The effect of rapamycin on intestinal damage.

<p>Rapamycin or vehicle was administered then intestinal I/R performed with 6 h of reperfusion. A. Representative histological images, magnification x 200. B. Histologic mucosal damage was calculated using the Chiu scoring system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041584#pone.0041584-Chiu1" target="_blank">[61]</a>. C. Representative images of TUNEL staining in Intestinal tissues. D. Quantitative results of TUNEL staining. Results are mean ± SEM, p<0.05 statistically significant, n = 5 mice per group.</p

The effect of p70S6K on cell growth, apoptosis and migration.

<p>p70S6K expressing plasmid (p70S6K group) and empty plasmid (Ctrl and Rap groups) transfected cells were cultured with FBS-free medium overnight under hypoxic conditions then either treated with vehicle (Ctrl and p70S6K groups) or rapamycin (100 nM) (Rap group) under normoxic or hypoxic conditions. A. Cell growth was measured using MTTassay. B. DNA fragmentation was then measured using a Cell Death Detection ELISA kit. C. Cell migration after wounding. Recovered surface area 24 h after wounding was calculated and quantitative results shown. D. Typical microscopic images of the wound area. The patterns of initial wounding (upper panel) and after 24 h (lower panel) are shown. Black lines indicate the margin of cell migration. All experiments were carried out triplicate. Results are mean ± SEM, p<0.05 statistically significant.</p

The effect of rapamycin on intestinal permeability.

<p>Rapamycin or vehicle was administered then intestinal I/R performed. Intestinal tissues were harvested for permeability using the ex-vivo isolated everted ileum sac method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041584#pone.0041584-Wattanasirichaigoon1" target="_blank">[64]</a>. Intestinal permeability was expressed as the mucosal-to-serosal clearance of FD4. Results are mean ± SEM, p<0.05 statistically significant, n = 5 mice per group.</p

The effect of rapamycin on animal survival.

<p>Rapamycin or vehicle was administered then intestinal I/R performed. Animals were observed for 7 days. Survival was analyzed using the Mantel–Haenzel log rank test. P values <0.05 were considered significant. n = 10 mice per group. There was a significant decrease in survival in rapamycin treated animals (p<0.01).</p

Stimulation of p70S6K in vitro and in vivo and inhibition of p70S6K by rapamycin in vivo.

<p>A. Stimulation of p70S6K. Cells were cultured with FBS-free medium overnight under hypoxic conditions and then treated with 10% FBS for indicated time-points under normoxic conditions. Cells were harvested and expression of phosphorylated p70S6K (p-p70S6K) measured. The experiments were carried out triplicate. B. Stimulation of p70S6K by I/R in vivo. The superior mesenteric artery was occluded for 60 min then the intestine was harvested after indicated periods of reperfusion and Western blot analysis performed. C. Confirmation of increased p70S6K activity in intestinal epithelium by immunohistochemistry. Rapamycin or vehicle treated animals underwent intestinal I/R with 6 h of reperfusion. The intestinal tissue was harvested for immunohistochemical staining for p-p70S6K. D. Confirmation of the inhibition of p70S6K by rapamycin using Western blot. Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041584#pone-0041584-g002" target="_blank">Figure 2C</a>. Intestinal tissues were harvested and Western blot analysis performed. For animal experiments, n = 5 mice per group.</p

The effect of rapamycin on cytokine levels in serum and intestinal tissues.

<p>Rapamycin or vehicle was administered then intestinal I/R performed. Blood and intestinal tissues were collected after 6 h of reperfusion for evaluation of TNF-α, IL-6 and IL-1β levels. A. Relative levels of TNF-α in serum. B. Relative levels of TNF-α in intestinal tissues. C. Relative levels of IL-6 in serum. D. Relative levels of IL-6 in intestinal tissues. E. Relative levels of IL-1β in serum. F. Relative levels of IL-1β in intestinal tissues. Results are mean ± SEM, p<0.05 statistically significant, n = 5 mice per group.</p

The effect of rapamycin on neutrophil infiltration.

<p>Rapamycin or vehicle was administered then intestinal I/R performed with 6 h of reperfusion. A. Myeloperoxidase (MPO) activity in intestinal tissues. B. Representative images of neutrophil staining in intestinal tissues. C. Quantification of positively stained neutrophils. Results are mean ± SEM, p<0.05 statistically significant, n = 5 mice per group.</p

Inhibition of ERK1/2 by U0126 increased the levels of proinflammatory cytokines in plasma.

<p>Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). The relatively levels of cytokines in plasma was determined using a commercial kit. A. The relatively level of cytokines TNF-α in plasma. B. The relatively level of cytokines IL-6 in plasma. C. The relatively level of cytokines IL-1β in plasma. Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.</p

Activities of ERK1/2 and p70S6K were inhibited by U0126 in vitro and in vivo.

<p>A. IEC-6 cells were cultured in medium without FBS for 12 hours under hypoxic conditions followed treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 20 minutes (n=3). Protein expression in cells was determined by Western blot analysis. B. IEC-6 cells were transfected with/without empty plasmid or p70S6K plasmid for 24 hours and cultured in medium without FBS for 12 hours under hypoxic conditions followed treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 20 minutes (n=3). Protein expression was determined by Western blot analysis. C. Mice were pretreated with U0126 or vehicle and then subjected to one hour ischemia followed by 6 hours reperfusion in the intestine (n=5). Protein expression in the intestine was determined by Western blot analysis. U=U0126, Sham-U=Sham-U0126, IR=I/R, IR-U=I/R-U0126.</p

Inhibition of ERK1/2 and p70S6K by U0126 decreased cell proliferation and migration, and promoted cell apoptosis in vitro (n=3).

<p>A. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in medium without FBS for 12 hours under hypoxic conditions followed by treatment with U0126 or vehicle for 1 hour and then stimulated with 10% FBS under normoxic conditions for 3 days. Cell proliferation was measured by MTT assay. B. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in medium without FBS in a 12-well plate for 12 hours under hypoxic conditions. The confluent cell monolayer was then scraped with a 1-ml pipette tip. Cells were then treated with U0126 or vehicle for 1 hour followed by adding 10% FBS under normoxic conditions for 24 hours. The recovery area was measured. C. After transfection with/without empty plasmid or p70S6K plasmid for 12 hours, IEC-6 cells were cultured in FBS-free medium with U0126 or vehicle under hypoxic conditions for 24 hours followed by incubation in normoxic conditions for 6 hours. Cell apoptosis was determined by evaluation of DNA fragmentation. U=U0126.</p