Myosin light chain kinase is involved in lipopolysaccharide-induced disruption of colonic epithelial barrier and bacterial translocation in rats

Abstract

Sepsis is associated with bacterial translocation (BT) and changes in colonic paracellular permeability (CPP), but the link between these effects is unknown. The present study aimed to identify whether changes in CPP after lipopolysaccharide (LPS) administration triggers BT, colonic inflammation, visceral pain, and sickness behavior and to evaluate the role of myosin light chain kinase (MLCK) in colonocyte cytoskeleton contraction. Rats received the MLCK inhibitor ML-7 alone or combined with LPS. CPP was measured for 6 hours after administration. Visceral pain, food intake, BT, electron microscopy of tight junctions of colonocytes, cytokine levels, and Western blotting of phosphorylated MLC from colonic mucosa were assessed in a time range of 0 to 3 hours after treatment. Sepsis increased CPP at 0 to 6 hours after LPS and associated with tight junction morphological changes, increased MLC phosphorylation, and mucosal release of proinflammatory cytokines. Massive BT, visceral hyperalgesia, and reduced food intake were also observed. Addition of ML-7 prevented all LPS-induced effects, except for changes in food intake. In conclusion, LPS-mediated effects on CPP include gut inflammation, BT, and visceral hyperalgesia. Inhibition of MLCK-dependent colonocyte cytoskeleton contraction by ML-7 prevents the LPS-induced alterations of CPP and its subsequent effects.

Figure 1

Influence of MLCK inhibitor (ML-7) on LPS-induced increase in CPP to ⁵¹Cr-EDTA from 0 to 6 hours after LPS administration. LPS injection (1 mg/kg, intraperitoneally) significantly increased 0- to 6-hour CPP. ML-7 injections (3 × 1 mg/kg/48 hours, intraperitoneally) reversed LPS-induced increase in permeability for the 0- to 6-hour period. ML-7 had no effect per se on CPP. Values are means ± SEM. *Significantly different (P < 0.05) from corresponding control values. ⁺Significantly different (P < 0.05) from LPS values.

Figure 2

Transmission electron micrographs of the TJ region of colonic enterocytes. A: In controls, TJs of cell membranes of adjacent cells are in close opposition. B: Typical TJ dilatations (arrows) observed 3 hours after LPS exposure. C: This effect was prevented by ML-7 pretreatment. Scale bar, 0.2 μm.

Figure 3

Proinflammatory cytokine levels in colonic mucosa 3 hours after LPS administration. Compared with corresponding control rats (LPS vehicle + M-7 vehicle), injection of LPS (1 mg/kg, intraperitoneally) significantly increased the level of TNF-α in colonic mucosa 1 hour after its administration but had no effect on IL-1β, IL-6, or IFN-γ release. Three hours after LPS injection, a significant increase in TNF-α, IL-1β, IL-6, and IFN-γ levels was detected in colonic mucosa of septic rats compared with corresponding controls. Compared with corresponding LPS groups, prior treatment with ML-7 had no effect on cytokine release 1 hour after LPS administration but had significantly reduced the IL-β and IFN-γ release 3 hours after LPS. However, MLCK inhibition failed to reduce the release of IL-6 and TNF-α. ML-7 had no effect per se on proinflammatory cytokine release. Values are means ± SEM. *Significant difference (P < 0.05) from corresponding control values. ⁺Significant difference (P < 0.05) from corresponding LPS values.

Figure 4

Representative Western immunoblots of phosphorylated MLC (pMLC). Results are representative of three animals per group. A: Time course pMLC levels were measured at 0.5, 1, 2, 3, and 6 hours after LPS challenge. pMLC levels increased 1 hour after LPS exposure and were maintained throughout 3 hours. B: ML-7 inhibited the increase of pMLC levels consecutive to LPS treatment. C: ML-7 had no effect per se on pMLC levels in basal conditions. The graphs represent relative mean area density of the total protein blots ± SEM.

Figure 5

Effect of ML-7 on LPS-induced visceral hypersensitivity in response to different volumes of rectal distension at 3 hours after LPS. LPS increased the abdominal response to distension at 3 hours after LPS but only for the lowest volume of distension (0.4 ml). ML-7 reduced the LPS-induced allodynia. ML-7 had no effect per se on visceral sensitivity. Values are means ± SEM. *Significantly different (P < 0.05) from corresponding control values. ⁺Significantly different (P < 0.05) from LPS values.

Figure 6

Effect of intraperitoneal administration of LPS on food intake with or without ML-7 pretreatment. Food intake was assessed 1.5 hours after LPS (1 mg/kg) administration during a 3-hour period. ML-7 pretreatment (3 × 1 mg/kg/48 hours, intraperitoneally) failed to reverse the LPS-induced effects on food intake. Results are expressed as means ± SEM. *Significantly different (P < 0.05) from corresponding control values (saline).

Figure 7

Diarrheic effect of LPS administration with or without ML-7 pretreatment. LPS administration (1 mg/kg, intraperitoneally) resulted in a significant emission of watery feces compared with controls. Prior treatment with ML-7 (3 × 1 mg/kg/48 hours, intraperitoneally) did not prevent the LPS-induced diarrheic effects. Results are expressed as means ± SEM. *P < 0.05 versus control group.