Role of myosin light chain kinase in intestinal epithelial barrier defects in a rat model of bowel obstruction

Abstract

BackgroundBowel obstruction is a common cause of abdominal emergency, since the patients are at increased risk of septicemia resulting in high mortality rate. While the compartmentalized changes in enteric microfloral population and augmentation of bacterial translocation (BT) have already been reported using experimental obstruction models, alterations in epithelial permeability of the obstructed guts has not been studied in detail. Myosin light chain kinase (MLCK) is actively involved in the contraction of epithelial perijunctional actinomyosin ring and thereby increases paracellular permeability. In the current study we attempt to investigate the role of MLCK in epithelial barrier defects using a rat model of simple mechanical obstruction.MethodsWistar rats received intraperitoneal injection of ML-7 (a MLCK inhibitor) or vehicle at 24, 12 and 1 hrs before and 12 hrs after intestinal obstruction (IO). The distal small intestine was obstructed with a single ligature placed 10 cm proximal to the ileocecal junction in IO rats for 24 hrs. Sham-operated rats served as controls.ResultsMucosal injury, such as villous blunting and increased crypt/villus ratio, was observed in the distal small intestine of IO rats. Despite massive enterocyte shedding, intestinal villi were covered with a contiguous epithelial layer without cell apoptosis. Increased transmural macromolecular flux was noticed in the distal small intestine and the proximal colon after IO. The bacterial colony forming units in the spleen and liver of IO rats were significantly higher than those of sham controls. Addition of ML-7 ameliorated the IO-triggered epithelial MLC phosphorylation, mucosal injury and macromolecular flux, but not the level of BT.ConclusionsThe results suggest that IO-induced premature enterocytic sloughing and enhanced paracellular antigenic flux were mediated by epithelial MLCK activation. In addition, enteric bacteria may undergo transcytotic routes other than paracellular paths to cross the epithelium.

Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion

“…The bacterial colony-forming units (CFUs) were calculated and normalized per gram of tissue (CFU/g). 43,44 Ussing Chamber Studies and Macromolecular Flux Assay Muscle-stripped intestinal tissues were opened along the mesenteric border and mounted in Ussing chambers (WPI Instruments, USA). The opening of the chamber (2 cm 2 ) exposed the tissue to 10 ml of circulating oxygenated Krebs buffer.…”

Role of the Barrier Dysfunction of the Intestines in the Development of Alimentary Allergy in Children

“…Сокращение актино-миозиновых филаментов, которое открывает межклеточные пространства, связано с фос-форилированием легкой цепи (ЛЦ) миозина посредством особого фермента -миозин-ЛЦ-киназы [37]. Помимо физического открытия плотных связей, миозин-ЛЦ-киназа также обеспечивает эндоцитоз белков плотных контактов в вакуольные апикальные пространства.…”

Commensal bacterial internalization by epithelial cells: An alternative portal for gut leakiness

“…Bacterial endocytosis by epithelial cells was observed after 6 h of obstruction, whereas TJ damage was seen only after 24 h. 15,29,57 The TJ damage in gut tissues after 24 h of obstruction was evidenced by occludin cleavage, ZO-1 destruction, and increased tissue conductance and macromolecular permeability. 15,29,57 Interestingly, continuous epithelial lining on villus surface with no sign of cell death was found at both time points, ruling out loss of cell viability as a cause of bacterial transcellular passage. Normal crypt/villus ratio was observed after 6 h in comparison to villus blunting without crypt hyperplasia after 24 h, suggesting cell sloughing on villus tip may partly contribute to TJ damage at the later time point.…”

“…21,22,26 We and others demonstrated that bacterial endocytosis by epithelial cells preceded TJ damage under inflammatory stress in a doseand time-dependent manner. 15,29,38,57 Therefore, we suspect that bacterial entry through intermicrovillous lipid rafts may trigger activation of intracellular NLRs by the sensing of endosomally derived bacterial structural components (unrelated to virulence factors). The NLRmediated immune responses may adversely cause aggravated inflammatory and oxidative stress, leading to secondary TJ damage in epithelial cells (Fig.…”

“…As many previously established models, such as chemically induced colitis and pathogen infection, are confounded by co-existing transcellular and paracellular bacterial influx, we attempted to overcome this limitation by developing novel animal models that displayed transcellular passage of bacteria without TJ disruption. Two models with enteric bacterial dysbiosis (changes in numbers or composition of gut microbiota) were established, including bowel obstruction 15,29,57 and infection with antibiotic-resistant enterobacteria. 16 The bowel obstruction model induced commensal bacterial overgrowth after loop ligation in the distal small intestine.…”