Small Intestine Barrier Dysfunction And Mechanism Of Medication Administration In Septic Rats

Backgroud and objectiveSepsis is a generalized inflammatory response induced by a systemic infection. Its complications leads to immunological dysfunction and deterioration of many organs function. In recent years, the gastrointestinal tract has assumed more importance in the septic patient. Previously, the gastrointestinal tract was regarded as an organ that contributed little to the pathophysiology of sepsis but it is now recognized that the small intestine and colon make important contributions to the maintenance of hypermetabolism in sepsis.This is due to changes in gastrointestinal structure and function that promote loss of intestinal barrier function. The small intestine barrier comprises both immunological and non-immunological protective components, the former being divided into local (Mesenteric lymph nodes, MLNs; Intestinal intraepithelial lymphocytes, IELs; etc) and systemic components (circulatory lymphocytes, spleen), and the latter comprises physical (healthy enterocyte and tight junction) as well as intraluminal bacteria. The physical mechanism is represented by the small intestinal epithelium. The structure of the intestinal epithelium with its tight junction disposal allows only the passage of very tiny molecules, preventing the bacterial or the macromolecular (lipopolysaccharides) transport.Under normal circumstances, the intestinal tract absorbs nutrients while maintaining bacteria within the intestinal lumen. In sepsis, the changes in the small intestinal permeability to macromolecular follow the same time-course as bacterial overgrowth and increased toxin production, the small intestine may allow increased bacterial infiltration into mesenteric lymph nodes (MLNs) and other extraintestinal sites (eg, spleen, lung, liver, blood). This process has been termed bacterial translocation. Paracellular bacterial transport may also be facilitated by changes in epithelial cell structure, particulary involving tight junction (TJ). Increased transcellular and paracellular bacterial transport across the epithelial cell layer have been demonstrated with both direct and indirect injury to the small intestinal mucosa.Passage of bacteria across the intestinal lumen alone is insufficient to produce the deleterious effects observed in the sepsis population; the survivability of the translocated bacteria also is essential. To this end, the host immune response becomes a vital mediator of pathogenicity.Because of a state of immunosuppression, sepsis patients have an inability to mount an adequate immune response to invading pathogens.This suppression is characterized by a decrease in splenic and peripheral blood T-cell proliferation.The intestine immune system, containing most of the lymphocytes is considered to be of great importance for maintained function and integrity of the intestine mucosa. The population of T cell found in the small intestine accounts for approximately 60% of the total body T-cell population. The suppression of T-cell proliferation has been a common finding after sepsis, and this suppression may allow bacterial growth in MLN and their passage to the spleen and circulation. In addition to supression of population and proliferation, T cells are directed by antigen-presenting cells (APCs), which present antigenic peptides from captured microbes to B and T lymphocytes either locally (in the intestine) or in the MLN (to which they migrate via afferent lymph vessels), to initiate a Th1 immune response. Central to the Th1 response is the secretion of IFN-γfrom the T cells, which activates macrophages to respond to the stimulus. During a normal Th1 response, T cells also secrete IL-4 (Th2 response), which acts to limit the Th1 response. This Th1/Th2 balance and other regulatory mechanisms may be disrupted in sepsis, allowing an abnormally robust inflammatory response. Impairment of cell immunity, then is acting on the epithelium, further disrupting the barrier and increasing permeability. In this manner, a vicious cycle is created in which barrier dysfunction allows further leakage of luminal contents, thereby triggering an immune response that can in turn feed back on the intestinal barrier to promote further leakiness.One of the common complications of sepsis is prolonged gastrointestinal stasis, or ileus. Sepsis inhibits gastrointestinal motility. Retention of food in the gastrointestinal tract can cause bacterial overgrowth, leading to bacterial translocation and multiple organ failure. These observations show that gut stasis may be a result of sepsis but may also cause sepsis, particularly persistent or recurrent sepsis. Microcirculatory dysfunction also plays a pivotal role in the pathophysiology of sepsis and contributes considerably to tissue hypoxia and multiple organ failure. Splanchnic hypoperfusion and redistribution of blood flow away from the mucosa lead to mucosal hypoxia in sepsis. Because of its particular vascular architecture with countercurrent oxygen exchange in the villi and extensive requirements of oxygen and nutrients for functional integrity, the bowel mucosa is especially vulnerable to perfusion deficits. Ensuing ischemia of the gut, resulting from microvascular derecruitment of vascular beds, may be specially relevant for the evolution of sepsis because the mucosal barrier may be disrupted and allow for translocation of bacteria and toxins across the intestinal wall, thereby maintaining a septic state and being a motor of sepsis.During host defense against microbial attack, mast cells play an important role because they typically concentrate at strategically important locations, such as mucosal surfaces. The intestinal mucosa consists of approximately 2-3% of mast cells within the lamina propria in healthy individuals. Mast cells can ingest and kill enteric pathogens such as Escherichia coli. Bacterial binding triggers mast cell degranulation. Abundant mucosal mast cells are observed in gastric tissue infected with Helicobacter pylori. Therefore, it is imaginable that intestinal mast cell activation, in some degree, may often happen physiologically but may not always be appropriate or beneficial to the host. Recently, Mast cells are considered a harmful role in bacterial infections through use of a septic model. This conclusion is supported by studies showing that dipeptidyl peptidaseⅠ(DPPI)-/- mice are protected from death due to sepsis induced by cecal ligation and puncture (CLP) and that mast cell DPPI enhances the likelihood of death from sepsis in syngeneic wild-type animals, notwithstanding the net protective role of mast cells suggested in prior studies in mast cell-deficient mice. In the intestine, mast cells are now thought to regulate other tissue functions of central importance for gut function, including the regulation of epithelial barrier, motility and visceral sensitivity, by releasing preformed mediators from their granules or by releasing de novo synthesized mediators on activation including lipid mediators and cytokines. Nitric oxide and RMCP-Ⅱhave been also identified as important mast cell mediators related to gastrointestinal tracts.RMCP-Ⅱ, a soluble rat mast cell chymase, originally described as an intracellular 'group-specific' protease and isolated from intestinal mucosa, was subsequently shown to be of mast cell origin. 14 The Heterogeneous expression of granule proteinases by mast cell subpopulations was initially described in rodent mast cells where it was shown, using specific antibodies, that rat mucosal mast cell (MMC) expressed the highly soluble beta chymase RMCP-Ⅱ, but lacked the insoluble and strongly basic beta chymase, RMCPⅠ. Conversely, connective mast cell (CTMC) contains RMCPⅠand lack RMCP-Ⅱ. An homologous, soluble chymase, MMCPⅠ, is uniquely expressed in mouse MMC that are predominantly located within mucosal epithelia. Similarly, in normal sheep, SMCP-Ⅰ, a dual-specific chymase/tryptase is expressed by MMC in the gut, but not by mast cells in the adjacent submucosa. Thus, in rodents and sheep, intestinal MMC have a distinct proteinase phenotype. Therefore, RMCPⅡis thought as a marker of intestinal mast cell degranulation.A small amount of NO from cNOS is produced on demand under physiological conditions and is often acting through a direct action on cells adjacent to the source of NO in the gastrointestinal mucosa. Such action is implicated for the defensive mechanisms operating in the gastrointestinal tract. On the other hand, a persistent and large quantity of NO produced in inflammatory cells from iNOS could induce nitrosative and oxidative stress in sepsis. These could promote and even initiate mucosal damage through an indirect reactive interaction with biological molecules.Because of the important contribution to the development of sepsis, the small intestine turns to be a new therapy target. Ethyl pyruvate (EP) is a stable lipophilic pyruvate derivative experimental agent. Ethyl pyruvate has been shown to ameliorate intestinal, renal, or hepatic injury when it is used as a therapeutic agent to treat rodents subjected to mesenteric ischemia and reperfusion, hemorrhagic shock, endotoxemia, or polymicrobial bacterial sepsis. Treatment with ethyl pyruvate also ameliorates organ dysfunction in murine models of acute pancreatitis and alcoholic hepatitis. In many of these models of acute critical illness, treatment with ethyl pyruvate down-regulates the expression of various pro-inflammatory genes, including inducible nitric oxide synthase (iNOS), tumor necrosis factor, cyclooxygenase-2, and interleukin (IL)—6.Ulinastatin ( UTI) is a glycoprotein with a molecular weight of about 24,000 Da and is extracted and purified from human urine. UTI has anti-inflammatory activity and suppresses the infiltration of neutrophils and the release of elastase and chemical mediators from neutrophils. Recent studies have shown that UTI may be cytoprotective against ischemia-reperfusion injury in the liver, kidney, heart, and lung. Furthermore, UTI inhibits the production of tumor necrosis factor (TNF)-αin lipopolysaccharide (LPS)-stimulated human monocytes and inhibits LPS-stimulated interleukin (IL)-8 gene expression in HL60 cells.Effect of EP and UTI on small intestine barrier (physical and immunological), motility and mucosal microcirculation in sepsis is few investigated. In the present study, a rat model of sepsis was induced by cecal ligation and puncture (CLP). Using this model, we evaluated change of the parameters of the small intestinal barrier and effect of EP and UTI in sepsis. Furthermore, the mechanism of medication on mast cell also was investigated in this experiment.Materials and methods1 Animals: Male Wistar rats, weighing 250 to 300g, were obtained from Shangdong Experimental Animal Center, and fed standard rat chow and water. The rats were allowed to acclimatize to our laboratory conditions for seven days and were subjected to a 12-hour day-night cycle living in mesh stainless steel cages at constant temperature. Because a higher mortality was expected with CLP, more animals were set in the septic group (n = 35) and the septic group with ethyl pyruvate administration (n = 35) and with ulinastatin administration (n=35) than in the sham-operated group (n=15). Rats were randomly assigned to every group. EP (injected intraperitoneally) administration was noted over a period of 4 days (at interval of 6h until the death of the animals). Ulinastatin (UTI, 50 000U/kg/d) was injected intraperitoneally once very day after CLP for 4 days.Surgical procedureSepsis in the experimental groups was induced by CLP, whereas the control group underwent a sham operation. Animals were anaesthetized with pentobarbital (50mg/kg, i.p.) and a 2-cm ventral midlined incision was made. The cecum was isolated and ligated with 4-0 silk avoiding intestinal obstruction. The cecum was then punctured twice with a 23-gauge needle, and a small amount of the bowel contents was extruded through the puncture holes. After returning the bowel to the abdomen, the midline incision was closed in two layers. Sham-operated animals underwent the same surgical procedure except that the cecum was neither ligated nor punctured.Four days after sepsis, or sham operation, immediately after reopening the abdomen, ascetic fluid was obtained and stored at -70℃until assayed. The 2cm distal ileum biopsies were removed. The MLNs and spleens were removed aseptically and placed in Petri dishes containing ice-cooled phosphate-buffered saline solution (PBS, pH 7.4).2 Methods(1) Survival analysis in every group.(2) Histopathological examination: Histological changes in ileal architecture were graded as described by Ozkan et al. Grade 1, normal structure of ileum; grade 2, hydropic degeneration and/or separation of the surface epithelial cells from lamina propria; grade 3, epithelial cells necrosis confined to the tips of the villi; grade 4, complete villi necrosis; grade 5, transmural necrosis.(3) Ultrastructural studies: observe enterocytes, mitochondria and mast cell change.(4) Bacterial translocation: Under strict sterile conditions, MLNs and spleens were collected, homogenized and inoculated onto a blood agar plate to incubated at 37℃for 24 to 48 hours. The bacterial colony forming units were counted.(5) Flow cytometric analysis: Single cell suspensions were prepared from MLNs and spleens. Three-color direct fluorescence staining was performed to label CD3, CD4 and CD8 positive cells. CD4 ~+ /CD8~+ T cell ratio was calculated by dividing the percentage of CD4~+ CD3~+ cells by the percentage of CD8~+ CD3~+ cells.(6) Immunohistochemistry: Immunohistochemically with CD4 and CD8 labelled tissues were examined(7) Splenocytes proliferation assay: spleen lymphocytes proliferation analysis with MTT method.(8) Cytokines ELISA: IFN-γand IL-4 concentrations in supernatants of cultural splenocytes were measured.(9) Microvascular perfusion in small intestine recorded with a laser Doppler flow monitor.(10) Small intestinal motility studies: small intestinal lumen pressure was analyzed(11) Ultrastructural observation of mast cell.(12) Level of RMCPⅡreleased into small intestinal lumen.(13) Concentration of NO_X in plasma and small intestine.(14) Small intestine tissue myeloperoxidase (MPO) activity. (15) Quantitative RT-PCR analysis of iNOS mRNA.3 StatisticsThe Kaplan-Meier and Log-Rank tests were used for comparing survival.Histopathological examination results were compared using Kruskall Wallis analysis. Other results were presented as means±SEM. Data were analyzed for significance by one-way ANOVA including the Neuman-Keuls test. Statistical analysis was performed by using the software SPSS 15.0 for windows. Two-tailed tests of significance were employed and significance was assumed at P<0.05.Results1 Survival rate: no animal died in the sham group. Survival rate was 37.1% in CLP group, 57.1% in the EP group, and 62.9% in the UTI group respectively.2 Histopathological analysis: The CLP group differed significantly from the sham group (P<0.001). A lesser degree of mucosal damage was seen in the EP group and UTI group as compared to the CLP group respectively (EP, P<0.05; UTI, P<0.05).3 Study of ultrastructural morphology: Only a low percentage of open TJ was observed in the sham group (3.5±1.9%) whereas this percentage was significantly higher in the CLP group (23±2.6%, P<0.001). EP and UTI administration significantly reduced the TJ opening percentage (EP, 16.5±3.4%, P<0.05; UTI, 13.5±3.4%).4 Bacterial translocation: No bacterial colony was observed in the sham group. The positive culture were found in the CLP group (13/13) EP group (16/20) and UTI group (17/22) respectively. Bacterial translocation results of the two groups were calculated by colony forming unit per gram (CFUs/g). The colonies in the EP group significantly decreased compared with that in the CLP group (14.4±5.9×10~3 CFUs versus 26.0±10.1×10~3 CFUs, P<0.05). Similarly, the colonies in the UTI group significantly also decreased (13.1±4. 0 ×10~3CFUs, P<0.01).5 Flow cytometric analysis: Proportion of CD4~+ T cells in the CLP group were significantly lower than those in the sham group. This phenomenon was ameliorated after EP administration. Proportions of CD8~+ T cells in splenocytes and MLNs did not show significant change among the three groups. CD4/CD8 T cell ratio decreased in the CLP group. Amelioration of the ratio in the EP group was observed. However, in the UTI group, this amelioration was not observed.6 Immunohistochemistry: CD8~+ T lymphocytes intraepithelial were significantly increased in the CLP group compared with that in the sham group, but EP administration can't attenuate this increase (P>0.05 versus CLP group). In lamina propria, there was a significant reduction in the number of CD4~+ T lymphocytes in the CLP group (P<0.01) compared with that in the sham group. An amelioration of CD4~+ cells was observed in the EP group (P<0.05) compared with that in the CLP group. In the UTI group, we can't observe this amelioration.7 Splenocyte proliferation assay: The PI in response to PHA were markedly suppressed in the CLP group (5.3±1.7) compared with the sham group (11.6±2.6, P<0.01). EP administration attenuated the suppression (7.1±2.0, P<0.05; versus the CLP group). However, UTI administration couldn't attenuate this suppression ( 6.1±2.4, P>0.05)c .8 Concentrations of Th1/Th2 cytokines in culture supernatant: In the supernatant of PHA-stimulated splenocytes, concentration of IFN-γin the CLP group (CLP, 1455.9±81.6 pg/ml; sham, 1910.5±121.1 pg/ml; P<0.001) was lower than in the sham group. EP administration changed this decrease (1705.6±128.3pg/ml, P<0.001, versus the CLP group). IL-4 level in the CLP group (CLP, 115.3±18.9 pg/ml; sham, 52.8±11.1 pg/ml P<0.001) was higher than that of the sham group. EP also changed this increase (86.4±17.2 pg/ml, P<0.01, versus the CLP group). In the UTI group, we didn't observe this amelioration ( IFN-γ, 1560.3±148.7, P>0.05; IL-4,102.2±28.3; P>0.05)9 Small intestine intraluminal pressure analysis: In the sham group, the jejunal musculature generated relatively regular large prolonged phasic contraction and prolonged large phasic contractile frequency averaged 1.6±0.7 per minute, the amplitude of jejunal pressure waves averaged 11.2±3.7 mmHg. At 4 days after CLP, jejunal contractile amplitude in the CLP group was significantly reduced (4.6±2.3 mmHg, P<0.01), large phasic contractile frequency of jejunum was significantly less frequent (0.7±0.2 per minute, P<0.01) than that in the sham group. The amplitude and contractile frequency of the EP group were ameliorated (8.1±2.6 mmHg, 1.4±0.5 per minute, P<0.05 versus the CLP group). Similarly, we observed this change (9.0±2.5 mmHg, P<0.05; 1.5±0.4 per minute, P<0.01) in the UTI group.10 Small intestinal mucosal microcirculation: Mucosal blood flow of jejunum was observed decrease in the CLP group (158.6±42.9 PU) compared with the sham group (269.9±34.3 PU, P<0.001). However, mucosal perfusion of jejunum showed a significant flow increase in the EP group (199.5±36. 8, P<0. 01) and the UTI group (230.8±51.9 PU, P<0.001) compared with CLP group.11 Ultrastructure of mast cells: In the sham group, there were very few degranulated mast cells (6.3±4.8%), while in the CLP group, the number of degranulated mast cells increased (32.5±6.4%, P<0.01 versus the sham group). In the UTI group, the magnitude of mast cell degranulation decreased (21.3±4.8%, P<0.05 versus the CLP group).In the EP group, we didn't find this decrease (28.7±4.9%, P>0.05).12 Level of RMCPⅡreleased into small intestinal lumen: RMCP-Ⅱlevel in jejunal lumen of the CLP group (28.6±7.8 ng/ml) increased significantly compared with the sham group (0.9±0.2 ng/ml, P<0.001). However in the UTI group, the RMCPⅡlevel was reduced compared with the CLP group (22.7±7.9 ng/ml, P<0.05).In the EP group, we didn't find this amelioration (29.1±8.6 ng/ml, P>0.05).13 Small intestine myeloperoxidase (MPO) activity analysis. CLP group showed a significant increase in the small intestine tissue levels of MPO (12.9±6.2 U/g tissue; versus the sham group, 2.1±1.9 U/g tissue; P<0.001). EP and UTI administration can result in a significant decrease in the activity of MPO ( EP: 8.6±4.4 U/g tissue,P<0.05;UTI: 6.4±4.5 U/g tissue,P<0.01) .14 Concentration of NO_X in plasma and small intestine: Increased of NO synthesis in plasma was time dependent in the CLP group, UTI administration blocked the NO_X elevation in the plasma at all times after CLP. At 4 day after CLP, the nitrite/nitrate level in plasma in CLP group is 322.2±42.1 nmol/L. EP and UTI administration can inhibit this increase (EP , 249.7±41.6μmol/L,P<0.05 ; UTI,236.2±31.3μmol/L,P<0.0 1). The nitrite/nitrate level of jejunum in the CLP group (63.5±11.4 nmol/g tissue) was significantly greater than that in the sham group (20.3±5.1 nmol/g tissue; P<0.001), and its elevation was suppressed by EP and UTI administration (EP, 46.1±9.8,P<0.01; UTI,43.3±12.7,P<0.01).15 Expression of iNOS mRNA in small intestine: Expression of iNOS mRNA was not detected in jejunum of the sham group. At 4 days after CLP, iNOS mRNA expression in jejunum of the CLP group increased significantly than that of the sham group (0.62±0.13, P<0.001). EP and UTI administration blocked the iNOS mRNA production effectively (EP: 0.37±0.11, P<0.01; UTI: 0.33±0.17, P<0.01).Conclusions and significances1 In this septic model induced by CLP, we found the small intestinal barrier failure: physical and immunological barrier, motility and microcirculation, the activity of mast cells, NO concentration in plasma and small intestine tissue, MPO activity and iNOS mRNA. 2 We found ethyl pyruvate and ulinastatin could ameliorate sepsis mortality, small intestinal structure, motility and microcirculation, some inflammatory mediator. These medications also can protect the small intestine by different mechanisms.(1) EP can inhibit bacterial translocation and ameliorate survival rate. EP administration can prevent small intestine physical and immunological barrier changes, especially immunological change. Distribution of CD4+ T cells in villi, proportions of CD4+ T cells in mesenteric lymph nodes and spleen, proliferative capacity of splenocytes were increased. IFN-γand IL-4 release were also modulated. EP also can attenuate small intestine microcirculation and motility and inhibit MPO, NO_X and iNOS mRNA increase. EP can't inhibit the mast cell activity.(2) UTI can inhibit bacterial translocation and ameliorate survival rate. UTI can ameliorate the small intestine physical barrier, but not the immunological barrier. UTI also can ameliorate the small intestine motility and mucosal microcirculation and reduce NO_X and iNOS mRNA expression. It is important that UTI can inhibit the mast cell activity amd RMCPⅡlevel in the small intestine lumen.