The global incidence of cardiac diseases is increasing, imposing a substantial socioeconomic burden on healthcare systems. The pathogenesis of cardiovascular disease is complex and not fully understood, and the physiological function of the heart is inextricably linked to well-regulated cardiac muscle movement. Myosin light chain kinase (MLCK) is essential for myocardial contraction and diastole, cardiac electrophysiological homeostasis, vasoconstriction of vascular nerves and blood pressure regulation. In this sense, MLCK appears to be an attractive therapeutic target for cardiac diseases. MLCK participates in myocardial cell movement and migration through diverse pathways, including regulation of calcium homeostasis, activation of myosin light chain phosphorylation, and stimulation of vascular smooth muscle cell contraction or relaxation. Recently, phosphorylation of myosin light chains has been shown to be closely associated with the activation of myocardial exercise signaling, and MLCK mediates systolic and diastolic functions of the heart through the interaction of myosin thick filaments and actin thin filaments. It works by upholding the integrity of the cytoskeleton, modifying the conformation of the myosin head, and modulating innervation. MLCK governs vasoconstriction and diastolic function and is associated with the activation of adrenergic and sympathetic nervous systems, extracellular transport, endothelial permeability, and the regulation of nitric oxide and angiotensin II. Additionally, MLCK plays a crucial role in the process of cardiac aging. Multiple natural products/phytochemicals and chemical compounds, such as quercetin, cyclosporin, and ML-7 hydrochloride, have been shown to regulate cardiomyocyte MLCK. The MLCK-modifying capacity of these compounds should be considered in designing novel therapeutic agents. This review summarizes the mechanism of action of MLCK in the cardiovascular system and the therapeutic potential of reported chemical compounds in cardiac diseases by modifying MLCK processes.

UNASSIGNED: Pulmonary hypertension (PH) is a refractory disease characterized by elevated pulmonary artery pressure and resistance. Drag-reducing polymers (DRPs) are blood-soluble macromolecules that reduce vascular resistance by altering the blood dynamics and rheology. Our previous work indicated that polyethylene oxide (PEO) can significantly reduce the medial wall thickness and vascular resistance of the pulmonary arteries, but the specific mechanism is still unclear.
UNASSIGNED: This study was designed to investigate the role and mechanism of PEO on intracellular calcium [Ca2 +] i and cytoskeletal proteins of endothelial cells (ECs) induced by low shear stress (LSS) in PH. Primary Pulmonary Artery Endothelial Cells (PAECs) were subjected to steady LSS (1 dyn/cm2) or physiological shear stress (SS) (10 dyn/cm2) for 20 h in a BioFlux 200 flow system. Calcium influx assays were conducted to evaluate the mechanisms of PEO on [Ca2 +] i. Subsequently, taking the key protein that induces cytoskeletal remodeling, the regulatory light chain (RLC) phosphorylation, as the breakthrough point, this study focused on the two key pathways of PEO that regulate phosphorylation of RLC: Myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK) pathways.
UNASSIGNED: Our current research revealed that PEO at LSS (1 dyn/cm2) significantly suppressed LSS-induced [Ca2 +] i and the expression level of transient receptor potential channel 1(TRPC1). In addition, ECs convert LSS stimuli into the upregulation of cytoskeletal proteins, including filamentous actin (F-actin), MLCK, ROCK, p-RLC, and pp-RLC. Further experiments using pharmacological inhibitors demonstrated that PEO at the LSS downregulated cytoskeleton-related proteins mainly through the ROCK and MLCK pathways.
UNASSIGNED: This study considered intracellular calcium and cytoskeleton rearrangement as entry points to study the application of PEO in the biomedical field, which has important theoretical significance and practical application value for the treatment of PH.

OBJECTIVE: Our previous studies have found that the composition ratio of Prevotella melaninogenica (Pm) on buccal mucosa surface of oral lichen planus (OLP) patients increased significantly compared with control. Furthermore, Pm could invade the epithelium of OLP patients. This study aimed to further explore the impact of Pm on oral keratinocytes.
METHODS: The Pm-human oral keratinocyte (HOK) co-culture model was established to detect monolayer permeability, zona occludens-1 (ZO-1) expression, and intracellular survival of Pm. We performed RNA-seq followed by identification of differentially expressed genes (DEGs) and Gene Ontology (GO) analysis, with a particular focus on myosin light chain kinase (MLCK). An MLCK inhibitor ML-7 was utilized in Pm-HOK co-culture model to assess its effects on monolayer permeability and ZO-1 expression.
RESULTS: HOK monolayer permeability was increased, and ZO-1 expression was decreased after co-culture (p < 0.05). Pm could survive in HOK cells. RNA-seq revealed MLCK was an upregulated common DEG. The expression of MLCK in the Pm-HOK co-culture model was upregulated. Inhibition of MLCK rescued the increased epithelial permeability, and ZO-1 expression was upregulated (p < 0.05).
CONCLUSIONS: MLCK may be involved in disrupting epithelial barrier function by Pm.

Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.

In severe acute pancreatitis (SAP), intestinal mucosal barrier damage can cause intestinal bacterial translocation and induce or aggravate systemic infections. Heme oxygenase-1 (HO-1) is a validated antioxidant and cytoprotective agent. This research aimed to investigate the effect and mechanism of HO-1 on SAP-induced intestinal barrier damage in SAP rats. Healthy adult male Sprague-Dawley rats were randomly separated into the sham-operated group, SAP group, SAP + Hemin group, and SAP + Znpp group. The rat model of SAP was established by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Hemin (a potent HO-1 activator) and Znpp (a competitive inhibitor of HO-1) were injected intraperitoneally in the selected groups 24 h before SAP. Serum and intestinal tissue samples were collected for analysis after 24 h in each group. Hemin pretreatment significantly reduced systemic inflammation, intestinal oxidative stress, and intestinal epithelial apoptosis in SAP by increasing HO-1 expression. Meanwhile, pretreatment with Hemin abolished the inhibitory effect on the expression of the tight junction proteins and significantly inhibited the activation of the MLCK/P-MLC signaling pathway. Conversely, ZnPP completely reversed these effects. Our study indicates that upregulation of HO-1 expression attenuates the intestinal mucosal barrier damage in SAP. The protective effect of HO-1 on the intestine is attributed to MLCK/p-MLC signaling pathway inhibition.

Aflatoxin B1 (AFB1) is a widespread mycotoxin in food and feed. Although the liver is the main target organ of AFB1, the intestine is the first exposure organ to AFB1. However, the mechanism by which AFB1 induced intestinal barrier dysfunction via regulating the farnesoid X receptor (FXR)-mediated myosin light chain kinase (MLCK) signaling pathway has rarely been studied. In vivo, AFB1 exposure significantly decreased the small intestine length and increased the intestinal permeability. Meanwhile, AFB1 exposure markedly suppressed the protein expressions of FXR, ZO-1, occludin, and claudin-1 and enhanced the protein expression of MLCK. In vitro, AFB1 exposure induced intestinal barrier dysfunction by the elevation in the FITC-Dextran 4 kDa flux and inhibition in the transepithelial electrical resistance in a dose-dependent manner. In addition, AFB1 exposure downregulated the mRNA and protein expressions of FXR, ZO-1, occludin, and claudin-1, redistributed the ZO-1 protein, and enhanced the protein expressions of MLCK and p-MLC. However, fexaramine (Fex, FXR agonist) pretreatment markedly reversed the AFB1-induced FXR activity reduction, MLCK protein activation, and intestinal barrier impairment in vitro and in vivo. Moreover, pretreatment with the inhibition of MLCK with ML-7 significantly alleviated the AFB1-induced intestinal barrier dysfunction and tight junction disruption in vitro. In conclusion, AFB1 induced intestinal barrier impairment via regulating the FXR-mediated MLCK signaling pathway in vitro and in vivo and provided novel insights to prevent mycotoxin poisoning in the intestine.

BACKGROUND: Intestinal barrier breakdown, a frequent complication of intestinal ischemia-reperfusion (I/R) including dysfunction and the structure changes of the intestine, is characterized by a loss of tight junction and enhanced permeability of the intestinal barrier and increased mortality. To develop effective and novel therapeutics is important for the improvement of outcome of patients with intestinal barrier deterioration. Recombinant human angiopoietin-like protein 4 (rhANGPTL4) is reported to protect the blood-brain barrier when administered exogenously, and endogenous ANGPTL4 deficiency deteriorates radiation-induced intestinal injury.
OBJECTIVE: To identify whether rhANGPTL4 may protect intestinal barrier breakdown induced by I/R.
METHODS: Intestinal I/R injury was elicited through clamping the superior mesenteric artery for 60 min followed by 240 min reperfusion. Intestinal epithelial (Caco-2) cells and human umbilical vein endothelial cells were challenged by hypoxia/ reoxygenation to mimic I/R in vitro.
RESULTS: Indicators including fluorescein isothiocyanate-conjugated dextran (4 kilodaltons; FD-4) clearance, ratio of phosphorylated myosin light chain/total myosin light chain, myosin light chain kinase and loss of zonula occludens-1, claudin-2 and VE-cadherin were significantly increased after intestinal I/R or cell hypoxia/reoxygenation. rhANGPTL4 treatment significantly reversed these indicators, which were associated with inhibiting the inflammatory and oxidative cascade, excessive activation of cellular autophagy and apoptosis and improvement of survival rate. Similar results were observed in vitro when cells were challenged by hypoxia/reoxygenation, whereas rhANGPTL4 reversed the indicators close to normal level in Caco-2 cells and human umbilical vein endothelial cells significantly.
CONCLUSIONS: rhANGPTL4 can function as a protective agent against intestinal injury induced by intestinal I/R and improve survival via maintenance of intestinal barrier structure and functions.

The neonicotinoid pesticide, imidacloprid (IMI), is frequently detected in the environment and in foods. It is absorbed and metabolized by the intestine; however, its effects on intestinal barrier integrity are not well studied. We investigated whether IMI disrupts the permeability of the intestinal epithelial barrier via in vivo tests on male Wistar rats, in vitro assays using the human intestinal epithelial cell line, Caco-2, and in silico analyses. A repeated oral dose 90-day toxicity study was performed (0.06 mg/kg body weight/day). IMI exposure significantly increased intestinal permeability, which led to significantly elevated serum levels of endotoxin and inflammatory biomarkers (tumor necrosis factor-alpha and interleukin-1 beta) without any variation in body weight. Decreased transepithelial electrical resistance with increased permeability was also observed in 100 nM and 100 μM IMI-treated Caco-2 cell monolayers. Amounts of tight junction proteins in IMI-treated colon tissues and between IMI-treated Caco-2 cells were significantly lower than those of controls. Increased levels of myosin light chain phosphorylation, myosin light chain kinase (MLCK), and p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) phosphorylation were found in IMI-exposed cells compared with control cells. Furthermore, the barrier loss caused by IMI was rescued by the MLCK inhibitor, ML-7, and cycloheximide. Pregnane X receptor (PXR, NR1I2) was inhibited by low-dose IMI treatment. In silico analysis indicated potent binding sites between PXR and IMI. Together, these data illustrate that IMI induces intestinal epithelial barrier disruption and produces an inflammatory response, involving the down-regulation of tight junctions and disturbance of the PXR-NF-κB p65-MLCK signaling pathway. The intestinal barrier disruption caused by IMI deserves attention in assessing the safety of this neonicotinoid pesticide.

Evidence 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.
In 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).
The 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.
In 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.

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.