Protective Effects And Mechanisms Of Somatostatin On The Intestinal Barrier Function

Background and ObjectiveThe intestinal lumen has the largest pool containing bacteria and endotoxin in the body. The integrity of epithelium is essential for protecting against luminal infection and providing nutrient transport.The intestinal barrier includes mechanical barrier, immune barrier, biological barrier and chemical barrier. The tight junction (TJ), a multiprotein complex at the apical end of lateral membrane, selectively regulates the paracellular transportation by permitting the iron and micromolecule to pass through and prenventing the endotoxin and microorganism. TJs consist of transmembrane proteins (occludin, claudins and junction adhesion molecule), intracellular plaque zonula occludens (ZO-1, ZO-2, ZO-3), cingulin and others. Defective TJs of the intestinal epithelium contributes to the intestinal hyperpermeability, which could result in the translocation of bacterial and endotoxins from the intestinal lumen followed by systemic inflammatory response syndrome (SIRS) and multiple organ dysfunctions (MODS). The tight junction is affected by cytokines and inflammatory mediators, such as IL-1β,TNF-α, IFN-r and PEA. Defective TJ of the intestinal epithelium has been shown to be an important pathogenic factor of many diseases, such as inflammation bowel disease, intestinal ischemia-reperfusion injury, severe acute pancreatitis, etc. Lipopolysaccharide (LPS), the major cell wall component of gram-negative bacteria, is a major pathogenic factor of bacteria infection. Under normal circumstances, intestinal bacteria and endotoxins can not pass through the intestinal epithelial barrier. When the intestinal mucosal barrier was injured, the pathogens and toxins can enter the body and stimulate the production of inflammatory mediators, then cause local or systemic inflammation. In return, the inflammatory reaction may further damage the intestinal barrier function. In addition, LPS can directly or indirectly destroy the tight junction by affecting the phosphorylation and dephosphorylation of tight junction proteins. Confirmation of multiple in vivo and in vitro experiments, LPS can damage the intestinal epithelial barrier function and down-regulate the expression of tight junction proteins.In recent years, studies have shown that P38-MAPK pathway is involved in the regulation of the intestinal mucosa barrier function. Several studies showed that ERK-MAPK affected the epithelial barrier and the tight junction; however, the results were inconsistent. Some studies showed that the activation of ERK/MAPK pathway could protect the integrity and function of intestinal epithelia, while other studies indicated that the activation of the ERK/MAPK pathway was involved in the occurrence and development of intestinal mucosal barrier dysfunction in some pathological conditions. The effect of ERK/MAPK pathway on intestinal mucosal barrier dysfunction induced by LPS does not be well understood.Serious complications of severe acute pancreatitis (SAP) include systemic infection, pancreatic and peripancreatic abscess. Death from SAP occurs mainly from the secondary infection of bacteria and endotoxins translocated from the gastrointestinal tract, which may result in multiple organ dysfunction syndrome (MODS) as a consequence of the systemic inflammatory response syndrome (SIRS). One study found that the cultured bacteria in SAP group were maily Escherichia Coli and other gram-negative bacteria, which suggested the occurrence of intestinal bacterial translocation. Ammori et al have shown the increased intestinal permeability in patients with severe acute pancreatitis72hours after onset of acute pancreatitis. While another study has shown that the intestinal permeability was significantly increased6h after induction of SAP and the translocation of endotoxin and bacterial occurred18h after induction of SAP. Now, it is konwn that the intestinal hypermeability in SAP is associated with the production of cytokines and inflammatory mediators, intestinal ischemia reperfusion injury and apoptosis. However the mechanism is still not completely clear. Studies have shown that the disturbance of the TJ structure could result in the high permeability of gastrointestinal tract in many other digestive diseases, such as inflammation bowel disease, enteritis, etc. However, little is know about whether the intestinal barrier dysfunction of SAP is associated with the disturbance of TJ proteins.Somatostatin (SST), a kind of neuroendocrine peptides, was first separated as a growth hormone inhibitor from the hypothalamus in1973by Brazeatu, and it is widely distributed in the body. Somatostatin is widely used in acute pancreatitis, prevention and treatment of pancreatic fistula after pancreatic operation, hemorrhage of upper digestive tract, etc. Besides the effects of inhibitory of pancreatic enzyme secretion and intestinal fluid secretion, relaxation of the sphincter of Oddi functions, more and more researches indicated that SST could inhibit the tumor cell proliferation, inhibit inflammatory exudation and leukocyte chemotaxis, and inhibit the production of inflammatory mediators and cytokines. The intestinal mucosa, which expresses various somatostatin receptors (SSTRs), is an important target organ of SST. SST exerts its biological effects via interacting with the specific receptors. One study showed that SST could limit intestinal ischemia-reperfusion injury in macaques via suppression of TLR4-NF-κB cytokine pathway. Matthias et al indicated that SST regulated the TJ permeability in cultured keratinocytes via directly interacting with the TJ protein MUPP1. Another study about multiple sclerosis suggested that the somatostatin (SST)-like immunoreactivity in the cerebrospinal fluid of patients with multiple sclerosis was lower. And treatment with SST could restore LPS-induced disruption of TJ protein and protect the blood brain barrier in multiple sclerosis. Furthermore, other studies have indicated that somatostatin could inhibit the apoptosis of intestinal epithelial cells, and protect the intestinal mucosal mechanical barrier. However, the effects of SST on the intestinal barrier function and tight junction remain uncertain.Objective:1. Using Caco-2monolayer cells as an in vitro model of the intestinal epithelial barrier, the effect of LPS on the expression of intestinal epithelial tight junction proteins was evaluated. The effect of LPS on cell proliferation was evaluated in IEC-6monolayers, which were derived from the rat small intestinal crypt epithelial cell.2. The aim of this part was to evaluate the protective effects of SST on LPS induced barrier dysfunction of Caco2cells and the possible mechanism; and further discuss the effect of SST on the proliferation of IEC-6cells;3. The aim of this part was to establish a rat model of SAP, and evaluate the protective effect of SST on the blood cytokines level and intestinal TJ injury of SAP rats.Materials and Methods1. Regents and Materials Caco-2cells and IEC-6cells (ATCC, Manassas, VA), somatostatin (Sigma-Aldrich), Stilamin (Merck Serono, German), rabbit anti-occludin (Abcam), rabbit anti-ZO-1(Santa cruz), transwell inserts (Corning). CCK8assay kit (Dojindo Company, Japan), D-Lactate Colorimetric Assay Kit (Biovision), TNF-a and IL-1β Immunoassay kits (R&D Systems), Wistar rats (Experiment Animal Center of Southern Medical University).2. Methods2.1The effects of LPS on cell proliferation of IEC-6cells and the expression of tight junction proteins in Caco2cells.Cell proliferation was measured by CCK8Assay Kit according to the manufacturer’s instructions. The cells were treated with different concentration of LPS (0.1,1,10,50,100μg/ml) and incubated for24h. After LPS treatment,10μl of the CCK-8solution was added to each filter, and the reaction was allowed to occur in standard culture condition for2hours. The absorbance was measured at450nm using a precision microplate reader. Human colon cancer cell Caco-2cells, as the intestinal barrier model, were exposed to different concentration of LPS (0.1,1,10,50,100μg/ml) for24-hour. Levels of TJ proteins occludin and ZO-1were analyzed by Western blot.2.2SST inhibited the anti-proliferation of LPS and protected the LPS-induced tight junction damage through suppression the ERK-MAPK pathway in Caco2cells.IEC-6monolayers were pretreated with different concentration of SST (1,10,100,1000nM)1h prior to LPS, then incubated for24h before the measurement of cell proliferation. After the experiment,10μl of the CCK-8solution was added to each filter, and the reaction was allowed to occur in standard culture condition for2hours. The absorbance was measured at450nm using a precision microplate reader. Caco2monolayers were pretreated with1nM SST or U01261h pror to LPS, and incubated for24h. Transepithelial electrical resistance (TER) and FITC-dextran permeability were measured to analyze barrier integrity. Expressions of TJ proteins, occludin and ZO-1, were analyzed by Western blot. Under SST and LPS treatment, the localization of tight junction proteins was analyzed by immunofluorescence microscopy, and the expressions of p-ERK and ERK were analyzed by Western blot.2.3Somatostatin analog ameliorates intestinal mucosa damage via suppression NF-κB pathway in experiment acute pancreatitisThe adult wistar rats were randomly divided into three groups:sham operation group (SO group), SAP group (5%sodium taurocholate was retrograde injected into biliopancreatic duct) and SAP+SST group (SST was administered intravenously via the caudal vein in a dose of5ug/kg/h after the SAP induction at every24-hour).Each group was divided into three time points:6h,24h,48h (n=8per time point per group). The blood, pancreatic tissue and terminal ielum were harvested. Levels of plasma D-lactate, TNF-a and IL-1β were determined. Pancreatic and intestinal mucosal morphology were measured by HE staining. Expression and localization of TJ proteins (occludin and ZO-1) were detected by Western blot analysis and immunofluorescence microscopy. The expression of NF-κB was analyzed by immunohistochemistry.2.4Statistical analysisSPSS statistical software (SPSS13.0, Chicago, IL) was used to analyse results. All data were expressed as means±SD. The statistical significance of the differences between groups was determined by one-factor analysis of variance (ANOVA). A two-tailed p value of less than0.05was considered statistically significant.Results 1. LPS inhibited the proliferation of IEC-6cells and down-regulated the expression of tight junction proteins in Caco2cells.LPS inhibited the proliferation of IEC-6monolayers in a dose-dependent way. Low concentration of LPS could not inhibit the proliferation in IEC-6monolayers. However, when increasing the concentration to50ug/ml and100ug/ml, LPS became to exhibit the anti-proliferation effect in IEC-6cells (p<0.05). Otherwise, LPS could not affect the proliferation of Caco2cells, even in high concentration of100ug/ml. The expression of occludin and ZO-1were obviously reduced in a dose-dependent way in LPS-treated group. The occludin was significantly decreased at the concentration of lug/ml LPS and above, while the ZO-1was decreased at all concentrations (p<0.01). The minimum expression was at the concentration of100ug/ml (p<0.001).2. SST inhibited the anti-proliferation of LPS and ameliorated the LPS-induced tight junction damage through suppression the ERK-MAPK pathway in Caco2cells.High-dose of LPS (100ug/ml) obviously inhibited the proliferation of IEC-6, we next evaluated the effect of SST on the cell proliferation in IEC-6monolayers.IEC-6monolayers were pretreated with different concentrations of SST (1nM-100nM)1h prior to LPS, then incubated for24h before the cell proliferation was measured. SST reversed LPS-induced inhibition of cell proliferation at the concentration of1nM (p<0.01), but not higher concentrations. In contrast, SST alone did not affect cell proliferation in IEC-6monolayers at any tested concentration.Aadministration of100μg/ml LPS for24h resulted in a significant increase in FITC-dextran permeability and decrease in TER (p<0.01compared with control group). When Caco2cells were pretreated with lnM SST, the increased paracellular permeability was lost (0.008±0.0006mg/ml,p<0.001compared to LPS group), and TER was increased compared with the LPS alone group (477.44±27.41vs318.67±4.04, respectively, p<0.001).. Western blot analysis revealed that treatment with SST significantly increased the expressions of tight junction proteins, occludin and ZO-1. The localization of occludin and ZO-1was determined by immunostaining. In the control group, occludin and ZO-1presented a continuous band of cells encircling the apical cellular junctions. LPS treatment caused a pronounced disruption in localization of occludin and ZO-1staining, characterized by decreased intensity staining and marked discontinuity localization of intercellular junctions. Pretreatment with SST, the localization and intensity of TJ proteins were more similar to the control group.We found that exposure to100g/ml LPS could increase the expression of p-ERK (p<0.05). When pretreated with1nM SST, the expression of p-ERK was decrease compared with LPS group. Then Caco2cells were pretreated with10uM U01261h prior to LPS, the increase in paracellular permeability and decease in TER were reversed (p<0.01), and the expression of tight junction proteins occludin and ZO-1was increased compared with LPS group.3. Somatostatin analog ameliorated intestinal mucosa damage via suppressing NF-κB pathway in experiment acute pancreatitisWe succeeded in establishing the model of rats with SAP. There was no statistically significant difference in survival rate among the three groups at each time point. The SAP rats were all suffered from pancreatic injury characterized by acinar cell necrosis, fat necrosis and inflammation infiltrate. Treatment of pancreatitis rats with Stilamin (SST) resulted in a significant amelioration of pancreatic injury compared with SAP group (p<0.01). Concentration of plasma D-lactate in SAP group was significantly higher than in SO group at the three time point after the induction of SAP (p<0.01). Then, we found that treatment with SST could decrease the levels of plasma D-lactate at the three time points. The difference between these two groups was not significant until48h after induction of SAP (p<0.05). At the three time points, levels of TNF-a and IL-1β were higher in the SAP group than in the SO group (p<0.01). Compared with the SAP group, the levels of IL-1β were significantly decreased in SST group at the three time points (p<0.01). Whereas TNF-a were decreased at the time points of24and48hours (p<0.01), but the difference between SAP and SST group was not that significant at6hours (p=0.046). The structure of intestinal mucosa was ruined in SAP group compared with the SO group and SST group. The intestinal damage, including incomplete mucosa, inflammation cells infiltration in propria, focal necrosis was observed in SAP rats. The ileal villus heights and crypts depths were shorter than those of the SO group and the SST group (p<0.01), there is no difference between SO and SST group (p>0.05).Western blot analysis showed that expressions of TJ proteins, occludin and ZO-1, were decreased in SAP group24h after modeling. While injected with Stilamin, the expressions of occludin and ZO-1were increased compared with SAP group. Expression and location of occludin and ZO-1was determined by immunofluorescence. In SO group, the occludin and ZO-1were located at the cellular borders at the surface of the intestinal mucosa epithelia. When at24hours after induction of SAP, occludin and ZO-1were both disrupted in the continuity and integrity. In SST group, the distribution and localization were trend to be normal compared with SAP group. The expression of NF-κB was analyzed by immunohistochemistry. The staining intensity of NF-κB was higher in the SAP group than in the SO at6h after the induction of SAP (p<0.01). The increased staining intensity was observed at24h and48h either. When treatment with SST, the intensity of NF-κB was decreased compared with that in SAP group (p<0.01). There was no statistical difference between the SO and SST groups (p>0.05).Conclusion1. LPS significantly inhibit the proliferation of intestinal epithelia and disrupt the tight junction, and cause intestinal epithelial barrier dysfunction.2. This study for the first time indicates that11nM SST shows the ability to improve the barrier function of IEC monolayers under LPS stimulation. Otherwise, when exposed to LPS, the phosphorylation of ERK1/2is activated, and this may be involved in the disruption effects of LPS on intestinal barrier function. SST significantly suppresses the activation of ERK. Therefore, it seems that SST regulates the intestinal barrier partly via the ERK-MAPK pathway in response to LPS challenge.3. This study suggests that increased intestinal permeability and damaged intestinal mucosa in SAP rats was accompanied by production of cytokines in plasma. The disturbance of TJ proteins of intestinal epithelia was observed24h after induction of SAP. The results suggest that down-regulation of TJ proteins might be a pathological factor in the development of intestinal barrier dysfunction in SAP. Furthermore, SST had a protective effect on the intestinal barrier and mucosa integrity during SAP. This effect was apparently mediated by inhibition of NF-κB activation, and suppression of the production of cytokines. SST may be an effective means for protecting the intestinal barrier.