Study On Protective Roles Of Platelet Activating Factor Receptor Antagonist In Intestinal Barrier Function During Endotoxemia In Young Rats

IntroductionChildren gastrointestinal dysfunction refers to the clinical syndrome caused by injury of one of the digestion, absorption and excretion. The clinical manifestation is abdominal distention, bowel sound disappearing, bloody vomiting, he-matemesis and melena based on critical illness. This prognosticates the condition aggravating and unfavorable prognosis. Additional the disease of gastrointestinal system, its etiology is often associated with some serious conditions, such as infection, trauma, shock, burns and asphyxia. The gastrointestinal tract was proposed as the " motor" organ of multiple system organ failure in 1980s. This illustrates gastrointestinal dysfunction play important roles in the development of critical illness.Severe infection is one of the common causes of gastrointestinal dysfunction in children with critical illness, the mechanism of which is close related to endo-toxemia and impairment of gut mucosal barrier function. Disruption of gastrointestinal epithelial barrier integrity may induce increasing of permeability of gastrointestinal wall, bacterial and endotoxin translocation, releasing inflammatory mediators, and lead to lethal sepsis and distant organ injury, finally result in multiple system organ failure. The original sign of impairment of gut mucosal is increasing of permeability. Before the marked change of morphology of gut mucosal , increasing of permeability of intestinal tract has happened. So increasing of permeability of gut mucosal can reflect injury of intestinal barrier earlier. Increasing of permeability of gut mucosal may provide a chance for absorption oftoxin in enteric cavity, which is the pathological foundation of bacterial translo-cation. Barrier of preventing bacterial translocation is comprised of follow: (D The mechanical barrier is comprised of continual epithelial of gut mucosal;(2) Cellular and humoral immunity provided by lymphoid tissue of intestinal tract;?Normal and massive commensal intestinal bacterial clusters, preventing the o-ver proliferating and permanent planting in gut mucosal of pathogenic microorganism. One or several of these were injuried, that will induce bacterial translocation. Many scholars have paid attention to the intestinal barrier function. The mechanical barrier is comprised of mucus of intestinal surface, microvilli, epithelial cells of gut mucosa, and the tight junction between them, and special structure of mucosa. Intercellular adhension in intestinal epithelia is composed of cell - cell junctions that include tight junctions ( TJ) , adherens junctions ( AJ) and desmosomes. The proteins of cell - cell junctions in intestinal epithelia include ZO - 1, occludin, cadherin, claudin and (B — catenin. These molecules directly or indirectly interact with many intracellular proteins, especially cytoskeleton proteins and comprise the TJ and AJ of epithelial cells. As a selective intercellular barrier, the main function is modulating the diffusion of molecules and ions.In recent years, the roles of platelet activating factor (PAF) to impairment of gastrointestinal mucosa has been paid increasing attention. PAF was regarded as central amplify mediator in inflammatory factors inducing gastrointestinal mucosal impairment. Intraperitoneally injection of lipopolysaccharide ( LPS) in neonatal rats may induce intestinal injury. Intestine can generate endogenous PAF by intraperitoneally injected with lipopolysaccharide. PAF may be the major effective factor in endotoxin roles. It has been confirmed that the changes of necrotizing enterocolitis (NEC) induced by LPS and PAF was alike. Bowel necrosis induced by LPS can be lightened by using PAF receptor antagonist WEB 2170. It was found that LPS may caused increasing PAF secretion, as well as an obviously increased expression of PAF receptor mRNA in peripheral blood lymphocyte of multiple organ dysfunction syndrome ( MODS) patients. PAF may cause injury of intestinal mucosal barrier and bacterial translocation in acute pancreatitis (AP). Using PAF receptor antagonist BB - 882 can lesson injury ofintestinal mucosal barrier and inhibit bacterial translocation. Hence, LPS make gastrointestine and systemic organs dysfunction through up - regulating expression of gastrointestinal PAF receptor and irritating generation of PAF.It has been verified that PAF participated gastric ulceration, NEC, ische-mic gastric and small intestinal mucosal impairment. But how does PAF play the roles in intestinal lesion during endotoxemia? Whether the intestinal function disorder was partly caused by injured intestinal barrier? PAF display its biologic activity through binding to a variety of cell types on PAF receptor (PAFR). In this study, we used endotoxemia model of 18 day -old young rats, observed ul-trastructural changes of small intestine epithelial cells, assessed diamine oxidase (DAO) of blood and intestinal tissue, figured out the intestine lesion index, measured the secretory immunoglobulin A ( slgA) —an immuno - barrier function index, and changes of ZO - 1, E - cadherin, (3 - catenin, and effect of PAF receptor antagonist to above. In order to explore the mechanism of PAF in intestinal lesion in endotoxemia through protective effects of PAF receptor antagonist for intestinal barrier function, and provide theory base for therapeutic approaches of clinical gastrointestinal dysfunction.Materials and Methods1. Animal modle ProtocolEighteen - day - old Wistar rats were randomly separated into 4 groups: LPS group and LPS plus PAF receptor antagonist group ( prevention group and treatment group) and control group (n =8, respectively) , removed the ileum at 1.5, 3, 6, 24, 48 and 72 h after LPS injection. LPS group and control group was injected intraperitoneally lipopolysaccharide 5 mg/kg and normal saline 1 mL/kg respectively. Prevention group and treatment group was injected intraperitoneally PAF receptor antagonist BN52021 5 mg/kg 30 minute before or 30 minute after LPS injection.2. Tissue PreparationAll rats were sacrified at each time point and then blood were collected via decapitation. Blood were centrifuged for 15 min in 3 500 r/min, taken superna-tant and stored at -20^ until use. Ileum speciments were dissected about 5 cm proximal to the ileo - cecal junction. Samples were filled with freezing saline, to rinse small intestine contents. The ileum used to observing morphology and im-munohistochemistry were fixed with 10% neutral buffered formalin solution. Samples used to transmission electron microscopy ( TEM) observation were fixed in 2. 5% glutaraldehyde. Samples used to determining reverse transcription -polymerase chain reaction (RT - PCR) were got into no - ribonuclease test tube as soon as possible, then quickly frozen in liquid nitrogen and then stored at -80^ until use. Samples used to evaluating radioimmunoassay were filled with 200 |xL normal saline, stayed overnight for 4^ , centrifuged for 15 min in 2 000 r/min, taken supernatant and stored at - 201 until use.3. Methods and evaluation3. 1 Ileum morphological examination3. 1. 1 Macroscopic exam: After abdominal cavity was opened, the small intestine was grossly examined for such sign as intestinal colour, edema, hemorrhage, distention and perforation. Weighing the weight of each rat before kill them at each time point, calculated the changes of weight by means of ( weight after experiment - weight before experiment) /weight before experiment.3.1.2 light microscopic exam: Fixed ileum were dehydrated, embedded in paraffin, microtome - sectioned at 4 - 5 jxm, and stained with hematoxylin and erosin for morphomatric measurement.3. 1. 3 Transmission electron microscopy exam: Small samples (1 mm3) were subsequently post - fixed in 1 % osmium tetroxide, dehydrated in a gradede series of ethanol, and embedded in epoxy resin (Epon 812) and ultra thin cross sections (0.05ji,m) prepared with ultramicrotome, and finally stained with ura-nyl acetate and lead citrate, observed with transmission electron microscopy.3.1.4 W/D measurement: The fresh ileum tissue blotted water using filter paper were weighed first wet weight ( W) , then put into 37 X, baking oven for 72h, weighed the tissue again as dry weight ( D). W/D ratio was calculated by means of % according to W - D/W.3.2 Protein and gene of ileum examination3. 2.1 Spectro photometer method;The content of DAO in ileum tissue andplasma was determined respectively.3.2. 2 Double antibody - PEG radioimmunoassay: The content of slgA in intestinal mucosa was measured.3.2.3 Immunohistochemistry: The location of ZO - 1, E - cadherin, |3 -catenin and their proteins expression were evaluated respectively.3. 2.4 Reverse transcription - polymerase chain reaction ( RT - PCR) : The expression levels of ZO - 1 mRNA, E - cadherin mRNA, p - catenin mRNA were determined respectively.4. Statistical analysisSoftware SPSS 10. 0 for Windows was used in all statistical tests. All data were expressed as mean ± standard deviation. Comparisons between two groups were performed with ANOVA LSD method (least significant difference). When P was less than 0.05, the difference was considered statistically significant.Results1. The pathological findings of the young rat ileum tissue 1. 1 At the organ level: After injected LPS, rats appeared lassitude, tremble , anorexy, loss of weight after 1.5 h, diarrhea, abdominal distention. Those who critical appeared convulsion, accelerated breathing, cyanosis at lip and extremities, body chilly, even dead. Death usually occured at 6 h to 12 h. In the antagonist group, condition was obviously improved and mortality decreased. Mortality of the LPS group, the prevention group and the treatment group was 19.54% (17/87), 7.41% (4/54), 3.57% (2/56) respectively. Congestion of intestinal wall and mesentery, intestinal canal distention and edema, effusion of the gut lumen increase, loose stools, seriously spot or plaque hemorrhage in intestinal wall, bloody purulent was observed. Edema and congestion was mild in intestinal canal at 1.5 h, seriously at 3 h - 6 h, with hypercoagulation of blood. After 48 h the gross changes turned to normal. The gross change of antagonist group were mild, only mild edema in the serious.1.2 At the light microscope level: Intestinal villi edema, capillary congestion of the lamina propria, extension of the subepithelial lympho channel, andpolymorphonuclear infiltration in enteric cavity were noted in the LPS group at 1.5, 3, 6 and 24 h after LPS injection. No obvious changes occurred at 48 h and 72 h. In the PAF receptor antagonist groups only villi edema were found at 1.5, 3, 6 and 24 h. Structure of ileum in control group was normal at each point.1. 3 Ultramicrostructural change of TEM: Microvilli, cytoplasmic organoids and tight junctions were intact in the control group. Enlargement of tight junctions were observed in experiment group and microvilli were thin, rare or dis-rupL,.shed. The rough eadoplasmic reticulum, mitochondria, and glycogen particles were damaged. Changes of TEM were extremely obvious in LPS group at 3h and 6 h. The above changes were alleviated in PAF receptor antagonist group.1.4 Intetine W/D ratio: The W/D ratio in experiment group was significantly higher than that in the control group at all time points. There were statistically significant at 3 h and 6 h, P <0. 05. The ratio in antagonist group was slightly lower than that in LPS group. The W/D ratio of prevention group was slightly higher than that of treatment group, but having no statistical difference.2. The content of DAO in ileum tissue and plasma: The content of DAO in ileum of LPS group was obviously lower than that of control group within 1.5 h and sustained through 72 h. The difference was significantly, P < 0. 01. The tendency of changes in prevention group and treatment group was the same as that in LPS group, the content of DAO at each time point was higher than that of LPS group, but no statistical difference.The content of DAO in plasma of LPS group was obviously higher than that of control group. The expression was extremely obvious at 6 h, P<0.01. The tendency of changes in prevention group and treatment group was the same as that of LPS group, the content of DAO at each time point was lower than that of LPS group, DAO of prevention group was slightly higher than that of treatment group, but no statistical difference.3. The change of slgA level as index of ileal immuno - barrier function;The slgA content was obviously decreased after 1.5, 3, 6, 24 and 48 h of LPS challenge compared with that in the control group (P <0.01). It reached to anadir at 6 h. The tendency of changes in the prevention group and treatment group was the same as that in the LPS group. The level of slgA in the antagonist group was higher than that in the LPS group at each time point. There was no statistical difference in the slgA level between the prevention group and the treatment group.4. The expression of ZO - 1, E - cadherin, p - catenin as index of ileal mechanical barrier function4.1 The staining of ZO -1 in villi of the control group was similar to a honeycomb, which reflected a continuous and uniform distribution localized at the apical portion of cell - to - cell contact of the enterocytes. In LPS group, the signals of ZO -1 were disrupted and irregularly distributed at the outer enterocytes periphery.The content of peripheral membrane protein ZO - 1 was obviously decreased in the LPS group than that in the control group in optical density average and ZO- 1 mRNA. The ZO - 1 content decreased most at 6 h. The tendency of changes in the prevention group and the treatment group was the same as that in the LPS group. The level of ZO - 1 protein and ZO - 1 mRNA in the antagonist group was higher than that in the LPS group at each time point. The level of ZO-1 protein and ZO - 1 mRNA in the prevention group was slightly lower than that in the treatment group. There was no statistical difference in the ZO - 1 protein and ZO - 1 mRNA level between the prevention group and the treatment group.4.2 Membranous E - cadherin staining was localized uniformly at the intercellular borders of histologically normal epithelial cells. In the LPS group showed a decrease or irregularly distributed in membranous E - cadherin expression.The level of paracellular adherence junctional protein E - cadherin was obviously decreased in the LPS group than that in the control group in average optical density and E - cadherin mRNA, P < 0. 01. The content of E - cadherin mRNA decreased most at 6 h and 24 h. The tendency of changes in the prevention group and the treatment group was the same as that in the LPS group. The level of E - cadherin protein and E - cadherin mRNA in the antagonist groupwas higher than that in the UPS group at each time point. Hie level of E - cadherin protein and E - cadherin mRNA in the prevention group was slightly lower than that in the treatment group. There was no statistical difference in the E -cadherin protein and E - cadherin mRNA level between the prevention group and the treatment group.4. 3 p - catenin immunohistochemical labeling was evident in the control rats where p - catenin labeling appeared mainly in the shape of lines defining the pericellular space between cells. This was in contrast to labeling in experimental rats, where p - catenin labeling were substantially reduced or irregularly distributed around many cells.The level of (3 - catenin was obviously decreased in the LPS group than that in the control group in optical density average and p - catenin mRNA, P < 0.01. The content of p - catenin mRNA decreased most at 3 h to 24 h. The tendency of changes in the prevention group and the treatment group was the same as that in the LPS group. The level of p - catenin protein and p - catenin mRNA in the antagonist group was higher than that in the LPS group at each time point. The level of p - catenin protein and p - catenin mRNA in the prevention group was slightly lower than that in the treatment group. There was no statistical difference in the p - catenin protein and p - catenin mRNA level between the prevention group and the treatment group.Conclusions1. Using PAF receptor antagonist BN52021 may relieve intestinal gross change, pathological and ultramicrostructural lesion, lessen intestinal edema, diminish the change of weight, decrease the mortality and protect intestinal mu-cosal barrier function, which indicate that PAF play important roles in intestinal lesion of endotoxemia model of young rats.2. The activity of DAO in plasma and ileum tissue changed synchronizely, so determining the activity of DAO in plasma may reflect the destruction of intestinal mucosal function earlier and sensitively.3. The level of protein and mRNA of ZO - 1, E - cadherin and p - cateninin endotoxemia model of young rats obviously reduced. Using PAF receptor antagonist BN52021 may relieve the decreasing extent of ZO -1, E - cadherin and (3 - catenin, which indicate that PAF may play roles in the pathogenesis of intestinal lesion in endotoxemia through destroying intestinal mechanical barrier function. Pretreatly and therapeutically interventing of PAF receptor antagonist BN52021 may protect intestinal barrier function through making the decreasing of junctional proteins of epithelial cells softenly.4. The contents of slgA decreased obviously in endotoxemia. Preventive and remedial use of PAF receptor antagonist BN52021 may soften intestinal injury through making the decreasing of slgA relievely, which indicate that PAF may play critical roles in the development of intestinal immuno - barrier function injury.