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Acute pancreatitis (acute pancreatitis, AP) clinical critical illness, most patients with mild pancreatitis, and is self-limiting, usually in the incidence future improvement; However, there are still about 20% -30% of patients showed for severe acute pancreatitis. Severe acute pancreatitis (severe acute pancreatitis, SAP) to systemic inflammatory response syndrome, multiple organ failure and local complications characterized clinical treatments continue to improve, but mortality is still as high as 15-30%. The early stages of multiple organ failure, and post-infection complications lead to the main reason for the high mortality of severe acute pancreatitis. Multiple organ failure is the result of a systemic inflammatory response, is worth noting that the excessive activation of macrophages, monocytes, neutrophils release of inflammatory cytokines may lead to injury of distant organs. Played an important role in the pathogenesis of severe acute pancreatitis, pancreatic local, as well as the systemic inflammatory response. In-depth understanding and study of severe acute pancreatitis inflammation reaction mechanisms, and explore methods for its treatment has been a hot research. SAP's an important question is not yet a specific serological or other indicators to diagnosis and prognosis, and then guide treatment. Clinical judged by serum amylase and C-reactive protein and other SAP prognosis, but the application of very limited value. Advanced glycation end products receptors (the receptor for advanced glycation end products, RAGE), a member of the multi-ligand receptor belonging to the immunoglobulin superfamily of cell surface molecules that are widely distributed in the monocytes - macrophages, endothelial cells, cell surface of smooth muscle cells, mesangial cells, tumor cells, astrocytes, T lymphocytes; advanced glycation end products receptor is important receptor-mediated HMGB1 cytokine activity. In addition and HMGB 1, its ligands also includes the advanced glycation end products, p amyloid peptide, S100 protein, such as. Interaction of RAGE with its ligand often leads to rapid and continuous cell activation and downstream transcription, a variety of cell signal ERK1 / 2 (p44/p42), p38 and of SAPK / JNK MAP kinase, Rho-GTP enzymes, phosphatidylinositol -3 - kinase and the JAK / STAT signaling pathway is activated in different cell types, and lasting through the nuclear factor kappaB (Nuclear Factor-kappaB, NF-kB) activation to maintain and expand the inflammatory response. It has been reported that, RAGE knock out mice can tolerate septic shock induced by cecal ligation and puncture that RAGE play an important role in the systemic acute inflammatory process. RAGE is a single transmembrane segment receptor, is divided into the three parts of the extracellular domain, a hydrophobic transmembrane region and intracellular domain; its extracellular portion contains one \The binding site is located in the \Recently, several RAGE variants found in humans and mice lungs recognized soluble RAGE (soluble fraction of of RAGE, sRAGE) and endogenous secretion of RAGE (endogenous for secretory of RAGE, esRAGE). Although they produce the mechanisms and molecular size is not the same, but because the ligand binding of extracellular region includes the lack of the transmembrane region, and therefore can compete with RAGE with RAGE ligand binding, and block the RAGE signal to the cells transfected guide. Soluble RAGE (sRAGE) present in the loop, is constituted by the ectodomain of RAGE; sRAGE including esRAGE and RAGE proteolytic cleavage, forms. SRAGE level rise in end-stage renal disease and acute lung injury. SRAGE levels decreased in rheumatoid arthritis, Alzheimer's disease, Alzheimer's disease and essential hypertension. The research suggests that severe acute pancreatitis combined organ failure, sRAGE levels were significantly different. The high mobility group protein B1 (high mobility group box-1, HMGB1) is one of the the RAGE major ligand is a DNA-binding protein, also known as gender protein, because of its rapid migration in the polyacrylamide gel electrophoresis is named, is found so far only in the extracellular nuclear protein induced cytokines and activation of inflammatory cells. HMGB1 structure having three domains, A, B, C zone, A and B are the major form of HMGB1 non-specific DNA binding domain; the C region may be interaction with other proteins to regulate the affinity of HMGB1 with DNA. HMGB1 is very widely distributed, were expressed in the cells of various organs and tissues (such as lymphoid tissue, brain, liver, lung, heart, spleen, kidney, etc.), it is released into the extracellular through two different pathways: activation of inflammatory cells active secretion and passive release of necrotic cells. Recent studies suggest that extracellular HMGB1 as a late inflammatory cytokines involved in the pathophysiology of sepsis and severe acute pancreatitis (severe acute pancreatits, SAP) from systemic inflammatory response syndrome (, systemic inflammatory response syndrome, SIRS) play a very important role to multiple organ dysfunction syndrome (multiple organ dysfunction syndrome, MODS), multiple organ failure (multiple organ failure, MOF) process. And other proinflammatory factors such as tumor necrosis factor-a (tumor necrosis factor-alpha, of TNF-a) and interleukin prime -1 (interleukin-1, of IL-1) of HMGB1 has appeared late but the duration of the long, thereby expanding the clinical treatment of the time window. Of severe acute pancreatitis with RAGE and its ligand HMGB1 research at home and abroad less clear RAGE role in the pathogenesis of severe acute pancreatitis, we intend to establish in rats with severe acute pancreatitis model, observed severe acute pancreatitis made RAGE and its ligands in pancreatic tissue of rats die after the success of HMGB1 changes in transcription and protein levels detected by ELISA serum sRAGE and HMGB1 levels of inflammatory mediators. This study not only provide new ideas for the clinical treatment of severe acute pancreatitis, but also provide reference for other inflammatory mechanisms involved in the acute injury disease. Rats with severe acute pancreatitis model purpose: preparing SAP animal models to understand the rat SAP course of law, to provide a basis for the next experimental study. Methods: 64 male Sprague-Dawley rats were randomly divided into eight groups, 20% L-arginine solution configured with saline, using intraperitoneal injection method, in accordance with the 250mg/100g dose interval of one hour repeated injections, preparation of SAP model, the control group, SD rats by intraperitoneal injection of normal saline. 6h after modeling, 18h, 24h, 36h, 48h, 72h, 96h sacrificed rat heart blood, at room temperature for centrifugal separation of serum, placed in separate bottles at -80 ℃ cryopreservation. Specimens from the pancreas, liver, lung, intestinal tissue. Serum amylase (serum amylase, AMY), staining of pancreatic tissue, lung pathology changes and rating. Results: 6h modeling, serum amylase levels began to rise, which SAP18h serum amylase levels were significantly higher than the other groups. As the disease progresses, the rat model of SAP at each time point the amount of ascites gradually increased, to achieve the highest level of SAP 96h ascites volume. Light microscope, a clear structure of the pancreatic tissue of the control group, the structural integrity of acini, interlobular septa clear, mild edema without inflammatory cell infiltration and hemorrhage, necrosis. Rats in 6h from pancreatic pathology score was significantly elevated SAP 6h group pancreatic tissue interstitial edema, light, moderate infiltration of inflammatory cells, mainly neutrophils acinar structures still good organizational structure without obviously damaged. SAP18h pancreas mesenchymal edema, a large number of inflammatory cell infiltration, mild substance of necrosis and hemorrhage. SAP 24h pancreatic acinar cell swelling, visible focal necrosis, acinar cell infiltration of inflammatory cells, red blood cells within the pancreatic parenchyma oozing more pancreatic necrosis, hemorrhage than 18h obvious. SAP 36h acinar structures destroy more lobular disorganized, during which flooded inflammatory exudate, necrosis of acinar structures in the region disappeared. SAP 48h acinar structures severely damaged, extensive damage to pancreatic tissue, marked hyperemia peripheral adipose tissue, and inflammatory cell infiltration. SAP 72h group necrosis aggravated acinar structures disappear, necrotic areas HE staining pink structure area, nucleus dissolved, the the leaf gap widened interstitial microvascular rupture, a large number of red blood cells overflow, flaky fat necrosis formation, visible of saponification spot. SAP 96h group pancreatic tissue necrosis achieve the highest degree of Light microscope, complete control rats alveolar wall, interstitial lung inflammatory cell infiltration, a clear structure of the lung tissue. SAP24h group of lung interstitial widened alveolar cavity pink exudate, accompanied by alveolar atrophy alveolar and interstitial massive neutrophil infiltration. SAP 36h group alveolar interstitium significantly widened alveolar spaces significant congestion, edema, infiltration of inflammatory cells. SAP 48h, SAP 72h, SAP 96h group alveolar structural disorder, alveolar wall rupture, alveolar hemorrhage significantly, significantly widened alveolar septa, and most of the alveolar and interstitial neutrophil infiltration, serious structural damage. Conclusion: intraperitoneal injection of 20% L-arginine solution preparation of rats with severe acute pancreatitis model, with a simple, fast, small animals traumatic, repeatability and stable model, a high success rate, etc., is a more The ideal model, to lay the foundation for further development of the SAP pathogenic mechanisms related research. Second, RAGE expression in rats with severe acute pancreatitis Objective: To study the rat SAP pathogenesis of pancreatic tissue RAGE and sRAGE serum content changes in the law. Methods: In accordance with the various time points the rats were sacrificed, and specimens from pancreatic tissue under sterile conditions, total mRNA was extracted, reverse transcriptase polymerase chain reaction (RT-PCR) amplification of rat RAGE, amplification product with the expected purposes Gene consistent. RAGE mRNA expression of pancreatic tissue using quantitative real-time PCR (real-time PCR) method to detect; immunohistochemistry (Immunohistochemisty, IHC) detection of RAGE protein expression in different periods of SAP rats pancreatic tissue; using ELISA (Enzyme -linked immunosorbent adsorption method, ELISA) serum in sRAGE level. Results: real-time quantitative PCR method to detect RAGE expression at the mRNA level, statistics showed that SAP 6h group SAP 18h group, SAP 24h group SAP36h group, SAP 48h group, SAP 72h group and SAP 96h group RAGE in pancreatic tissue The expression amount significantly higher than the control group (P lt; 0.05), SAP 24h group and SAP 36h group RAGE in pancreatic tissue relative expression level was significantly higher than the other when the phase group (P lt; 0.05), after there was a reduction. IHC SAP 48h pancreatic tissue inflammation cell membrane and cytoplasm in the expression of RAGE protein levels, no expression in the control group. This experiment by RAGE ELISA kit serum sRAGE content, results showed that the sRAGE lower concentrations in the serum of the control group, SAP the 6h group of sRAGE concentration began to increase, compared with the control group, a significant difference (P lt; 0.05); SAP18h group sRAGE concentration was significantly higher, SAP 24h group sRAGE concentration peak, compared with the other groups, a significant difference (P lt; 0.05); 24 h after serum sRAGE concentration began to decline, but SAP 36h group and SAP 48h group sRAGE concentration remained at a relatively high level, SAP 96h group sRAGE concentration decreased to near baseline levels, compared with the control group, differences remained statistically significant (P lt; 0.05). Conclusion: RAGE in severe acute pancreatitis during the development of an important role in tissue and serum was time-dependent expression of RAGE, RAGE expression in the serum may partly reflect SAP course. , HMGB1 expression in rats with severe acute pancreatitis purpose: study rats SAP pathogenesis of pancreatic tissue and serum RAGE ligand HMGB1 protein variation: follow the rats were sacrificed at each time point, under sterile conditions to stay pancreatic tissues, extraction of total mRNA, and reverse transcriptase polymerase chain reaction (RT-PCR) amplification of rat HMGB1, gene amplification product with the intended purpose consistent. HMGB1 mRNA expression using real-time PCR method to detect pancreatic tissue; IHC detection HMGB1 protein expression in rat pancreatic tissue of SAP in different time slots group; using ELISA serum HMGB1 levels. Results: Real-time quantitative PCR method to detect HMGB1 mRNA levels, the statistics showed the SAP 6h group, SAP18h group, SAP 24h group, SAP 36h group, SAP 48h group, SAP 72h groups and SAP 96h group HMGB1 pancreatic tissue expression was significantly higher than that in the control group (P lt; 0.05), SAP 36h group relative expression levels of HMGB1 in the pancreatic tissue was significantly higher than the other groups (P lt; 0.05). IHC HMGB1 protein SAP 36h group rat pancreatic acinar cells and inflammatory cell cytoplasm, the nucleus and pancreatic interstitial protein levels, no expression in the control group. This experiment by HMGB1 ELISA kit serum HMGB1 levels, results showed that the control group, serum HMGB1 concentration is low, SAP the 6h group of HMGB1 concentration began to increase, compared with the control group, a significant difference (P lt; 0.05); SAP18h group SAP 24h HMGB1 concentration was significantly higher HMGB1 concentration SAP36h group reached the peak, compared with the other groups, a significant difference (P lt; 0.05); 36h after serum HMGB1 concentration began to decline, but SAP 48h group and SAP 72h Group HMGB1 concentrations remained at a relatively high level SAP 96h group the HMGB1 concentration decreased to near baseline levels, compared with the control group, differences remain statistically significant (P lt; 0.05). Conclusion: HMGB1 may be as late inflammatory cytokines involved in the systemic inflammatory response of severe acute pancreatitis, serum phase later its receptor RAGE. Through the above research, the subject of the following conclusions: 1 by intraperitoneal injection of L-arginine successfully established rat model of severe acute pancreatitis. 2, RAGE severe acute pancreatitis during the development of an important role in RAGE expression in tissue and serum showed a time-dependent manner, the expression of RAGE in serum may partly reflect SAP course. 3, HMGB1 may be as late inflammatory cytokines involved in the systemic inflammatory response of severe acute pancreatitis, and the serum phase late its receptor RAGE, both expected as diagnostic markers of SAP.
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