg/kg; Tocris Bioscience), a selective α7nAChR antagonist, was dissolved in saline and intraperitoneally administered 5 minutes prior to nicotine treatment.

Techniques: Expressing, Activity Assay, Transfection, Western Blot, Translocation Assay, Control, Binding Assay

Journal: The Journal of infectious diseases

Article Title: Stimulation of the α7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation.

doi: 10.1093/infdis/jit669

Figure Lengend Snippet: Figure 4. Stimulation of α7 nicotinic acetylcholine receptor by nicotine suppresses Toll-like receptor 4 (TLR4) expression through PI3K/Akt activation in lipopolysaccharide (LPS)-treated RAW264.7 cells. A and C, PI3K and Akt activation. The cells were pretreated with methyllycaconitine (MLA; 30 μM) for 30 minutes, then treated with nicotine (30 μM) for 1 hour, and finally LPS (1 μg/mL) for 1 hour. B and D, PI3K and Akt activation. The cells were pretreated with wortmannin (20 nM) or LY294002 (20 μM) for 30 minutes, then treated with nicotine (30 μM) for 1 hour, and finally LPS (1 μg/mL) for 1 hour. TLR4 protein (E) and mRNA (F) expression. The cells were pretreated with MLA (30 μM) or wortmannin (20 nM) for 30 minutes, then treated with nicotine (30 μM), and finally LPS (1 μg/mL) for 1 hour and 6 hours prior to measuring TLR4 protein and mRNA expression, respectively. Densitometry was performed, and fold changes in protein and mRNA expression are shown below the representative bands. Results are expressed as the mean ± standard error of mean of 3 independent experiments. P < .05, P < .01 (Bonferroni test; significant difference from control group). yyP < .01 (Bonferroni test; significant difference from LPS group). xP < .05, xxP < .01 (Bonferroni test; significant difference from LPS + nicotine group).

Article Snippet: Methyllycaconitine (MLA, 5 mg/kg; Tocris Bioscience), a selective α7nAChR antagonist, was dissolved in saline and intraperitoneally administered 5 minutes prior to nicotine treatment.

Techniques: Expressing, Activation Assay, Control

Journal: The Journal of infectious diseases

Article Title: Stimulation of the α7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation.

doi: 10.1093/infdis/jit669

Figure Lengend Snippet: Figure 5. Nicotine activates the α7 nicotinic acetylcholine receptor/Akt pathway during sepsis. A and B, PI3K and Akt activity. Mice were treated with methyllycaconitine (MLA; 5 mg/kg) 5 minutes prior to nicotine (400 μg/kg; 0 hour after cecal ligation and puncture [CLP]) exposure. Liver tissue was ob- tained 6 hours after CLP. Sham-operated animals were subjected to laparotomy and intestinal manipulation; however, the cecum was neither ligated nor punctured. The p-PI3K/PI3K (A) and p-Akt/Akt (B) ratios were determined by Western blot analysis. C, Survival rate. Mice were treated with wortmannin (1 mg/kg) 1 hour before nicotine (400 μg/kg; 0, 24, 48, and 72 hours after CLP) treatment. D and E, PU.1 and TLR4 protein expression in liver. Mice were treated with wortmannin (1 mg/kg) 1 hour before nicotine (400 μg/kg; 0 hour after CLP) treatment. Six hours after CLP, nuclear and whole extracts were prepared for Western blot analysis of PU.1 (D) and TLR4 (E), respectively. F and G, Serum tumor necrosis factor-alpha (TNF-α ) and interferon-gamma (IFN- γ ) levels. Mice were treated with wortmannin (1 mg/kg) 1 hour before nicotine (400 μg/kg; 0 hour after CLP) treatment. Six hours after CLP, serum TNF-α (F) and IFN-γ (G) levels were measured, respectively. Densitometry was performed, and fold changes in protein and mRNA expression are shown below the representative bands. Results are expressed as the mean ± standard error of mean of 6–8 animals per group. P < .05, P < .01 (Bonferroni test; significant difference from sham group). yP < .05, yyP < .01 (Bonferroni test; significant difference from CLP group). xP < .05, xxP < .01 (Bonferroni test; significant difference from CLP + nicotine group).

Article Snippet: Methyllycaconitine (MLA, 5 mg/kg; Tocris Bioscience), a selective α7nAChR antagonist, was dissolved in saline and intraperitoneally administered 5 minutes prior to nicotine treatment.

Techniques: Activity Assay, Ligation, Western Blot, Expressing