Phosphorylated‑myosin light chain mediates the destruction of small intestinal epithelial tight junctions in mice with acute liver failure

Abstract

Tight junction dysregulation and epithelial damage contribute to intestinal barrier loss in patients with acute liver failure (ALF); however, the regulatory mechanisms of these processes remain poorly understood. The aim of the present study was to investigate the changes of intestinal tight junction and intestinal mucosa in mice with ALF and their mechanisms. In the present study, ALF was induced in mice through an intraperitoneal injection of D‑galactosamine and lipopolysaccharide (D‑GalN/LPS), and the morphological changes of the liver or small intestine were analyzed using hematoxylin and eosin staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The intestinal tissues and isolated serum were analyzed using western blotting, immunofluorescence staining and ELISA. D‑GalN/LPS‑induced mice exhibited signs of hepatocyte necrosis, alongside inflammatory cell infiltration into the liver tissue and partial microvilli detachment in the small intestinal mucosa. TEM demonstrated that the intestinal epithelial tight junctions were impaired, whereas SEM micrographs revealed the presence of abnormal microvilli in D‑GalN/LPS‑induced mice. In addition, the expression levels of phosphorylated (p)‑myosin light chain (MLC), MLC kinase (MLCK) and Rho‑associated kinase (ROCK) were significantly increased in the D‑GalN/LPS‑induced mice compared with those in the control mice, whereas the subsequent inhibition of MLCK or ROCK significantly reduced p‑MLC expression levels. Conversely, the expression levels of occludin and zonula occludens‑1 (ZO‑1) were significantly decreased in the D‑GalN/LPS‑induced mice, and the inhibition of MLCK or ROCK significantly increased occludin and ZO‑1 protein expression levels compared with those in the control group. Changes in the serum levels of tumor necrosis factor‑α (TNF‑α) and interleukin (IL)‑6 were similar to the trend observed in p‑MLC expression levels. In conclusion, the findings of the present study suggested that in a D‑GalN/LPS‑induced ALF model, TNF‑α and IL‑6 signaling may increase MLCK and ROCK expression levels, further mediate phosphorylation of MLC, which may result in tight junction dysregulation and intestinal barrier dysfunction.

Keywords: myosin light chain; myosin light chain kinase; Rho‑associated kinase.

Figure 1.

Representative images of the hematoxylin and eosin stained liver sections, Scale bar=50 µm. Black arrows indicate inflammatory cells, white arrows indicate necrotic hepatocytes. D-GalN/LPS, D-galactosamine and lipopolysaccharide; MLCK, myosin light chain kinase; ROCK, Rho-associated kinase.

Figure 2.

D-GalN/LPS-induced mice exhibit severe damage to the intestinal mucosa. (A) Representative images of hematoxylin and eosin-stained sections of the small intestine. Scale bar=50 µm. (B) Representative micrographs obtained from the scanning electron microscopy of microvilli tubules. Scale bar=100 µm. Arrows indicate microvilli tubules. D-GalN/LPS, D-galactosamine and lipopolysaccharide; MLCK, myosin light chain kinase; ROCK, Rho-associated kinase.

Figure 3.

Tight junctions are dilated in D-GalN/LPS-induced mice. Transmission electron microscopy was used to analyze the ultrastructure of the intestine in control, D-GalN/LPS, D-GalN/LPS+MLCK inhibition and D-GalN/LPS+ROCK inhibition groups. Arrows indicate tight junctions (scale bar=2.0 µm). D-GalN/LPS, D-galactosamine and lipopolysaccharide; MLCK, myosin light chain kinase; ROCK, Rho-associated kinase.

Figure 4.

Expression levels of p-MLC are increased by D-GalN/LPS and decreased by MLCK or ROCK inhibition. (A) Immunofluorescence staining was used to analyze the expression level of p-MLC in the control, D-GalN/LPS, D-GalN/LPS + MLCK inhibition and D-GalN/LPS + ROCK inhibition groups; red staining indicates p-MLC, blue staining indicates the nucleus (magnification, ×200). (B) Western blot analysis of intestinal expression levels of p-MLC and MLC. (C) Relative expression of p-MLC compared with GAPDH. GAPDH was used as an internal control. (D) Relative expression of MLC compared with GAPDH. (E) Relative expression of p-MLC compared with MLC. Data were analyzed by one-way ANOVA followed by Tukey's test. *P<0.01 vs. the control group; ^#P<0.05 vs. the D-GalN/LPS group. p-, phosphorylated; MLC, myosin light chain; D-GalN/LPS, D-galactosamine and lipopolysaccharide; MLCK, myosin light chain kinase; ROCK, Rho-associated kinase.

Figure 5.

Tight junction protein expression levels in the control, D-GalN/LPS, D-GalN/LPS + MLCK inhibition and D-GalN/LPS + ROCK inhibition groups. (A) Western blot analysis of intestinal MLCK, ROCK, occludin and ZO-1 expression levels. (B) Semi-quantification of western blotting demonstrated that the expression levels of MLCK and ROCK were significantly increased, whereas ZO-1 and occludin expression levels were significantly decreased in the D-GalN/LPS-induced mice. Data were analyzed by one-way ANOVA followed by Tukey's test. *P<0.01 vs. the control group; ^#P<0.05 vs. the D-GalN/LPS group. D-GalN/LPS, D-galactosamine and lipopolysaccharide; MLCK, myosin light chain kinase; ROCK, Rho-associated kinase; ZO-1, zonula occludens-1.

Figure 6.

Serum TNF-α and IL-6 levels are increased in D-GalN/LPS-induced mice. The inhibition of MLCK expression levels using ML-7, or the inhibition of ROCK expression levels using Y-27632, significantly reduced serum TNF-α and IL-6 levels. Data were analyzed by one-way ANOVA followed by Tukey's test. *P<0.05 vs. control group; ^#P<0.05 vs. D-GalN/LPS group. TNF; tumor necrosis factor; IL, interleukin; D-GalN/LPS, D-galactosamine and lipopolysaccharide; MLCK, myosin light chain kinase; ROCK, Rho-associated kinase.