Myosin Light Chain Kinase Protein Expression Research Articles

Dexmedetomidine protects against burn-induced intestinal barrier injury via the MLCK/p-MLC signalling pathway

BackgroundEvidence suggests that sedative dexmedetomidine can prevent intestinal dysfunction. However, the specific mechanisms of its protective effects against burn-induced intestinal barrier injury remain unclear. We aimed to explore the possible positive effects of dexmedetomidine on burn-induced intestinal barrier injury and the effects the myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) signalling pathway in an experimental model of burn injury. MethodsIn this study, the plasma concentration of fluorescein isothiocyanate-labelled dextran (FITC-dextran) was measured. Histological changes were evaluated using haematoxylin and eosin (HE) staining. Tight junction proteins were evaluated by western blot and immunofluorescence analyses to assess the structural integrity of intestinal tight junctions. The level of inflammation was detected by enzyme-linked immunosorbent assay (ELISA). ResultsThe results shows that the increase in intestinal permeability caused by burn injury is accompanied by histological damage to the intestine, decreases in the expression of the tight junction proteins Zonula Occludens-1 (ZO-1) and Occludin, increases in inflammatory cytokine levels and elevation of both MLCK protein expression and MLC phosphorylation. After dexmedetomidine treatment, the burn-induced changes were ameliorated. ConclusionsIn conclusion, dexmedetomidine exerted an anti-inflammatory effect and protected tight junction complexes against burn‑induced intestinal barrier damage by inhibiting the MLCK/p-MLC signalling pathways.

Wogonoside alleviates colitis by improving intestinal epithelial barrier function via the MLCK/pMLC2 pathway

BackgroundIntestinal epithelial barrier dysfunction, which involves myosin light chain kinase (MLCK) activation, contributes to the occurrence and progression of inflammation in inflammatory bowel disease (IBD). Wogonoside helps maintain intestinal homeostasis in mice with dextran sulfate sodium (DSS)-induced colitis, but it is unclear whether it modulates intestinal barrier function. PurposeHere, we demonstrate that wogonoside protects against intestinal barrier dysfunction in colitis via the MLCK/pMLC2 pathway both in vivo and in vitro. MethodsCaco-2 cell monolayers treated with the proinflammatory cytokine TNF-α showed barrier dysfunction and were assessed in the absence and presence of wogonoside for various physiological, morphological, and biochemical parameters. Colitis was induced by 3% DSS in mice, which were used as an animal model to explore the pharmacodynamics of wogonoside. We detected MLCK/pMLC2 pathway proteins via western blot analysis, assessed the cytokines IL-13 and IFN-γ via ELISA, tested bacterial translocation via fluorescence in situ hybridization (FISH) and a proper sampling of secondary lymphoid organs for bacterial culture. In addition, the docking affinity of wogonoside and MLCK was observed with DS2.5 software. ResultsWogonoside alleviated the disruption of transepithelial electrical resistance (TER) in TNF-α exposured Caco-2 cell; FITC-dextran hyperpermeability; loss of the tight junction (TJ) proteins occludin, ZO-1 and claudin-1 in Caco-2 cell monolayers; and bacterial translocation in colitic mice. Moreover, wogonoside reduced the levels of the proinflammatory cytokines IL-13 and IFN-γ to maintain intestinal immune homeostasis. Transmission electron microscopy (TEM) confirmed that wogonoside ameliorated the destruction of intestinal epithelial TJs. Wogonoside not only inhibited the cytoskeletal F-actin rearrangement induced by TNF-α, stabilized the cytoskeletal structure, suppressed MLCK protein expression, and reduced MLC2 phosphorylation. In addition, the results of molecular docking analysis showed that wogonoside had a high affinity for MLCK and formed hydrogen bonds with the amino acid residue LYS261 and π bonds with LYS229. ConclusionCollectively, our study indicates that wogonoside alleviates colitis by protecting against intestinal barrier dysfunction, and the potential mechanism may involve regulation of TJs via the MLCK/pMLC2 signaling pathway. Meanwhile, our study also explains the success of S. baicalensis in the treatment of ulcerative colitis (UC).

MMP-9-induced increase in intestinal epithelial tight permeability is mediated by p38 kinase signaling pathway activation of MLCK gene.

Matrix metalloproteinase-9 (MMP-9) has been implicated as being an important pathogenic factor in inflammatory bowel disease (IBD). MMP-9 is markedly elevated in intestinal tissue of patients with IBD, and IBD patients have a defective intestinal tight-junction (TJ) barrier manifested by an increase in intestinal permeability. The loss of intestinal epithelial barrier function is an important contributing factor in the development and prolongation of intestinal inflammation; however, the role of MMP-9 in intestinal barrier function remains unclear. The purpose of this study was to investigate the effect of MMP-9 on the intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved by using in vitro (filter-grown Caco-2 monolayers) and in vivo (mouse small intestine recycling perfusion) systems. MMP-9 caused a time- and dose-dependent increase in Caco-2 TJ permeability. MMP-9 also caused an increase in myosin light-chain kinase (MLCK) gene activity, protein expression, and enzymatic activity. The pharmacological MLCK inhibition and siRNA-induced knockdown of MLCK inhibited the MMP-9-induced increase in Caco-2 TJ permeability. MMP-9 caused a rapid activation of the p38 kinase signaling pathway and inhibition of p38 kinase activity prevented the MMP-9-induced increase in MLCK gene activity and the increase in Caco-2 TJ permeability. MMP-9 also caused an increase in mouse intestinal permeability in vivo, which was accompanied by an increase in MLCK expression. The MMP-9-induced increase in mouse intestinal permeability was inhibited in MLCK-deficient mice. These data show for the first time that the MMP-9-induced increase in intestinal TJ permeability in vitro and in vivo was mediated by the p38 kinase signal transduction pathway upregulation of MLCK gene activity and that therapeutic targeting of these pathways can prevent the MMP-9-induced increase in intestinal TJ permeability. NEW & NOTEWORTHY MMP-9 is highly elevated in patients with IBD. IBD patients have compromised intestinal TJ barrier function manifested by an increase in intestinal permeability and intestinal inflammation. This study shows that MMP-9, at clinically achievable concentrations, causes an increase in intestinal TJ permeability in vitro and in vivo. In addition, a MMP-9-induced increase in intestinal TJ permeability was mediated by an increase in MLCK gene and protein expression via the p38 kinase pathway.

The possible association between AQP9 in the intestinal epithelium and acute liver injury‑induced intestinal epithelium damage.

The present study aimed to investigate the expression and function of aquaporin (AQP)9 in the intestinal tract of acute liver injury rat models. A total of 20 Sprague Dawley rats were randomly divided into four groups: Normal control (NC) group and acute liver injury groups (24, 48 and 72 h). Acute liver injury rat models were established using D-amino galactose, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Tbil) and albumin were determined using an automatic biochemical analyzer. Proteins levels of myosin light chain kinase (MLCK) in rat intestinal mucosa were investigated via immunohistochemistry. Pathological features were observed using hematoxylin and eosin (H&E) staining. MLCK, AQP9 and claudin-1 protein expression levels were detected via western blotting. Levels of ALT and AST in acute liver injury rats were revealed to steadily increase between 24 and 48 h time intervals, reaching a peak level at 48 h. Furthermore, TBil levels increased significantly until 72 h. Levels of ALT were revealed to significantly increase until the 48 h time interval, and then steadily decreased until the 72 h time interval. The acute liver injury 72 h group exhibited the greatest levels of MLCK expression among the three acute liver injury groups; however, all three acute liver injury groups exhibited enhanced levels of MLCK expression compared with the NC group. Protein levels of AQP9 and claudin-1 were enhanced in the NC group compared with the three acute liver injury groups. H&E staining demonstrated that terminal ileum mucosal layer tissues obtained from the acute liver injury rats exhibited visible neutrophil infiltration. Furthermore, the results revealed that levels of tumor necrosis factor-α, interleukin (IL)-6 and IL-10 serum cytokines were significantly increased in the acute liver injury groups. In addition, AQP9 protein expression was suppressed in acute liver injury rats, which induced pathological alterations in terminal ileum tissues may be associated with changes of claudin-1 and MLCK protein levels.

P071 MECHANISM OF MMP-9 ACTIVATION OF MYOSIN LIGHT CHAIN KINASE (MLCK) MEDIATED DISRUPTION OF INTESTINAL TIGHT JUNCTION BARRIER

Matrix Metalloproteinases 9 (MMP-9) has been implicated to have a key pathogenic factor in inflammatory bowel disease (IBD). MMP-9 is elevated in intestinal tissue of patients with inflammatory bowel disease (IBD). IBD patients have a defective intestinal tight junction (TJ) barrier manifested by an increase in intestinal permeability. The role of MMP-9 in intestinal barrier function remains unclear. The purpose of this study was to delineate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ barrier. Caco-2 monolayers were used as an in-vitro model system to examine our aims. Transepithelial resistance (TER) and inulin flux were measured to assess Caco-2 TJ permeability. Western blot analysis was performed for protein expression; real-time PCR for mRNA levels; Luciferase assay for promoter activity; molecular methods for micro-RNA and antisense transfections. 1. MMP-9 induced increase in Caco-2 TJ permeability was associated with an increase in myosin light chain kinase (MLCK) mRNA and protein expression. 2. MMP-9 induced increase in MLCK gene and protein expression was regulated in part by p38 kinase signaling pathway. 3. Inhibition of p38 kinase by pharmacologic inhibitor (SB203580) or by siRNA silencing prevented the MMP-9 induced increase in MLCK promoter, mRNA and protein expressio and increase in Caco-2 TJ permeability. 4. Our data show that MMP-9 caused activation of p38 kinase-dependent transcription factor ATF-2. 5. SiRNA induced silencing of ATF-2 prevented the MMP-9 mediated increase in MLCK promoter, mRNA and protein expression and the increase in Caco-2 TJ permeability. Our data demonstrated the involvement of p38 kinase signaling pathway in mediating the MMP-9 induced activation of MLCK gene activity and subsequent increase in Caco-2 TJ permeability.

Tu1374 1,25-Dihydroxyvitamin D3 Preserves Intestinal Epithelial Barrier Function From TNF-α Induced Injury via Suppression of MLCK-Mlc Phosphorylation Signaling Pathway

Intestinal barrier dysfunction occurs inmultiple enteropathies. Some known proinflammatory cytokines, such as TNF-a, INF-γ, play a pivotal role as mediator between various pathogens and gut epithelium. In our previous work, TNF-α injured Caco-2 monolayers were used to screen potential barrier protective reagents. 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) turned out to be a very effective protector. This is consistent with literatures which report that 1,25(OH)2D3 has anti-inflammation effect in bowel diseases. To elucidate the underlying mechanism, we performed this work. Given the fact that tight junction (TJ) is the important intercellular structure related with intestinal epithelial barrier function, and that the key role of myosin light chain (MLC) phosphorylation in regulating TJ. The TJ status and MLC phosphorylation was studied.Methods Experiments were conducted on Caco-2 monolayers. Barrier dysfunction was generated by 10ng/ml TNF-α treatment for up to 48h. Barrier function was represented by trans-epithelial electrical resistance (TEER), and macro molecule tracer, FITC-Dextran 40,000 Da (FD-40) trans-membrane flux. Morphological changes of intercellular TJ were visualized by immunofluorescence staining of ZO-1 and Occludin. The expression level of phosphorylated MLC (P-MLC), and its upstream MLC kinase (MLCK) were determined by immunoblotting. The nuclear translocation and activation of NF-kB p65 was analyzed by Electrophoretic Mobility Shift Assay (EMSA) and immunofluorescent staining. Results TNF-α decreased TEER by ~60% after 48h treatment, 10-7M 1,25(OH)2D3 reversed TEER to ~80% of original level. Consistently, TNF-α increased FD-40 flux through monolay by nearly 5 folds, 1,25(OH)2D3 repressed the number to about2 folds. TJ structure altered significantly following TNF-α treatment. 1,25(OH)2D3 ameliorated TNF-α-induced TJ damage. The MLC phosphorylation, as well as MLCK protein expression were induced by TNF-α. These increases were significantly inhibited by 1,25(OH)2D3 treatment. Additionally, 1,25(OH)2D3 suppressed the nuclear translocation and activation of NF-kB p65.Conclusion 1,25(OH)2D3 attenuates TNF-α-induced intestinal epithelial barrier dysfunction by inhibiting the signaling pathway ofMLCK-dependentMLCphosphorylation via NF-kB p65 pathway.

Myosin Light Chain Kinase Inhibitor Inhibits Dextran Sulfate Sodium-Induced Colitis in Mice

Myosin light chain kinase (MLCK) plays a central role in the mechanisms of barrier dysfunction, and intestinal epithelial MLCK protein expression is upregulated in active ulcerative colitis (UC). ML-7, a MLCK inhibitor, has been used in many MLCK studies. However, the effect of ML-7 has never been estimated in colitis models. The aim of this study was to determine whether ML-7 can treat UC. Experimental colitis was induced and ML-7 was administered by intraperitoneal injection. The disease activity index (DAI) scores were evaluated and colon tissue was collected for the assessment of histological changes, myeloperoxidase (MPO) activity, and tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-13 and interleukin (IL)-17 levels. The small intestinal mucosa was ultrastructurally examined, epithelial MLCK protein expression and enzymatic activity were determined, and intestinal permeability was assayed using FITC-dextran 4000 (FD-4) and Evans blue (EB). ML-7 was found to be significantly effective in reducing the DAI scores and histological index scores, and decreasing MPO activity and TNF-α, IFN-γ, IL-13 and IL-17 levels. The small intestinal epithelial MLCK protein expression and enzymatic activity were downregulated by ML-7. The epithelial cells and intercellular tight junctions were ameliorated, and the amount of FD-4 in blood and EB permeating into the intestine were decreased by ML-7 in colitis mice. ML-7 has a significant anti-colitis effect in colitis mice. It is mainly associated with the inhibition of the epithelial MLCK protein expression, resulting in ameliorated intestinal mucosal permeability.

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.

Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light‐chain kinase (MLCK) and MLC phosphatase (MLCP)

To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of β-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to β-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone.

Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-alpha-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-alpha-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-alpha-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-alpha-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-alpha increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-alpha-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-alpha-induced activation. Glucocorticoids inhibit the TNF-alpha-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-alpha-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

Molecular mechanism of tumor necrosis factor-α modulation of intestinal epithelial tight junction barrier

A TNF-alpha-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions. Previous studies from our laboratory suggested that the TNF-alpha-induced increase in intestinal TJ permeability was mediated by an increase in myosin light chain kinase (MLCK) protein expression. However, the molecular mechanisms that mediate the TNF-alpha increase in intestinal TJ permeability and MLCK protein expression remain unknown. The purpose of this study was to delineate the intracellular and molecular mechanisms that mediate the TNF-alpha-induced increase in intestinal TJ permeability; using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. To examine the molecular mechanisms involved in the TNF-alpha regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-alpha treatment of filter-grown Caco-2 monolayers transfected with plasmid vector containing the MLCK promoter region produced an increase in MLCK promoter activity and MLCK transcription. The TNF-alpha-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation, and the inhibition of NF-kappaB activation prevented the TNF-alpha-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-alpha-induced activation of MLCK promoter was mediated by binding of the activated NF-kappaB p50/p65 dimer to the downstream kappaB binding site (-84 to -75) on the MLCK promoter region; deletion of the kappaB binding site prevented the TNF-alpha increase in promoter activity. Additionally, siRNA silencing of NF-kappaB p65 also prevented the TNF-alpha increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was mediated by NF-kappaB p50/p65 binding and activation of the MLCK promoter. NF-kappaB p50/p65 activation of the MLCK promoter then leads to a stepwise increase in MLCK transcription, expression and activity, and MLCK-mediated opening of the intestinal TJ barrier.

Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression

TNF-alpha plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-alpha-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-alpha-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-alpha (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-alpha increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-alpha-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-alpha increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-alpha increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-alpha increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-alpha increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg(2+)-myosin ATPase, and metabolic energy prevented the TNF-alpha increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-alpha-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-alpha increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

Gut edema alters expression of tight junction proteins

Abstract Introduction: We have previously shown that gut edema without ischemia/reperfusion injury increases intestinal permeability in the ileum. Intestinal permeability is regulated by tight junction proteins which control the movement of macromolecules between epithelial cells. The proteins occludin and claudin-1 are the structural components and myosin light chain kinase is the regulatory component of the tight junction which is reinforced by the actin cytoskeleton. We hypothesized that gut edema increases permeability by decreasing expression of tight junction proteins. Methods: At laparotomy, rats were given 80cc/kg of 0.9% saline + superior mesenteric vein partial occlusion (Edema) or sham surgery. At 12 hrs, ileum was harvested for evaluation of occludin, claudin-1, myosin light chain kinase (MLCK) and mature, filamentous actin (F-actin) by deconvolution microscopy. Tissue water was determined using wet to dry weight ratio. Data are expressed as mean +/− SEM, t-test, 4–5/group. Results: Gut edema significantly increased the wet to dry ratio compared to sham and was associated with a significant increase in occludin and claudin-1 protein expression. MLCK protein expression and F-actin were significantly decreased. ∗Wet to dry ratioOccludin (pixel units)Claudin-1 (pixel units)F-actin (pixel units)MLCK (pixel units)Sham3.22 ± 0.092251 ± 291906 ± 211973 ± 1151962 ± 307Edema3.69 ± 0.07∗3786 ± 330∗3356 ± 777∗602 ± 83∗666 ± 113∗∗Denotes a P Conclusions: Gut edema disrupts the cytoskeleton ring by decreasing the presence of mature, filamentous actin. This disruption does not rely on increased expression of MLCK, but may contribute to the increased expression of structural proteins claudin and occludin seen in gut edema.