Eicosapentaenoic Acid Enhances Heat Stress-impaired Function Of Intestinal Epithelial Tight Junction Barrier

Background and ObjectiveThe intestinal epithelial barrier prevents the entry of luminal pathogens such as bacteria and endotoxins into blood circulation. As the most important component of the epithelial barrier, a functional tight junction (TJ), which forms a continuous circumferential seal at the apical-most portion of cells, is required for the maintenance of the barrier function of the intestine 1,2. TJ breakdown contributes to intestinal hyperpermeability. Moreover, bacterial products from the intestinal lumen entering into the circulation cause systemic inflammatory response syndrome and multiple organ failure 3. Heat stroke is a severe illness characterized by an elevated core body temperature above 40℃ and central nervous system dysfunction associated with mucosal damage 4. Intestinal barrier function plays an important role in the pathophysiology of heat stroke in rat models, and the Caco-2 and T84 cells monolayer exposed to heat stress. Heat stress impairs the intestinal barrier integrity by increasing intestinal permeability and reducing epithelial resistance5’ 6. It has been reported that intestinal permeability to endotoxin or lipopolysaccharides (LPS) from the gut entering the circulation increases in heat-stressed animals 7,8. On the contrary, anti-LPS antibodies protect against the transition from heat stress to heatstroke 9. Therefore, protecting the integrity of the intestinal barrier is an important goal in the prevention of heatstroke.Previous studies have found that the supplementation of n-3 fatty acids (PUFAs), which include eicosapentaenoic acid (EPA,20:5 n-3) and docosahexaenoic acid (DHA,22:6 n-3), effectively prevents the disruption of TJ structure, decrease of transepithelial electrical resistance (TEER) and elevation of flux of FITC-dextran (FD4) induced by inflammatory factors, thus reducing the permeability of the intestinal cells 10 11. N-3 PUFAs have shown potential beneficial effects on the immune response in experimental models of rheumatoid arthritis, inflammatory bowel disease and psoriasis by down-regulating the production of pro-inflammatory signals and supporting the intestinal barrier 12. The evidence demonstrates that EPA up-regulates the expression of TJ protein occludin, but AA exerts an down-regulatory effect in endothelial cells 13. Moreover, it has also been demonstrated that PUFAs down-regulate endotoxin translocation from gut into systemic circulation in rat models 14.In spite of these findings, the individual effects of n-3 PUFAs on TJ in intestinal epithelial cells with heat exposure have not been investigated. We therefore hypothesized that the supplementation of n-3 PUFAs before heat exposure would reverse the heat stress-related increase of TJ permeability and reorganization of the TJ proteins. This would improve organ function by protecting the gut barrier and decreasing plasma endotoxin levels. In this study we examined the effect of n-3 fatty acids on the heat stress-induced dysfunction of the intestinal epithelial barrier in Caco-2 monolayers. Caco-2 cells were used as a model to form typical TJ structure similar to mature intestinal epithelium in vitro 15.Objectives:1. Dysfunction of the intestinal epithelial tight junction (TJ) barrier is known to have an important etiologic role in the pathophysiology of heat stroke. This part is aimed at investigating that heat stress-induced dysfunction of intestinal tight junction.2. This part is aimed at discussing the effect of eicosapentaenoic acid (EPA) on TJ dysfunction of intestinal epithelium induced by heat stress.3. N-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), play a role in maintaining and protecting the TJ structure and function. This part is aimed at investigating whether n-3 PUFAs could alleviate heat stress-induced dysfunction of intestinal tight junction.4. The aim of this part was to establish a rat model of heatstroke, and evaluate the protective effect of EPA pre-treatment on heat-induced intestinal TJ injury.2 Materials and Methods Regents and MaterialsCaco-2 cells (ATCC, Manassas, VA), PUFAs (Sigma-Aldrich), Horseradish peroxidase (Sigma), mouse anti-occludin (BD Biosciences), rabbit anti-ZO-1 (Invitrogen), transwell inserts (Corning), Trizoland PrimeScript RT reagent kit (TAKARA), SYBR Green I Maser kit (Roche), cell proliferation kit (Beyotime), Limμlus Amebocyte Lysate (LAL) Kinetic-QCL(CAMBREX), D-Lactate Colorimetric Assay Kit (Biovision).Methods1 The effect of heat stress on intestinal barrier function in human intestinal epithelial Caco-2 CellsHuman intestinal epithelial Caco-2 cell monolayers, as the intestinal barrier model, were exposed to different temperatures (37-43℃) for 1h. Transepithelial electrical resistance (TEER) and Horseradish Peroxidase (HRP) flux permeability were measured to evaluate barrier integrity. Messenger RNA levels were determined by quantitative real time polymerase chain reaction (Q-PCR).Level of TJ protein occludin was analyzed by Western blot.2 The dose-effect of eicosapentaenoic acid on heat-stress induced intestinal epithelial barrier function in Caco-2 CellsHuman intestinal epithelial Caco-2 cells were pre-incubated with various concentration of EPA (25、50、100、150 μmol/L) and then exposed to heat 43 ℃ for designated time. Cell Counting Kit-8 was detected the cellular proliferation and toxicity. The barrier integrity of TJ was measured by transepithelial electrical resistance (TEER) and Horseradish Peroxidase (HRP) permeability. Level of TJ protein occludin was analyzed by Western blot. Change of cytoskeleton using coomassie blue staining was observed by microscopy.3 Eicosapentaenoic acid enhances heat stress-impaired intestinal epithelial barrier functionHuman intestinal epithelial Caco-2 cells were pre-incubated with EPA, DHA or arachidonic acid (AA, n-6 PUFA) and then exposed to heat stress. Transepithelial electrical resistance (TEER) and Horseradish Peroxidase (HRP) permeability were measured to analyze barrier integrity. Levels of TJ proteins, occludin and ZO-1, were analyzed by Western blot and localized by immunofluorescence microscopy. mRNA levels were determined by Q-PCR. TJ morphology was observed by transmission electron microscopy.4 EPA reduces intestinal tight junction injury induced by heat stroke in ratsMale Sprague Dawley rats were randomized to 5 groups (n=6):control, heat stroke groups (control, EPA, DHA and corn oil group). Rats were fed saline, EPA, DHA or corn oil (1g/kg) via oral gavage for 21 days. Intestinal permeability was determined by HRP flux, mucosal morphology was measured by HE staining and transmission electron microscopy. Plasma levels of endotoxin and D-lactate were determinede. TJ ultrastructure was by. Expression and location of TJ proteins occludin and ZO-1 were detected by Western blot analysis. Their location in the epithelium was observed by immunofluorescence microscopy.Statistical analysis.Data were summarized as mean ± standard error of the mean (S.E.M.). Data were compiled with the software package SPSS 13.0. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA). Paired-sample t test was used applied to analyze the difference of the levels of plasma endotoxin in portal vein and aortaventralis after the heat exposure in rats model. A probability (p)-values less than 0.05 were considered statistically significant.Results1. Compared with the 37 ℃ group, the TEER decreased and the permeability for HRP increased significantly after heat exposure (P< 0.01) in 39℃,41 ℃ and 43℃ group. The expression of occludin increased from 37℃ to 41℃ and reached maximal levels at 41℃. However, its expression decreased at higher temperature and prolonged time at 43℃. Cell apoptosis increased from 37℃ to 43℃.2. At 50μM concentration, EPA showed the most effective proliferation and no cytotoxicity compared with other concentration groups (P< 0.01).50 μM EPA effectively attenuated the decrease in TEER and impairment of intestinal permeability induced by heat exposure (P< 0.01, compared with other concentration groups). The expression of occludin was significantly elevated by 50 μ M EPA. The distortion of cytoskeleton were also effectively prevented by pretreatment with 50 u M EPA.3. After 1h of 43℃ heat stress, there was a significant decrease in TEER in the Caco-2 monolayer cells. EPA prevented the decrease of TEER induced by heat stress (compared with 43℃ group, P< 0.01) and reversed the increase of paracellular permeability induced by heating (compared with 43℃ group, P< 0.01), while AA and DHA do so to a lesser extent in TEER and HRP flux. Western blot and Q-PCR analysis revealed that treatment with EPA significantly increased levels of mRNA and protein in occludin and ZO-1, while DHA was less effective and AA wasn’t. The expression of occludin and ZO-1 was significantly elevated by EPA, while DHA was less effective and AA was no effective. The junctional localization of occludin and ZO-1 was determined by immunostaining. In the control group at 37℃, occludin and ZO-1 presented a continuous band of cells encircling the apical cellular junctions. Heat exposure under 43℃ for 1h caused a pronounced disruption in junctional localization of occludin and ZO-1 staining, characterized by decreased intensity staining and marked discontinuity localized to the structures of intercellular junctions. In the EPA group, the localization and intensity of TJ proteins were more similar to the 37℃ cells. In contrast, compared with the 43 ℃ group, the localization and intensity of TJ proteins changed only slightly in the DHA group but did not change significantly in the AA group. Heat exposure resulted in the disruption of TJ ultrastructure in Caco-2 monolayers. In the 37℃ control Caco-2 monolayers, tight junctions were intact between the adjoining cells. After heat exposure, TJs became markedly "open" with shortening of the strand length. TJ membranes lost fusion and had less electron-dense material. In EPA-incubated cells, the TJ strands displayed intact ultrastructure. However, DHA-treated cells had non-continuous TJ strands. AA treatment only slightly alleviated the decrease in length of tight junctions.4. Changes in intestinal permeability were determined by the measurement of serum endotoxin, serum D-lactate and intestinal permeability to HRP. Endotoxin level of portal vein and aortaventralis in all post-heatstroke groups significantly increased (compared with normal control group, P< 0.01). The value in EPA and DHA pretreatment groups was significantly lower than the heatstroke group (P< 0.01), but was more closely in EPA group and the normal control group. There was no significant difference between corn oil group and the control group. The results of serum D-Lactate showed that EPA and DHA pretreatment can reduce the D-Lactate concentration after heatstroke, while corn oil had no effect. In vitro, permeability of HRP flux from intestinal Lumina permeability significantly increased after heatstroke. EPA and DHA pre-gavage can obviously decrease the elevated degree of HRP flux (P< 0.01), while corn oil group had no difference.Intestinal mucosal villi pathological structure after heat stroke changed too. There were structural disorder and shedding of epithelial cells in the heatstroke group. In the EPA group there were only the hyperemic mucosa and mild edema, epithelial cells arranging in neat rows, little shedding and almost complete villous structure. In DHA group obvious cell shedding was found. In corn oil group more epithelial cell sheddings were showed. Heatstroke resulted in the disruption of TJ ultrastructure. After heatstroke TJ became "open", and lost connections in some parts with microvilli rupture in cell membrane. In EPA group intercellular TJs were intact, and no obvious decline in density. In DHA group, shortened TJ length was found, while the structure is almost complete. In corn oil group there was no obvious improvement of TJ structure.Western blot analysis showed that expression of TJ protein in occludin and ZO-1 decreased after heatstroke. EPA and DHA pretreatment could increase the protein expression, and the enhancement of EPA was more obvious. There was no difference between corn oil group and heatstroke group. Expression of occludin and ZO-1 was determined by immunostaining. Heatstroke caused protein change characterized by obviously decreased expression, noncontinuous distribution and even large deletion. In EPA group and DHA group, proteins expression were better with continuous and dense distribution, but they decreased with obvious shedding in corn oil group.Conclusion1. Heat stress can induce damage of tight junction, so cause intestinal epithelial barrier dysfunction.2. This study indicates that 50 μ M EPA is potent in protecting against heat-induced permeability dysfunction and epithelial barrier damage of tight junction.3. This study indicates for the first time that EPA is more potent than DHA in protecting against heat-induced permeability dysfunction and epithelial barrier damage of tight junction in human intestinal epithelial Caco-2 cells.4. The supplementation of EPA before heat exposure reverses the heat stress-related intestinal epithelium TJ disruption. EPA shows potential beneficial effects on tight junction in intestinal epithelia barrier. DHA is less effective in protecting TJ than EPA.