Horseradish Peroxidase Permeability Research Articles

ERRα protects against sepsis-induced acute lung injury in rats

BackgroundSepsis-induced acute lung injury (ALI) is associated with poor survival rates. The identification of potential therapeutic targets for preventing sepsis-induced ALI has clinical importance. This study aims to investigate the role of estrogen-related receptor alpha (ERRα) in sepsis-induced ALI.MethodsLipopolysaccharide (LPS) was used to simulate sepsis-induced ALI model in rat pulmonary microvascular endothelial cells (PMVECs). The effects of ERRα overexpression and knockdown on LPS-induced endothelial permeability, apoptosis and autophagy were determined by horseradish peroxidase permeability assay, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, flow cytometry, immunofluorescence staining, RT-PCR and Western Blotting. The rat model with sepsis-induced ALI was established by cecal ligation and puncture in anesthetized rats to verify the results of in vitro experiments. Animals were randomly assigned to receive intraperitoneal injection of vehicle or ERRα agonist. Lung vascular permeability, pathological injury, apoptosis and autophagy were examined.ResultsOverexpression of ERRα ameliorated LPS-induced endothelial hyperpermeability, degradation of adherens junctional molecules, upregulation of bax, cleaved caspase 3 and cleaved caspase 9 levels, downregulation of anti-apoptotic protein Bcl-2 level, and promoted the formation of autophagic flux, while the knockdown of ERRα exacerbated LPS-induced apoptosis and inhibited the activation of autophagy. Administration of ERRα agonist alleviated the pathological damage of lung tissue, increased the levels of tight junction proteins and adherens junction proteins, and decreased the expression of apoptosis-related proteins. Promoting the expression of ERRα significantly enhanced the process of autophagy and reduced CLP-induced ALI. Mechanistically, ERRα is essential to regulate the balance between autophagy and apoptosis to maintain the adherens junctional integrity.ConclusionERRα protects against sepsis-induced ALI through ERRα-mediated apoptosis and autophagy. Activation of ERRα provides a new therapeutic opportunity to prevent sepsis-induced ALI.

Human antigen R (HuR) and Cold inducible RNA-binding protein (CIRP) influence intestinal mucosal barrier function in ulcerative colitis by competitive regulation on Claudin1.

To investigate the effects of RNA-binding proteins cold-inducible RNA binding protein (CIRP) and human antigen R (HuR) on expression of Claudin1 and mucosal barrier function in ulcerative colitis (UC). The clinical specimens of UC patients and healthy volunteers were collected. In the clinical experiments, the expressions of CIRP, Claudin1, and HuR, along with their correlations in tissues of UC patients were analyzed by qRT-PCR, Western blot and Pearson correlation coefficient, respectively. The chi-square test was utilized to assess the relevance between CIRP/HuR/Claudin1 level and clinicopathological characteristics of UC patients. The in vitro and in vivo models of UC were established by lipopolysaccharide treatment or dextran sulfate sodium injection. For cell experiments, after loss- and gain-of-function, the roles of CIRP or HuR in the apoptosis and proliferation of enterocytes were examined by flow cytometry and CCK-8 assay. The intestinal epithelial barrier function was inspected after determination on transepithelial electrical resistance value, horseradish peroxidase permeability and expressions of tight junction proteins (Occludin, ZO-1, and JAM-1). The relationship between HuR, CIRP, and Claudin1 was performed by RNA immunoprecipitation and dual-luciferase reporter gene assay. For in vivo experiments, the disease activity index score, weight loss and colon length of mice were assessed to observe the effect of CIRP or HuR on the UC mouse models. Histological analysis of colon tissues was conducted by H&E staining. FITC-dextran tracking was applied to inspect the intestinal mucosal barrier function of UC mouse models. In this study, high expression of CIRP and low expressions of HuR and Claudin1 were observed in patients, cells and mouse models of UC. The expressions of CIRP, HuR, and Claudin1 were correlated with the severity of patients with UC. There was a negative correlation between CIRP and Claudin1, and as a positive correlation between HuR and Claudin1. Claudin1 can be suppressed by CIRP, while enhanced by HuR. HuR and CIRP can competitively bind to Claudin1. HuR upregulation or CIRP downregulation promoted proliferation, suppressed apoptosis and ameliorated the damage of the barrier function in enterocytes. The in vivo experiments verified that the ameliorated damage of the intestinal mucosal barrier function in UC mice occurred with HuR overexpression or CIRP knockdown. CIRP and HuR confer pivotal effect on the intestinal mucosal barrier function of UC through competitively binding to Claudin1 mRNA.

Inhibitory effect of miR-429 on expressions of ZO-1, Occludin, and Claudin-5 proteins to improve the permeability of blood spinal cord barrier in vitro

To explore the feasibility and mechanism of inhibiting miR-429 to improve the permeability of the blood spinal cord barrier (BSCB) in vitro, and provide a new gene therapy target for enhancing the spinal cord microenvironment. First, the immortalized human brain microvascular endothelial cell line (hCMEC/D3) was transfected with the anti-miR-429 antagonist (antagomiR-429) and its negative control (antagomiR-429-NC), respectively. The miR-429 expression of hCMEC/D3 cells was observed by fluorescence microscopy and real-time fluorescence quantitative PCR to verify the transfection efficiency of antagomiR-429. Then the effect of miR-429 on BSCB permeability was observed in vitro. The experiment was divided into 4 groups. The blank control group (group A) was constructed of normal hCMEC/D3 cells and Ha-sc cells to prepare the BSCB model, the hypoxia-induced group (group B), the hypoxia-induced+antagomiR-429-NC group (group C), and the hypoxia-induced+antagomiR-429 group (group D) were constructed of normal, antagomiR-429-NC transfected, and antagomiR-429 transfected hCMEC/D3 cells and Ha-sc cells to prepare the BSCB models and hypoxia treatment for 12 hours. The permeability of BSCB in vitro was measured by horseradish peroxidase (HRP) permeability. Real-time fluorescence quantitative PCR, Western blot, and immunofluorescence staining were used to observe the expressions of ZO-1, Occludin, and Claudin-5. The antagomiR-429 and antagomiR-429-NC were successfully transfected into hCMEC/D3 cells under a fluorescence microscope, and the transfection efficiency was about 90%. Real-time fluorescence quantitative PCR results showed that the relative expression of miR-429 in antagomiR-429 group was 0.109±0.013, which was significantly lower than that of antagomiR-429-NC group (0.956±0.004, P<0.05). HRP permeability measurement, real-time fluorescence quantitative PCR, and Western blot results showed that the HRP permeability of groups B and C were significantly higher than those of groups A and D ( P<0.05), and the relative expressions of ZO-1, Occludin, and Claudin-5 proteins and mRNAs were significantly lower in groups B and C than in groups A and D ( P<0.05) and in group D than in group A ( P<0.05); there was no significant difference between groups B and C ( P>0.05). Immunofluorescence staining showed that the immunofluorescence of ZO-1, Occudin, and Claudin-5 at the cell membrane boundary in group D were stronger than those in groups B and C, but not as strong as that in group A. Inhibition of miR-429 expression can promote the expressions of ZO-1, Occludin, and Claudin-5 proteins in microvascular endothelial cells, thereby improving the increased permeability of BSCB due to hypoxia.

MiRNA-125a-5p attenuates blood-spinal cord barrier permeability under hypoxia in vitro.

Disruption of the blood-spinal cord barrier (BSCB) results in secondary injury and apoptosis of neurons, leading to permanent neurological dysfunction after spinal cord injury. In this study, we evaluate the role of miRNA-125a-5p in the BSCB under hypoxia. The miRNA-125a-5p mimics group showed lower horseradish peroxidase (HRP) permeability and endothelial cell death rates compared with the transfection control group. By contrast, the miRNA-125a-5p inhibitor group demonstrated higher HRP permeability and endothelial cell death rates than the transfection control group. The expressions of ZO-1, occludin, VE-cadherin and their mRNA significantly increased in miRNA-125a-5p-overexpressing cells. By contrast, a remarkable reduction in ZO-1, occludin, and VE-cadherin expression and their mRNA were observed in miRNA-125a-5p-inhibited cells. MiRNA-125a-5p appears to reduce the permeability of the BSCB by up regulating the expression of ZO-1, occludin, and VE-cadherin and their mRNA, and against hypoxia-induced apoptosis of spinal cord microvascular endothelial cells. Taken together, our results clearly indicate that miRNA-125a-5p plays an important role in protecting the functions of the BSCB under hypoxia.

Lactobacillus plantarum L9 but not Lactobacillus acidophilus LA reduces tumour necrosis factor induced bacterial translocation in Caco-2 cells.

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis and multiple organ dysfunction syndromes. Inflammatory cytokines increase paracellular permeability that allows increased luminal bacteria to translocate across mucosal epithelium and further deteriorate the gut barrier. In order to reduce this risk, the prophylactic use of probiotics has been recently addressed. In this paper, we investigate the protective role toward tumour necrosis factor (TNF)-α induced non-pathogenic Escherichia coli translocation across Caco-2 monolayers of Lactobacillus strains. According to our experimental data, Lactobacillus plantarum L9 and Lactobacillus acidophilus LA have good capacities to adhere to Caco-2 cells. Addition of L. plantarum L9 and L. acidophilus LA to the enterocyte monolayer surface result in significant inhibition of E. coli adhesion and cell internalisation. However, L. plantarum L9 and L. acidophilus LA did not inhibit the growth of the non-pathogenic E. coli B5 after 24 h incubation. Exposure to TNF-α for 6 h caused a dramatic increase in E. coli B5 translocation across Caco-2 cells, which was uncoupled from increases in paracellular permeability. Pretreatment with L. plantarum L9 prevent TNF-α induced transcellular bacterial translocation and IL-8 production in Caco-2 cells. L. plantarum L9 also did not affect the integrity of the monolayers, as indicated by lactate dehydrogenase release, horseradish peroxidase permeability, and transepithelial electrical resistance. L. plantarum L9 showed the potential to protect enterocytes from an acute inflammatory response and therefore could be good potential prophylactic agents in counteracting bacterial translocation.

Anti-inflammatory mechanism of ulinastatin: Inhibiting the hyperpermeability of vascular endothelial cells induced by TNF-α via the RhoA/ROCK signal pathway

ObjectiveUlinastatin reduces the high permeability of vascular endothelial cells induced by tumor necrosis factor alpha (TNF-α). This study investigated the molecular mechanism behind this effect, with the aim of understanding the action of ulinastatin in sepsis therapy and exploring novel therapeutic strategies for sepsis patients. MethodsA TNF-α treated human umbilical vein endothelial cell line (EA.hy926) was employed as an inflammation model. Horseradish peroxidase permeability assays and an epithelial voltmeter method were used to measure the permeability of EA.hy926 cells. Immunocytochemistry was used to assay the expression of p-MYPT1 and the distribution and morphology of F-actin; the expression of the key molecules related to vascular endothelial permeability (RhoA, ROCK2, MYPT1, p-MYPT1 and VE-cadherin) was detected by immunocytochemistry assays, western blotting and quantitative real-time polymerase chain reaction. ResultsAfter incubation with TNF-α or septic serum, the transendothelial electrical resistance of EA.hy926 cells decreased and the permeability of the cells increased significantly (all P<0.05). The expression of p-MYPT1 was higher and VE-cadherin was lower compared with the control group, and F-actin was redistributed, with the formation of additional stress fibers in the cells. Ulinastatin treatment moderated these phenomena. The immunocytochemistry assays and western blots showed that the expression of RhoA and ROCK2 was significantly upregulated in cells treated with TNF-α (P<0.05); however, ulinastatin could inhibit the high expression of these two proteins. Under treatment with TNF-α and ulinastatin, compared with normal EA.hy926 cells, overexpression of RhoA upregulated expression of RhoA, ROCK2 and p-MYPT1, downregulated expression of VE-cadherin, and restored the hyperpermeability of vascular endothelial cells due to TNF-α treatment (P<0.05). ConclusionsUlinastatin inhibited the hyperpermeability of vascular endothelial cells induced by TNF-α. This inhibitory effect of ulinastatin may be related to the RhoA/ROCK signaling pathway.

Oxidative stress regulates mitogen‑activated protein kinases and c‑Jun activation involved in heat stress and lipopolysaccharide‑induced intestinal epithelial cell apoptosis.

Heat stress and gut-derived endotoxinemia are common causes of multiple organ dysfunction syndrome in heat stroke patients. Evidence has demonstrated that cell apoptosis in the small intestine serves an important role in the pathogenesis of heatstroke, which leads to increased intestinal permeability to endotoxin or lipopolysaccharides (LPS) from the gut entering the circulation. However, little is known about the potential underlying mechanisms mediating heat stress combined with LPS-induced intestinal epithelial cell apoptosis. In the present study, LPS combined with heat stress induced production of reactive oxygen species (ROS), mitochondrial membrane potential disruption and cell apoptosis, which eventually led to increased intestinal permeability and reduced epithelial resistance in the IEC-6 cell line. Inductions in ROS, mitochondrial membrane potential disruption and cell apoptosis were detected by using an ROS assay kit, 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazo carbocyanine iodide dye kit and annexin V-fluorescein isothiocyanate apoptosis kit, respectively. The effect of ROS on mitogen activated protein kinases (MAPKs) and c-Jun activation was investigated using the antioxidant drug, butylated hydroxyanisole (BHA) by western blotting. The results of the present study demonstrated that ROS is essential to activate p38, extracellular signal-regulated kinase (ERK) and c-Jun, but not c-Jun N-terminal kinase (JNK), in LPS combined with heat stress treated cells. Furthermore, ROS, and activation of p38, JNK and c-Jun, were revealed to serve pro-apoptosis roles which aggravated damage to epithelial barrier integrity, as assessed by flow cytometry using Annexin V-fluorescein isothiocyanate staining and pretreatment of cells with specific inhibitors of ROS, JNK, p38 and c-Jun (BHA, SP600125, SB203580 and c-Jun peptide, respectively). Transepithelial electrical resistance and horseradish peroxidase permeability were detected in cells treated with LPS combined with heat stress, which revealed that ERK serves an anti-apoptosis role, as determined by pretreatment of cells with PD98059, a specific inhibitor of ERK. In conclusion, these findings suggested a novel role of the ROS signaling pathway which involved activation of MAPKs and c-Jun, following LPS combined with heat stress-induced IEC-6 cell apoptosis and impairment of the epithelial barrier. These results may facilitate understanding of pathological conditions involving ROS, such as heat stroke.

Maternal antibiotic-induced early changes in microbial colonization selectively modulate colonic permeability and inducible heat shock proteins, and digesta concentrations of alkaline phosphatase and TLR-stimulants in swine offspring.

Elevated intake of high energy diets is a risk factor for the development of metabolic diseases and obesity. High fat diets cause alterations in colonic microbiota composition and increase gut permeability to bacterial lipopolysaccharide, and subsequent low-grade chronic inflammation in mice. Chronic inflammatory bowel diseases are increasing worldwide and may involve alterations in microbiota-host dialog. Metabolic disorders appearing in later life are also suspected to reflect changes in early programming. However, how the latter affects the colon remains poorly studied. Here, we hypothesized that various components of colonic physiology, including permeability, ion exchange and protective inducible heat shock proteins (HSP) are influenced in the short- and long-terms by early disturbances in microbial colonization. The hypothesis was tested in a swine model. Offspring were born to control mothers (n = 12) or mothers treated with the antibiotic (ATB) amoxicillin around parturition (n = 11). Offspring were slaughtered between 14 and 42 days of age to study short-term effects. For long-term effects, young adult offspring from the same litters consumed a normal or a palm oil-enriched diet for 4 weeks between 140 and 169 days of age. ATB treatment transiently modified maternal fecal microbiota although the minor differences observed for offspring colonic microbiota were nonsignificant. In the short-term, consistently higher HSP27 and HSP70 levels and transiently increased horseradish peroxidase permeability in ATB offspring colon were observed. Importantly, long-term consequences included reduced colonic horseradish peroxidase permeability, and increased colonic digesta alkaline phosphatase (AP) and TLR2- and TLR4-stimulant concentrations in rectal digesta in adult ATB offspring. Inducible HSP27 and HSP70 did not change. Interactions between early ATB treatment and later diet were noted for paracellular permeability and concentrations of colonic digesta AP. In conclusion, our data suggest that early ATB-induced changes in bacterial colonization modulate important aspects of colonic physiology in the short- and long-terms.

Early maternal separation induces alterations of colonic epithelial permeability and morphology.

Early maternal separation could lead to significant intestinal barrier and epithelial dysfunction. However, the exact mechanism remains to be elucidated and need to be investigated. Neonatal C57BL/6 mice were subjected to maternal separation: Maternal separation (MS) daily 3h between postnatal day (PND) 5-9, single separation (SS) 3h on PND 9 and no separation (NS). Colon and ileum permeability was measured by Ussing chamber. Severity of morphological changes in the colon was evaluated by blinded grading of histological stained sections. Trans-epithelial resistance of colon and ileum did not change indicating that the tissues remained intact during the course of the experiment. Permeability of trans-cellular tracer Horseradish peroxidase (HRP) was significantly increased in the colon of MS compared to SS and NS (p<0.05 for SS and p<0.001 for NS), but there was no difference in para-cellular permeability of fluorescein isothiocyanate-conjugated dextran (FD4). However, there was no change in permeability of both HRP and FD4 in the ileum. MS and SS groups had marked intestinal epithelium morphology changes in comparison to controls (p<0.05). These preliminary observations indicate that neonatal maternal separation increases colonic trans-cellular permeability. This increase may be caused by the change of the transmural colonic morphology. The underlying mechanism is unknown and further investigation is necessary as it is of relevance to the development of early intestinal diseases such as necrotizing enterocolitis.

Activation of VEGF/Flk-1-ERK Pathway Induced Blood-Brain Barrier Injury After Microwave Exposure.

Microwaves have been suggested to induce neuronal injury and increase permeability of the blood-brain barrier (BBB), but the mechanism remains unknown. The role of the vascular endothelial growth factor (VEGF)/Flk-1-Raf/MAPK kinase (MEK)/extracellular-regulated protein kinase (ERK) pathway in structural and functional injury of the blood-brain barrier (BBB) following microwave exposure was examined. An in vitro BBB model composed of the ECV304 cell line and primary rat cerebral astrocytes was exposed to microwave radiation (50 mW/cm(2), 5 min). The structure was observed by scanning electron microscopy (SEM) and the permeability was assessed by measuring transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) transmission. Activity and expression of VEGF/Flk-1-ERK pathway components and occludin also were examined. Our results showed that microwave radiation caused intercellular tight junctions to broaden and fracture with decreased TEER values and increased HRP permeability. After microwave exposure, activation of the VEGF/Flk-1-ERK pathway and Tyr phosphorylation of occludin were observed, along with down-regulated expression and interaction of occludin with zonula occludens-1 (ZO-1). After Flk-1 (SU5416) and MEK1/2 (U0126) inhibitors were used, the structure and function of the BBB were recovered. The increase in expression of ERK signal transduction molecules was muted, while the expression and the activity of occludin were accelerated, as well as the interactions of occludin with p-ERK and ZO-1 following microwave radiation. Thus, microwave radiation may induce BBB damage by activating the VEGF/Flk-1-ERK pathway, enhancing Tyr phosphorylation of occludin, while partially inhibiting expression and interaction of occludin with ZO-1.

Detrimental Effect of Electromagnetic Pulse Exposure on Permeability of In Vitro Blood-brain-barrier Model

ObjectiveTo study the effect of electromagnetic pulse (EMP) exposure on permeability of in vitro blood-brain-barrier (BBB) model. MethodsAn in vitro BBB model, established by co-culturing brain microvascular endothelial cells (BMVEC) and astroglial cells (AC) isolated from rat brain, was exposed to EMP at 100 kV/m and 400 kV/m, respectively. Permeability of the model was assayed by measuring the transendothelial electrical resistance (TEER) and the horseradish peroxidase (HRP) transmission at different time points. Levels of BBB tight junction-related proteins were measured at 0, 1, 2, 4, 8, 12, 16, 20, 24 h after EMP exposure by Western blotting. ResultsThe TEER level was lower in BBB model group than in control group at 12 h after EMP, exposure which returned to its normal level at 24 h. The 24 h recovery process was triphasic and biphasic respectively after EMP exposure at 100 kV/m and 400 kV/m. Following exposure to 400 kV/m EMP, the HRP permeability increased at 1-12 h and returned to its normal level at 24 h. Western blotting showed that the claudin-5 and ZO-1 protein levels were changed after EMP exposure. ConclusionEMP exposure at 100 kV/m and 400 kV/m can increase the permeability of in vitro BBB model and BBB tight junction-related proteins such as ZO-1 and claudin-5 may change EMP-induced BBB permeability.

Corticotropin releasing hormone activates CD14+ cells to induce endothelial barrier dysfunction.

Endothelial barrier dysfunction is associated with the pathogenesis of a number of disorders in the body, but the aetiology is unclear. We have investigated the mechanism of the psychological stress mediator, corticotropin releasing hormone (CRH), on compromising the endothelial barrier function. Human endothelial cell line, Hmvec cells, was cultured in monolayers as a model of endothelial barrier. Human peripheral CD14+ cells were collected to be used as effector immune cells. The transepithelial resistance (TER) and permeability of horseradish peroxidase (HRP) were used as indicators of endothelial barrier function. Apoptosis in Hmvec cells were analysed by flow cytometry. Human CD14+ cells expressed both receptors (R) of CRH, the CRH-R1 and CRH-R2. Exposure to CRH induced CD14+ cells to release tumour necrosis factor (TNF)-α. CRH-activated CD14+ cells decreased TER and increased the permeability to HRP in co-cultured Hmvec monolayers. Co-culture with CRH-activated CD14+ cells increased the apoptosis in Hmvec cells. We conclude that CRH can activate CD14+ cells to produce TNF-α and compromise endothelial barrier function by inducing apoptosis of the endothelial cells.

High glucose enhances permeability of human pulmonary microvascular endothelial cells by lipopolysaccharide stimulation in vitro and effect of DDAH/NOS/NO imbalance on its pathogenesis

To investigate the damage to endothelial cells incubated in high concentration of glucose challenged by lipopolysaccharide (LPS), and the likely mechanisms of injury. Human pulmonary microvascular endothelial cells (PMVECs) were divided into the following groups: normal glucose group (NG), normal glucose + LPS stimulation group (NGL), high glucose stimulation group (HG), and high glucose + LPS stimulation group (HGL). The cells were incubated with normal glucose (5.5 mmol/L, contained 10% calf serum) or high glucose (33 mmol/L) for 5 days to form a monolayer of cells before LPS stimulation (10 mg/L) for 24 hours. The microfilaments (F-actin) were investigated by immuno-fluorescence, and the number and size change in fenestrae were examined by scanning electron microscopy. The permeability of vascular endothelial cell was assessed by trans-PMVEC horseradish peroxidase (HRP) flux. Western blotting was used to determine the expressions of dimethylarginine dimethylaminohydrolase 2 (DDAH2), inducible nitricoxide synthase (iNOS) and endothelial nitricoxide synthase (eNOS). Nitric oxide (NO) was assessed by Griess method. When stimulated with LPS, cells incubated with high glucose showed obvious microfilament rearrangement, a larger average diameter and increased number of F-actin, as well as higher HRP permeability on the hyperglycemic PMVECs compared with PMVECs cultured with normal glucose [(53.62±6.70)% vs. (23.63±3.92)%, P<0.01]. Furthermore, high glucose down-regulated DDAH2 expression (arbitrary units, AU, 0.33±0.08 vs. 0.77±0.14 , P<0.01) and up-regulated LPS-stimulated iNOS production (1.40±0.29 vs. 1.04±0.09, P<0.01), as well as increased LPS-stimulated nitrite/nitrate and stable NO end products compared with normal (20.36±2.25 μmol/L vs. 7.99±0.33 μmol/L, P<0.01) and reduction of eNOS levels was observed (0.67±0.09 vs. 0.91±0.17, P<0.05). It demonstrated that, in vitro high glucose deteriorate LPS-stimulated F-actin rearrangement and hyperpermeability of an endothelial monolayer, and the worsened imbalance of the NO pathway may lead to endothelial damage in microcirculation.

Effects of electromagnetic pulse exposure on the permeability of inner blood-retinal barrier model in vitro

To establish the inner blood-retinal barrier (BRB) model in vitro by co-culturing RF/6A cells and C6 cells and to investigate the effects of EMP (200 kV/m, 200 pulses) exposure on the permeability of the inner BRB model in vitro. RF/6A cells and C6 cells were co-cultured on transwell, and the characteristic of the inner BRB model was assessed by detecting transendothelial electrical resistance (TEER) and the permeability of horseradish peroxidase (HRP). The co-cultured model was exposed or sham exposed to the EMP (200 kV/m 200 pulses) for 0.5, 3, 6, 12, 24 h in vitro, then TEER and the permeability of HRP were measured for studying the effects of EMP on the permeability of inner BRB model in vitro. TEER value (145 Ωcm(2)) of the co-culturing inner BRB model significantly increased, as compared to that of RF/6A cells alone model (P < 0.05) on the 6th day after inoculation. There was significant difference of permeability of HRP between the co-culturing inner BRB model and RF/6A cells alone model (P < 0.05). The ability of inhibiting large molecular materials in the co-culturing inner BRB model enhanced. The TEER value decreased and the permeability of HRP increased as compared to the sham group at 0.5, 3, 6 h after the exposure. The inner BRB model by co-culturing RF/6A cells and C6 cells in vitro is efficient and suitable to study the alterations of the restricted permeability function of the inner BRB. EMP (200 kV/m for 200 pulses) could induce the enhanced permeability of the inner BRB model in vitro.

Lactulose as a marker of intestinal barrier function in pigs after weaning1

Intestinal barrier function in pigs after weaning is almost exclusively determined in terminal experiments with Ussing chambers. Alternatively, the recovery in urine of orally administered lactulose can be used to assess intestinal permeability in living animals. This experiment was designed to study the barrier function of the small intestine of pigs over time after weaning. The aim was to relate paracellular barrier function (measured by lactulose recovery in the urine) with macromolecular transport [measured by horseradish peroxidase (HRP) using Ussing chambers] and bacterial translocation to assess whether lactulose recovery is related to possible causes of infection and disease. Forty gonadectomized male pigs (6.7 ± 0.6 kg) were weaned (d 0) at a mean age of 19 d, fitted with urine collection bags, and individually housed. Pigs were dosed by oral gavage with a marker solution containing lactulose (disaccharide) and the monosaccharides l-rhamnose, 3-O-methylglucose, and d-xylose at 2 h and at 4, 8, and 12 d after weaning. The recovery of sugars in the urine was determined over 18 h after each oral gavage. The day after each permeability test, the intestines of 10 pigs were dissected to determine bacterial translocation to the mesenteric lymph nodes and jejunal permeability for HRP in Ussing chambers. Recovery of l-rhamnose in urine was affected by feed intake and by the time after weaning (P ≤ 0.05). Recovery of lactulose from the urine was greater (P ≤ 0.05) at 4, 8, and 12 d after weaning compared with the first day after weaning and was negatively correlated with feed intake (r = -0.63, P ≤ 0.001). The mean translocation of aerobic bacteria to the mesenteric lymph nodes was greater at 5 and 13 d after weaning compared with d 1 (P ≤ 0.05). Lactulose recovery showed no correlation with permeability for HRP nor with bacterial translocation (P > 0.05). Although both lactulose recovery and bacterial translocation increased over time after weaning, lactulose recovery did not correlate with the permeability for HRP nor bacterial translocation within a pig (P > 0.05). Therefore, we conclude that lactulose recovery in the urine of pigs after weaning is not associated with risk factors for infections. However, it appears to be possible to measure paracellular barrier function with orally administered lactulose in pigs shortly after weaning. Further studies will reveal whether this variable is relevant for the long-term performance or health of pigs after weaning.

Tanshinone IIA protects the human blood–brain barrier model from leukocyte-associated hypoxia-reoxygenation injury

To investigate the in vitro effect of tanshinone IIA on leukocyte-associated hypoxia-reoxygenation injury of human brain–blood barrier (BBB), we established the BBB model by culturing purified primary human brain microvascular endothelial cells (HBMVEC) to confluence on cell culture insert. BBB was identified by tight junction, transendothelial electrical resistance (TEER) and the permeability of BBB to horseradish peroxidase (HRP). The effect of tanshinone IIA on the permeability of BBB was tested at 2 h after hypoxia and 1 h after reoxygenation with or without the supernatants of activated leukocytes. The effect of tanshinone IIA on leukocytes activation was analyzed by detection of MMP-9, cytokines and reactive oxygen species. The results showed that BBB formed by confluent HBMVECs had no cellular gap. Immunofluorescent staining for ZO-1 confirmed that the cells were connected by tight junction. Moreover, the BBB model had a higher TEER and a lower permeability for HRP than confluent HUVECs. The permeability of BBB for HRP was enhanced by hypoxia-reoxygenation and further greatly enhanced by adding the supernatants of activated leukocytes before reoxygenation. But such an effect was reversed by addition of tanshinone IIA before hypoxia. Moreover, tanshinone IIA could decrease the levels of MMP-9, TNF-α, IL-1α, IL-2, IFN-γ and reactive oxygen species in leukocytes. In conclusion, tanshinone IIA can protect BBB against leukocyte-associated hypoxia-reoxygenation injury by attenuating the activation of leukocytes and inhibiting the injury effects of leukocytic products. Tanshinone IIA may be a novel therapeutic agent for cerebral ischemia–reperfusion injury.

Proteolysis of submembrane cytoskeletal proteins ankyrin-G and αII-spectrin following diffuse brain injury: a role in white matter vulnerability at Nodes of Ranvier.

A high membrane-to-cytoplasm ratio makes axons particularly vulnerable to traumatic injury. Posttraumatic shifts in ionic homeostasis promote spectrin cleavage, disrupt ankyrin linkages and destabilize axolemmal proteins. This study contrasted ankyrin-G and αII-spectrin degradation in cortex and corpus callosum following diffuse axonal injury produced by fluid percussion insult. Ankyrin-G lysis occurred preferentially in white matter, with acute elevation of all fragments and long-term reduction of a low kD form. Calpain-generated αII-spectrin fragments increased in both regions. Caspase-3 lysis of αII-spectrin showed a small, acute rise in cortex but was absent in callosum. White matter displayed nodal damage, with horseradish peroxidase permeability into the submyelin space. Ankyrin-G-binding protein neurofascin and spectrin-binding protein ankyrin-B showed acute alterations in expression. These results support ankyrin-G vulnerability in white matter following trauma and suggest that ankyrin-G and αII-spectrin proteolysis disrupts Node of Ranvier integrity. The time course of such changes were comparable to previously observed functional deficits in callosal fibers.

Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal

BackgroundEnprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products.ResultsEnprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1β and TNF-α) and up-regulated IFN-γ, IL-2 and IL-10.ConclusionOur results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

Ultrastructure and Fluid Flow Physiology of Fetal Trabecular Meshwork Cells

Purpose: To assess the ultrastructural and fluid flow characteristics of cultured trabecular meshwork (TM) cells derived from fetal sources. Methods: Fetal eyes were carefully dissected to isolate the developing TM tissue for culture. Immunostaining was used to assess the expression of the junction-associated proteins zonula occludens-1 (ZO-1) and occludin. Fetal and adult TM cells were grown to confluence on permeable membranes for both flow and ultrastructural studies. Fluid flow resistance was measured by permeation of horseradish peroxidase (hrp) activity and hydraulic conductivity (HC) experiments. The effects of dexamethasone (Dex) on permeability and HC were also evaluated. Results: ZO-1 and occludin are expressed in the TM region of tissue sections and at cell borders in cultured fetal and adult TM cells. Transmission electron microscopy demonstrated that cultured TM cells possessed numerous mitochondria, electron-dense bodies, surface microvilli, and adherens and gap junctions. The permeation of hrp across fetal TM cell monolayers (0.030 ± 0.010) and of adult TM cells (0.031 ± 0.010) had similar values for absorbance at 470 nm (p = 0.83, 95% CI: −0.004, 0.005). Dex treatment significantly reduced the permeability to 0.022 ± 0.008 (p = 0.002) and 0.018 ± 0.009 (p = 0.004) for fetal and adult TM cells, respectively. The average HC (μ l/min/mmHg/cm2) of fetal cells (2.78 ± 1.03) and of the adult cells (2.15 ± 1.31) was not significantly different (p = 0.24, 95% CI: −1.01, 0.26). Dex treatment significantly reduced HC in both fetal (1.24 ± 0.72, p = 0.0004) and adult (1.29 ± 0.29, p = 0.00001) TM. Conclusions: Cultured fetal TM cells exhibited similar expression of junctional proteins and ultrastructural features as their adult counterparts. The permeability and HC of the fetal cells were similar to their older adult counterparts. Dex treatment induced increased fluid flow resistance in both cell types. These cells may serve as a source for in vitro studies of meshwork physiology.

HEAT SHOCK STRESS AMELIORATES CYTOKINE MIXTURE-INDUCED PERMEABILITY BY DOWNREGULATING THE NITRIC OXIDE AND SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION PATHWAYS IN CACO-2 CELLS

Proinflammatory cytokines are known to impair intestinal barrier function and to activate signaling pathways, whereas heat shock responses prevent cytokine-induced mucosal damage. We hypothesized that heat shock response blocks the effects of proinflammatory cytokines by regulating nitric oxide (NO) production and the activities of the Janus kinase/signal transducer and activator of transcription (STAT) pathway. A monolayer of Caco-2 cells were pretreated with sodium arsenite (SA, 500 micromol/L) for 1 h, followed by a 1-h recovery, and then stimulated with a cytokine mixture (cytomix: tumor necrosis factor alpha [10 ng/mL], interferon beta [1000 U/mL], and interleukin [IL] 1beta [1 ng/mL]) for 24 h. The permeability of horseradish peroxidase and fluorescein isothiocyanate-conjugated Dextran and transepithelial resistance and potential difference were measured in Ussing chambers. Interleukin-6, IL-8, NO, inducible NO synthase mRNA, STAT activity, and suppressor of cytokine signaling (SOCS) expression were measured in medium or cell lysates. Cytomix resulted in increased epithelial permeability of both fluorescein isothiocyanate-conjugated Dextran and horseradish peroxidase; whereas treatment of Caco-2 cells with SA 500 micromol/L blocked the cytomix-induced permeability changes. In addition, SA treatment decreased cytomix-induced NO production and inducible NO synthase mRNA expression and decreased the levels of STAT1, STAT3, SOCS1, and SOCS3. The SA treatment also decreased cytomix-induced IL-6 and IL-8 production in a dose-dependent manner. In conclusion, cytomix increased epithelial permeability, which is associated with increased NO and STAT activities. The SA treatment ameliorated cytomix-induced permeability, possibly through the downregulation of the NO and Janus kinase/STAT pathways.