Oral insulin up-regulates Toll-like receptor 4 expression and enhances intestinal recovery following lipopolysaccharide-induced gut injury in a rat

Abstract

In the present study, we evaluated the protective effect of oral insulin (OI) on intestinal mucosa following lipopolysaccharide-induced intestinal damage in a rat. Male Sprague-Dawley rats were divided into three experimental groups: Sham rats, LPS-rats that were treated with lipopolysaccharide (LPS), and LPS-INS rats that were treated with OI given in drinking water 72 h before and following injection of LPS. Intestinal structural changes, enterocyte proliferation, enterocyte apoptosis, and mucosal expression of Toll-like receptor 4 (TLR4) were determined 24 h after the last LPS injection. LPS-INS animals showed a significantly greater bowel and mucosal weight in jejunum and ileum, mucosal DNA and protein in jejunum and ileum, villus height in ileum, crypt depth in jejunum and ileum, cell proliferation rates in jejunum, and significantly lower apoptotic index in ileum compared to LPS- animals. LPS rats demonstrated 50% increase in TLR4 expression in jejunum compared to sham animals. Treatment with OI resulted in a three-fold increase in TLR4 expression in jejunum, compared to LPS animals. In conclusion, OI improves intestinal recovery after LPS endotoxemia in a rat.

Fig. 1

Effect of lipopolysaccharide endotoxemia and oral insulin on enterocyte proliferation and apoptosis. Values are mean ± SEM. LPS lipopolysaccharide, INS insulin. *P<0.05 LPS versus Sham rats and ^† P<0.05 LPS-INS versus LPS rats

Fig. 2

Effect of lipopolysaccharide endotoxemia and oral insulin on crypt cell proliferation. 5-BrdU incorporation into proliferating cells was detected with a goat anti-BrdU antibody. The representative sections demonstrate that cell proliferation decreased following LPS administration compared to sham animals. Following administration of oral insulin, LPS-rats demonstrated a nonmarked increase in a number of proliferating cells compared to LPS-nontreated animals

Fig. 3

Effect of lipopolysaccharide endotoxemia and oral insulin on enterocyte apoptosis. Immunohistochemistry for caspase-3 was used to determine enterocyte apoptosis. As expected, Sham rats demonstrate a normal histologic architecture and single apoptotic cells. LPS rats show extended subepithelial space (Park score 2–3) and a significant increase in the number of the apoptotic cells. LPS-INS rats exhibited a less marked subepithelial space and a decrease in the number of apoptotic cells

Fig. 4

Effect of lipopolysaccharide endotoxemia and oral insulin on expression of Toll-like receptor 4 (TLR4). LPS rats demonstrated a 55% increase in TLR4 expression in jejunum compared to sham animals. Pretreatment with oral insulin resulted in a two-fold increase in TLR4 expression in jejunum compared to LPS-animals. There was no significant change in the TLR4 expression in ileum between the three experimental groups. Values are mean ± SEM. LPS lipopolysaccharide, INS insulin, TLR4 Toll-like receptor 4. *P<0.05 LPS versus Sham rats. ^† P<0.05 LPS-INS versus LPS rats