Myosin Light Chain Kinase Signaling Research Articles

New perspectives on the role of vitamin D in the mucosal barrier system of fish gills infected with Flavobacterium columnare

The mucosal barrier of vertebrates comprises a unique array of physical elements and bioactive molecules that act in concert to protect the host against pathogens. Vitamin D (VD), an essential nutrient for animals, along with its receptor (VDR), plays a major role in immunity. However, the correlation between VD/VDR and the mucosal barrier system remains unclear. Here, we employed the fish gill as a model and evaluated the role of VD as a dietary supplement in modifying this typical mucosal barrier system. Varies levels of VD (15.2, 364.3, 782.5, 1167.9, 1573.8, and 1980.1 IU/kg) were formulated and fed to grass carp (Ctenopharyngodon idella) for 70 d. Subsequently, fish were challenged with Flavobacterium columnare, a pathogen in epithelial tissues. Following infection, dietary VD alleviated gill rot and damage caused by F. columnare. Further investigations demonstrated that VD improved physical barrier function by elevating the antioxidant capacity and tight junction barriers, and moderated excessive apoptosis. The promoted physical barrier function was related to nuclear factor E2-related factor 2 and myosin light-chain kinase signaling. Additionally, VD treatment enhanced the immune barrier function by boosting disease resistance, producing antibacterial compounds and immunoglobulins, downregulating pro-inflammatory cytokines, and upregulating the gene expression of anti-inflammatory cytokines, which correlated with nuclear factor kappa B and the target of rapamycin signaling pathways. Thus, VD strengthened pathogen defenses in the gill mucosal barrier system. This finding supports the rationale for VD intervention in infectious diseases and will be critical for the viability of sustainable practices in aquaculture.

Regulation of mouse digestive function, intestinal mucosal barrier function, and inflammatory reaction by lycium barbarum polysaccharide pathway through myosin light chain kinase

This research investigated the impacts of lycium barbarum polysaccharide (LBP) on the digestive function, intestinal mucosal barrier function, inflammatory response, and myosin light chain kinase (MLCK) signaling pathway in immunosuppressed mice. 70 mg/kg cyclophosphamide was injected into abdomen for the preparation of immune suppression model. Healthy BALB/c mice served as control for the analysis of the differences in gastrointestinal motility and absorptive capacity, intestinal mucosal barrier function, the phagocytic ability of abdominal macrophages, serum immune factor and inflammatory factor levels, and the activation status of the MLCK signaling pathway after continuous gavage with 100 mg/kg LBP. Results revealed a decrease in d-xylose content, phagocytic rate, index of abdominal macrophages, and spleen index in the serum and urine of model mice compared to those of controls. In addition, levels of IgA, IgG, IgM, IL-6 (interleukin-6), IL-12, and interferon-γ (IFN-γ) decreased, while MLCK and myosin light chain (MLC) levels rose (P < 0.01). Versus those in Model group, urine d-xylose content, phagocytic rate, index of abdominal macrophages, spleen index, and the levels of IgA, IgG, IgM, IL-6, IL-12, and IFN-γ of mice undergoing the gavage with LBP increased, while MLCK and p-MLC levels declined (P < 0.05). In conclusion, LBP improved digestive absorption and immune function of immunosuppressed mice and regulated intestinal mucosal barrier immune system by inhibiting MLCK signaling pathway activation.

Emerging role of vitamin D3 in alleviating intestinal structure injury caused by Aeromonas hydrophila in grass carp (Ctenopharyngodon idella)

Bacterial pathogens destroy the structural integrity of functional organs in fish, leading to severe challenges in the aquaculture industry. Vitamin D3 (VD3) prevents bacterial infections and strengthens immune system function via vitamin D receptor (VDR). However, the correlation between VD3/VDR and the structural integrity of functional organs remains unclarified. This study aimed to investigate the influence of VD3 supplementation on histological characteristics, apoptosis, and tight junction characteristics in fish intestine during pathogen infection. A total of 540 healthy grass carp (257.24 ± 0.63 g) were fed different levels of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) for 70 d. Subsequently, fish were challenged with Aeromonas hydrophila, a pathogen that causes intestinal inflammation. Our present study demonstrated that optimal supplementation with VD3 (1) alleviated intestinal structural damage, and inhibited oxidative damage by reducing levels of oxidative stress biomarkers; (2) attenuated excessive apoptosis-related death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling (P < 0.05); (3) enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling (P < 0.05); and (4) elevated VDR isoform expression in fish intestine (P < 0.05). Overall, the results demonstrated that VD3 alleviates oxidative injury, apoptosis, and the destruction of tight junction protein under pathogenic infection, thereby strengthening pathogen defenses in the intestine. This finding supports the rationale for VD3 intervention as an essential practice in sustainable aquaculture.

Aflatoxin B1 damaged structural barrier through Keap1a/Nrf2/ MLCK signaling pathways and immune barrier through NF-κB/ TOR signaling pathways in gill of grass carp (Ctenopharyngodon idella)

Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 μg/kg diet.

Melatonin Potentiates Sensitivity to 5-Fluorouracil in Gastric Cancer Cells by Upregulating Autophagy and Downregulating Myosin Light-Chain Kinase.

5-Fluorouracil is an effective chemotherapeutic drug for gastric cancer. However, the acquisition of chemotherapeutic resistance remains a challenge in treatment. Melatonin can enhance the therapeutic effect of 5-fluorouracil; however, the underlying mechanisms are not well understood. We investigated the effects of combinations of melatonin and 5-fluorouracil on the proliferation, migration and invasion of gastric cancer cells. Melatonin significantly potentiated the 5-fluorouracil-mediated inhibition of proliferation, migration and invasion in gastric cancer cells, which potentiates sensitivity to 5-FU by promoting the activation of Beclin-1-dependent autophagy and targeting the myosin light-chain kinase (MLCK) signaling pathway. Previous studies have shown that autophagy might be associated with the MLCK signaling pathway. The autophagy inhibitor, 3-methyladenine, effectively rescued the migratory and invasive capabilities of gastric cancer cells, while also reducing expression level of MLCK and the phosphorylation level of MLC. This indicates that autophagy is involved in tumor metastasis, which may be related to inhibition of the MLCK signaling pathway. Our findings indicate that melatonin can improve the effectiveness of 5-fluorouracil in gastric cancer and could be used as a supplemental agent in the treatment of gastric cancer with 5-fluorouracil.

AGEs induce endothelial cells senescence and endothelial barrier dysfunction via miR-1-3p/MLCK signaling pathways

Advanced glycation end products (AGEs) disturb endothelial barrier function and contribute to age-related diseases. As microRNAs (miRNAs) are potential therapeutic agents, targeting AGEs-associated signaling using miRNAs in endothelial cells may be an effective intervention strategy for age-related vascular disorders. This study investigated the effects of AGEs on the endothelial cell senescence and barrier function in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with AGEs and transfected with miRNA-1-3p mimics to induce overexpression of miR-1-3p. Senescence-associated β-galactosidase (SA-β-Gal) staining and senescence-related proteins P53, P21, and P16 were detected to evaluate the endothelial cell senescence. The expression levels of myosin light chain kinase (MLCK) signaling and transendothelial electric resistance (TEER) were used to indicate endothelial barrier function. AGEs significantly increased SA-β-gal staining-positive cells accompanied by the upregulation of P53, P21, and P16 expression. AGEs also damaged endothelial barrier function by decreasing TEER and increasing zonula occludens protein 1, p-MLC/MLC, and MLCK. miRNA-1-3p was significantly reduced in HUVECs treated with AGEs. miR-1-3p overexpression decreased MLCK signal and improved AGEs-induced endothelial barrier function impairment. Meanwhile, miR-1-3p overexpression ameliorated oxidative stress and endothelial cell senescence induced by AGEs. AGEs induced endothelial cell senescence and endothelial barrier dysfunction by regulating miR-1-3p/MLCK signaling pathway.

Lactobacillus reuteri 1 Enhances Intestinal Epithelial Barrier Function and Alleviates the Inflammatory Response Induced by Enterotoxigenic Escherichia coli K88 via Suppressing the MLCK Signaling Pathway in IPEC-J2 Cells.

Intestinal epithelial barrier injury disrupts immune homeostasis and leads to many intestinal disorders. Lactobacillus reuteri (L. reuteri) strains can influence immune system development and intestinal function. However, the underlying mechanisms of L. reuteri LR1 that regulate inflammatory response and intestinal integrity are still unknown. The present study aimed to determine the effects of LR1 on the ETEC K88-induced intestinal epithelial injury on the inflammatory response, intestinal epithelial barrier function, and the MLCK signal pathway and its underlying mechanism. Here, we showed that the 1 × 109 cfu/ml LR1 treatment for 4 h dramatically decreased interleukin-8 (IL-8) and IL-6 expression. Then, the data indicated that the 1 × 108 cfu/ml ETEC K88 treatment for 4 h dramatically enhanced IL-8, IL-6, and tumor necrosis factor-α (TNF-α) expression. Furthermore, scanning electron microscope (SEM) data indicated that pretreatment with LR1 inhibited the ETEC K88 that adhered on IPEC-J2 and alleviated the scratch injury of IPEC J2 cells. Moreover, LR1 pretreatment significantly reversed the declined transepithelial electrical resistance (TER) and tight junction protein level, and enhanced the induction by ETEC K88 treatment. Additionally, LR1 pretreatment dramatically declined IL-8, IL-17A, IL-6, and TNF-α levels compared with the ETEC K88 group. Then, ETEC K88-treated IPEC-J2 cells had a higher level of myosin light-chain kinase (MLCK), higher MLC levels, and a lower Rho-associated kinase (ROCK) level than the control group, while LR1 pretreatment significantly declined the MLCK and MLC expression and enhanced ROCK level in the ETEC K88-challenged IPEC-J2 cells. Mechanistically, depletion of MLCK significantly declined MLC expression in IPEC-J2 challenged with ETEC K88 compared to the si NC+ETEC K88 group. On the other hand, the TER of the si MLCK+ETEC K88 group was higher and the FD4 flux in the si MLCK+ETEC K88 group was lower compared with the si NC+ETEC K88 group. In addition, depletion of MLCK significantly enhanced Claudin-1 level and declined IL-8 and TNF-α levels in IPEC-J2 pretreated with LR1 followed by challenging with ETEC K88. In conclusion, our work indicated that L. reuteri LR1 can decline inflammatory response and improve intestinal epithelial barrier function through suppressing the MLCK signal pathway in the ETEC K88-challenged IPEC-J2.

The role of orally ingested milk fat globule membrane on intestinal barrier functions evaluated with a suckling rat pup supplementation model and a human enterocyte model

Milk fat globule membrane (MFGM), the membrane surrounding secreted fat droplets in milk, contains components involved in a wide range of bioprocesses including cell proliferation and differentiation. The intestine is relatively immature and permeable at birth. Since MFGM is partly resistant to digestion in infancy, we hypothesized that orally ingested MFGM promotes intestinal development by enhancing intestinal barrier functions in early life. An established suckling rat model was used; Sprague–Dawley rats were bred, and litters were culled to 10 pups/dam. Pups were supplemented orally with MFGM (0, 100, or 300 mg/kg/d) from postnatal day 1–20. Intestine samples were collected for histology, real-time quantitative PCR, immunoblotting, and immunohistochemistry analysis. Additionally, differentiated Caco-2 cells were used to assess effects of MFGM on the human intestinal barrier. Control and MFGM-supplemented rat pups showed similar growth. Intestinal differentiation and expression of tight junction proteins in jejunum and colon were significantly increased by orally ingested MFGM, and MFGM supplementation significantly activated PI3K/Akt/mTOR, mitogen-activated protein kinases, and myosin light chain kinase signaling pathways, suggesting that MFGM promotes intestinal development by triggering various signaling pathways. In human enterocytes (polarized Caco-2 cells), MFGM (400 µg/mL for 72 h) decreased permeability, as revealed by increased transepithelial electrical resistance. In Caco-2 cells, MFGM also enhanced expression of tight junction proteins, including claudin-4 and ZO-2. In conclusion, orally ingested MFGM may exert beneficial roles in intestinal development by activating various cell signaling pathways to upregulate tight junction proteins and thereby increasing intestinal barrier functions.

Role of prostacyclin and thromboxane A2 in pulmonary hyper-permeability induced by mechanical ventilation in rabbits

To explore the role of prostacyclin (PGI2) and thromboxane A2 (TXA2) in lung hyper-permeability induced by mechanical ventilation (MV) in rabbits. Forty-eight healthy Japanese white rabbits were randomly allocated to vehicle treatment group (group V), tranylcypromine (a PGI2 synthase inhibitor) treatment group (group T), dazoxiben (a TXA2 synthase inhibitor) treatment group (group D), vehicle-treated MV group (group VM), tranylcyprominetreated MV group (group TM) and dazoxiben-treated MV group (group DM). The contents of PGI2 and TXA2 in the lung tissues and TNF-α level in BALF and lung tissues were measured by ELISA. The lung wet/dry weight (W/D) ratio, lung permeability index and pulmonary expressions of myosin light chain kinase (MLCK) protein and mRNA were detected to evaluate the pulmonary permeability. The severities of lung injury were assessed by lung histological scores. The measured parameters did not differ significantly among the rabbits receiving different treatments without MV. In rabbits in group VM, the contents of PGI2 and TXA2 in the lungs, TNF-α in BALF and lung tissues, PGI2/TXA2 ratio, lung W/D ratio, lung permeability index, pulmonary expressions of MLCK protein and mRNA and histological scores of the lungs all increased significantly (P < 0.05) as compared with those in group V, group T and group D. In rabbits undergoing MV, inhibition of PGI2 production by tranylcypromine significantly decreased the PGI2/TXA2 ratio (P < 0.05), further enhanced the production of TNF-α in the BALF and lung tissue (P < 0.05), and worsened lung hyper-permeability and lung injury (P < 0.05), while treatment with dazoxiben significantly reduced TXA2 production in the lung tissue (P < 0.05), increased the PGI2/TXA2 ratio (P < 0.05) and decreased TNF-α production in the BALF and lung tissue (P < 0.05), thus resulting in alleviated lung hyperpermeability and lung injury (P < 0.05). PGI2 plays a protective role against MV-induced lung hyper-permeability and lung injury by downregulating TNF-α/MLCK signaling pathway, while TXA2 can exacerbate MV-induced lung hyperpermeability in rabbits by up-regulating TNF-α/ MLCK signaling pathway.

CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.

Cis 9, trans 11, but not trans 10, cis 12 CLA isomer, impairs intestinal epithelial barrier function in IPEC-J2 cells and mice through activation of GPR120-[Ca2+]i and the MLCK signaling pathway.

This study aimed to investigate the effects of conjugated linoleic acid (CLA) on intestinal epithelial barrier function and explore the underlying mechanisms. IPEC-J2 cells and mice were treated with different CLA isomers. The intestinal epithelial barrier function determined by transepithelial electrical resistance (TEER), the expression of tight junction proteins, and the involvement of G-protein coupled receptor 120 (GPR120), intracellular calcium ([Ca2+]i) and myosin light chain kinase (MLCK) were assessed. In vitro, c9, t11-CLA, but not t10, c12-CLA isomer, impaired epithelial barrier function in IPEC-J2 by downregulating the expression of tight junction proteins. Meanwhile, c9, t11-CLA isomer enhanced GPR120 expression, while knockdown of GPR120 eliminated the impaired epithelial barrier function induced by c9, t11-CLA isomer. In addition, c9, t11-CLA isomer increased [Ca2+]i and activated the MLCK signaling pathway in a GPR120-dependent manner. However, chelation of [Ca2+]i reversed c9, t11-CLA isomer-induced MLCK activation and the epithelial barrier function impairment of IPEC-J2. Furthermore, inhibition of MLCK totally abolished the impairment of epithelial barrier function induced by c9, t11-CLA. In vivo, dietary supplementation of c9, t11-CLA rather than t10, c12-CLA isomer decreased the expression of intestinal tight junction proteins and GPR120, increased intestinal permeability, and activated the MLCK signaling pathway in mice. Taken together, our findings showed that c9, t11-CLA, but not t10, c12-CLA isomer, impaired intestinal epithelial barrier function in IPEC-J2 cells and mice through activation of GPR120-[Ca2+]i and the MLCK signaling pathway. These data provided new insight into the regulation of the intestinal epithelial barrier by different CLA isomers and more references for CLA application in humans and animals.

Effects of advanced glycation end products on endothelial cell senescence and endothelial barrier dysfunction

Objective To investigate the effects and mechanism of advanced glycosylation end products(AGEs)on endothelial cell senescence and endothelial barrier dysfunction. Methods Human umbilical vein endothelial cells(HUVECs)were isolated and cultured.The cells were randomized into three groups: the control group(normal medium), the bovine serum albumin-treated group(BSA control group)and AEGs group(treated with AEGs-BSA). Senescence of HUVECs were detected by senescence-associated β-galactosidase(SA-beta-Gal)staining.The mRNA and protein expressions of senescence-related genes of p53, p21 and p16 in each group were determined by reverse transcription and real-time PCR(RT-qPCR)and Western blot.Reactive oxygen species(ROS)level was determined by dichlorodihdrofluorescence diacetate(DCFH-DA). The transendothelial electric resistance(TER)were measured by endothelial electric resistance meter.The protein levels of myosin light chain kinase(MLCK), phosphorylated myosin light chain(p-MLC), myosin light chain(MLC)were detected by Western blot. Results Compared with the control group and the BSA control group, the AGEs group showed the significantly increased positive rate of senescence-associated SA-beta-Gal staining(67.30±0.75% vs.7.81±0.35% and 7.64±0.91%, respectively, P 0.05), while TER was lower in the AEGs group than in control group and the BSA group[(27.0±4.2)Ω·cm2vs. (48.0±6.3)Ω·cm2 and (42.0±7.8)Ω·cm2, P 0.05], while was increased in the AEGs group versus control group and the BSA group[(69.31±8.47)% vs.(38.36±8.55)% and (41.67±6.93)%, P<0.05). The protein expression levels of MLK and p-MLC/MLC were higher in the AGEs group than in the control group and the BSA group(P<0.05). Conclusions AGEs may lead to endothelial cell senescence and endothelial barrier dysfunction by promoting ROS production and oxidative stress, and by regulating MLCK signaling pathway. Key words: Glycosylation end products, advanced; Endothelial cell; Aging

Endocytosis of CF in marginal cells of stria vascularis regulated by ROCK and MLCK signaling cascade, but not G-proteins

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis.Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope.Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade.Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization.

Deoxynivalenol decreased the growth performance and impaired intestinal physical barrier in juvenile grass carp (Ctenopharyngodon idella)

Deoxynivalenol (DON) is one of the most common mycotoxin contaminants of animal feed worldwide and brings significant threats to the animal production. However, studies concerning the effect of DON on fish intestine are scarce. This study explored the effects of DON on intestinal physical barrier in juvenile grass carp (Ctenopharyngodon idella). A total of 1440 juvenile grass carp (12.17 ± 0.01 g) were fed six diets containing graded levels of DON (27, 318, 636, 922, 1243 and 1515 μg/kg diet) for 60 days. This study for the first time documented that DON caused body malformation in fish, and histopathological lesions, oxidative damage, declining antioxidant capacity, cell apoptosis and destruction of tight junctions in the intestine of fish. The results indicated that compared with control group (27 μg/kg diet), DON: (1) increased the reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) content, and up-regulated the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1: Keap1a but not Keap1b), whereas decreased glutathione (GSH) content and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes (except GSTR in MI) and NF-E2-related factor 2 (Nrf2), as well as the protein levels of Nrf2 in fish intestine. (2) up-regulated cysteinyl aspartic acid-protease (caspase) -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl2-associated X protein (Bax), Fas ligand (FasL) and c-Jun N-terminal protein kinase (JNK) mRNA levels, whereas down-regulated B-cell lymphoma-2 (bcl-2) and myeloid cell leukemia-1 (Mcl-1) mRNA levels in fish intestine. (3) down-regulated the mRNA levels of ZO-1, ZO-2b, occludin, claudin-c, -f, -7a, -7b, -11 (except claudin-b and claudin-3c), whereas up-regulated the mRNA levels of claudin-12, -15a (not -15b) and myosin light chain kinase (MLCK) in fish intestine. All above data indicated that DON caused the oxidative damage, apoptosis and the destruction of tight junctions via Nrf2, JNK and MLCK signaling in the intestine of fish, respectively. Finally, based on PWG, FE, PC and MDA, the safe dose of DON for grass carp were all estimated to be 318 μg/kg diet.

Dietary zinc deficiency reduced growth performance, intestinal immune and physical barrier functions related to NF-κB, TOR, Nrf2, JNK and MLCK signaling pathway of young grass carp (Ctenopharyngodon idella)

Our study investigated the effects of dietary zinc (Zn) deficiency on growth performance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (244.14 ± 0.40 g) were fed graded levels of zinc lactate (10.71, 30.21, 49.84, 72.31, 92.56, 110.78 mg Zn/kg diet) and one zinc sulfate group (56.9 mg Zn/kg diet) for 60 days. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. These results indicated that compared with optimal dietary Zn level, dietary Zn deficiency (10.71 mg/kg diet) decreased the production of antibacterial compounds, up-regulated pro-inflammatory cytokines related to nuclear factor kappa B (NF-κB) and down-regulated anti-inflammatory cytokines related to target of rapamycin (TOR) in three intestinal segments of young grass carp (P < 0.05), suggesting that dietary Zn deficiency could impair intestinal immune barrier of fish; decreased the activities and mRNA levels of antioxidant enzymes related to NF-E2-related factor 2 (Nrf2), up-regulated the mRNA levels of caspase-3, -7, -8, -9 related to p38 mitogen activated protein (p38 MAPK) and c-Jun N-terminal protein kinase (JNK), down-regulated the mRNA levels of tight junction complexes (TJs) related to myosin light chain kinase (MLCK) in three intestinal segments of young grass carp (P < 0.05), demonstrating that dietary Zn deficiency could injury intestinal physical barrier of fish. Besides, the Zn requirements (zinc lactate as Zn source) based on percent weight gain (PWG), against enteritis morbidity, acid phosphatase (ACP) activity in the proximal intestine (PI) and malondialdehyde (MDA) content in the PI of young grass carp was estimated to be 61.2, 61.4, 69.2 and 69.5 mg/kg diet, respectively. Finally, based on specific growth rate (SGR), feed efficiency (FE) and against enteritis morbidity of young grass carp, the efficacy of zinc lactate relative to zinc sulfate were 132.59%, 135.27% and 154.04%, respectively.

The decreased growth performance and impaired immune function and structural integrity by dietary iron deficiency or excess are associated with TOR, NF-κB, p38MAPK, Nrf2 and MLCK signaling in head kidney, spleen and skin of grass carp (Ctenopharyngodon idella)

This study was conducted to investigate the effects of dietary iron on the growth, and immune function and structural integrity in head kidney, spleen and skin as well as the underlying signaling of young grass carp (Ctenopharyngodon idella). Total 630 grass carp (242.32 ± 0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 mg/kg (diets 2–6 were added with ferrous fumarate) and 73.50 mg/kg (diet 7 was added with ferrous sulfate) diet for 60 days. Then, a challenge test was conducted by infection of Aeromonas hydrophila for 14 days. The results firstly showed that compared with optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated the mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65, IκB kinases β (IKKβ) and eIF4E-binding protein (4E-BP) in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired immune function in head kidney and spleen of fish. Secondly, iron deficiency down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), and inhibitor of apoptosis protein (IAP), and decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction complexes, and up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), Fas ligand (FasL), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12 and myosin light chain kinase (MLCK), and increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired structural integrity in head kidney and spleen of fish. Thirdly, iron deficiency increased skin hemorrhage and lesion morbidity, and impaired immune function and structural integrity in skin of fish. Fourthly, iron excess decreased growth and impaired the immune function and structural integrity in head kidney, spleen and skin of fish. Besides, in young grass carp, based on PWG and ability against skin hemorrhage and lesion, the efficacy of ferrous fumarate relative to ferrous sulfate was 140.32% and 126.48%, respectively, and the iron requirements based on PWG, ability against skin hemorrhage and lesion, ACP activities and MDA contents in head kidney and spleen were estimated to be 75.65, 87.03, 79.74, 78.93, 83.17 and 82.14 mg/kg diet (based on ferrous fumarate), respectively.

From the Cover: MechanisticInsights in Cytotoxic and Cholestatic Potential of the Endothelial Receptor Antagonists Using HepaRG Cells.

Several endothelin receptor antagonists (ERAs) have been developed for the treatment of pulmonary arterial hypertension (PAH). Some of them have been related to clinical cases of hepatocellular injury (sitaxentan [SIT]) and/or cholestasis (bosentan [BOS]). We aimed to determine if ambrisentan (AMB) and macitentan (MAC), in addition to BOS and SIT, could potentially cause liver damage in man by use of human HepaRG cells. Our results showed that like BOS, MAC-induced cytotoxicity and cholestatic disorders characterized by bile canaliculi dilatation and impairment of myosin light chain kinase signaling. Macitentan also strongly inhibited taurocholic acid and carboxy-2',7'-dichlorofluorescein efflux while it had a much lower inhibitory effect on influx activity compared to BOS and SIT. Moreover, these three drugs caused decreased intracellular accumulation and parallel increased levels of total bile acids (BAs) in serum-free culture media. In addition, all drugs except AMB variably deregulated gene expression of BA transporters. In contrast, SIT was hepatotoxic without causing cholestatic damage, likely via the formation of reactive metabolites and AMB was not hepatotoxic. Together, our results show that some ERAs can be hepatotoxic and that the recently marketed MAC, structurally similar to BOS, can also cause cholestatic alterations in HepaRG cells. The absence of currently known or suspected cases of cholestasis in patients suffering from PAH treated with MAC is rationalized by the lower therapeutic doses and Cmax, and longer receptor residence time compared to BOS.

Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury.

Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions, but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt tight junction barrier function via MLCK activation and myosin light chain (MLC) phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 h followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and FITC-dextran flux. Tight junction proteins and MLCK signaling were assessed by Western blotting, real-time PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) by using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared with control cells, as determined by TER and dextran fluxes (P < 0.05). The tight junction protein occludin was internalized during A/R injury and relocalized to the region of the tight junction after 4 h of recovery. MLC phosphorylation was significantly increased by A/R injury (P < 0.05), and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.

1,25-Dihydroxyvitamin D Protects Intestinal Epithelial Barrier by Regulating the Myosin Light Chain Kinase Signaling Pathway.

The myosin light chain kinase (MLCK) pathway controls intestinal epithelial barrier permeability by regulating the tight junction. 1,25-dihydroxyvitamin D (1,25(OH)2D3)-vitamin D receptor (VDR) signaling protects the epithelial barrier, but the molecular mechanism is incompletely understood. MLCK activation and barrier permeability were studied using monolayers of HCT116, Caco-2, and SW480 cells treated with tissue necrosis factor α with or without 1,25(OH)2D3. The MLCK pathway was analyzed in normal and inflamed colonic biopsies from patients with ulcerative colitis. Colonic mucosal barrier permeability and MLCK activation were also investigated using trinitrobenzene sulfonic acid-induced colitis models in vitamin D analog paricalcitol-treated wild-type mice and mice carrying VDR deletion in colonic epithelial cells. Tissue necrosis factor α increased cell monolayer permeability and induced long isoform of MLCK expression and myosin II regulatory light chain (MLC) phosphorylation, and 1,25(OH)2D3 blocked tissue necrosis factor α-induced increases in monolayer permeability and MLCK-MLC pathway activation by a VDR-dependent fashion. 1,25(OH)2D3 directly suppressed long MLCK expression by attenuating NF-κB activation, and chromatin immunoprecipitation assays confirmed that 1,25(OH)2D3 disrupted p65 binding to 3 κB sites in long MLCK gene promoter. In human ulcerative colitis biopsies, VDR reduction was associated with increases in long MLCK expression and MLC phosphorylation. In trinitrobenzene sulfonic acid colitis models, paricalcitol ameliorated colitis, attenuated the increase in mucosal barrier permeability, and inhibited long MLCK induction and MLC phosphorylation. In contrast, mice with colonic epithelial VDR deletion exhibited more robust increases in mucosal barrier permeability and MLCK activation compared with wild-type mice. These data demonstrate that 1,25(OH)2D3-VDR signaling preserves the mucosal barrier integrity by abrogating MLCK-dependent tight junction dysregulation during colonic inflammation.

Exogenous phospholipids supplementation improves growth and modulates immune response and physical barrier referring to NF-κB, TOR, MLCK and Nrf2 signaling factors in the intestine of juvenile grass carp (Ctenopharyngodon idella).

This study was conducted to investigate the effects of dietary phospholipids (PL) on the growth performance, intestinal enzyme activity and immune response and intestinal physical barrier of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juvenile grass carp with an average initial weight of 9.34 ± 0.03 g were fed six semi-purified diets containing 0.40% (unsupplemented control group), 1.43%, 2.38%, 3.29%, 4.37% and 5.42% PL for 2 months. Results indicated that 3.29% PL increased lysozyme (LZ) and acid phosphatase (ACP) activities and complement component 3 (C3) content (P < 0.05), up-regulated the mRNA relative expression levels of interleukin 10, transforming growth factor β1 (TGF-β1), inhibitor protein κBα (IκBα), target of rapamycin (TOR) and casein kinase 2 (CK2) (P < 0.05), and down-regulated tumor necrosis factor α (TNF-α), interleukin 1β, nuclear factor κB p65 (NF-κB p65), IκB kinase β (IKKβ) and IκB kinase γ (IKKγ) mRNA relative expression levels (P < 0.05) in the intestine, suggesting that optimum PL could improve fish intestinal immunity. In addition, 3.29% PL increased the activities of anti-superoxide anion (ASA), anti-hydroxyl radical, copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx) and glutathione reductase (GR), the content of glutathione (P < 0.05), and the mRNA relative expression levels of occludin, zonula occludens 1 (ZO-1), claudin 3, claudin 12, claudin b, claudin c, SOD1, GPx, GR and NF-E2-related factor 2 (Nrf2) and decreased malondialdehyde (MDA), protein carbonyl (PC) and ROS content (P < 0.05), the mRNA relative expression levels of Kelch-like-ECH-associated protein 1a (Keap1a), myosin light chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) in the intestine, indicating that the optimum PL could improve fish intestinal physical barrier. Finally, based on the PWG, C3 content in the DI, ACP activity in the DI, intestinal PC content and intestinal ASA activity, the optimal dietary PL levels for juvenile grass carp (9.34-87.50 g) were estimated to be 3.46%, 3.79%, 3.93%, 3.72%, and 4.12%, respectively.