| BakegroundAcute pancreatitis, characterized by interstitial edema, vacuolezation, inflammation, and acinar cell necrosis, is commonly caused by excessive ethanol consumption, biliary tract disease, certain medications and invasive procedures of the biliary and pancreatic ducts. The pathophysiology of acute pancreatitis is poorly understood and the clinical course is unpredictable. For example, patients who appear to have minimal disease at presentation may develop systemic sequelae such as adult respiratory distress syndrome (ARDS) and multiple organ failure and die. Various cytokines have been implicated in the local inflammatory changes of acute pancreatitis as well as the systemic effects. Activation of polymorphonuclear granulocytes (PMN) monocytes / macrophages, associated with release of the pro-inflammatory cytokines, IL-1, IL-6, and TNF- α in the pancreatic parenchyma, is an early events during acute pancreatitis and has been shown to contribute to the severity of the disease process.Conversely, the antiinflammatory cytokines, IL-4,-10 and TGF- β ,have been shown to decrease the severity of the AP in experimental models. However, recent studies utilizing novel geneknock-out models suggest that agents play only a contri- butory role in this disease process. Cyclooxygenase (COX) catalyzes the rat limiting step in PGs production; COX-2 is normally undectable in most tissues and its activity is inhibited by the selective inhibitors such as NS-398 and (SC-58125) celecoxib. The expression of the COX-2 has been shown to play an important role in many processes, such as inflammatory bowel disease, rheumatoid arthritis, asthma and the fact that COX-2 plays an important role in the inflammatory response of a number of tissues and is regulated by factors that alter the course of pancreatitis suggested a potential role for this isoform in the induction or progression of AP. Therefore, the purpose of this portion of this study was to determine whether COX-2 expression was altered following induction of AP and whether the manipulation of its activity using COX-2 selective inhibitors results in the attenuation of the severity of the microcirculation disturbance of the AP. ObjectiveTo investigate the expression and distribution of the COX-2 the pancreas of rat and explore the effects of COX-2 on microcirculatory permeability and by using its selective inhibitors (NS-398) resulting in the attenuation of the severity of the microcirculation disturbance of the AIP. MethodsThe acute interstitial pancreatitis was induced by CAE, and 120 of randomized male /female Wistar rats weighing 250g~300g were divided into 3 groups:(DNormal control (N.C., groupl,n=24); ?Acute interstitial pancreatitis (AIP, and the 2nd,4th, 6th,8th,16th,and 24th is the AIP2-hour, 4-hour,6-hour, 8-hour, 16-hour and 24-hour, respectively, group2, n=72); (3)NS-398 in vitro experimental group (group3,n=24).The experimental group received caerulin by subcutaneous injection 5.5 u g.Kg"1 the first hour and 7.5 u g.Kg'1 the hour next, While, the normal control group received same amount injection but sterile normal saline (0.9% sodium chloride) instead. Spectrophotometry, RT-PCR and western blot were used to determine the level of mRNA and protein of the COX-2, serum amylase (AMS) and plasma myeloperoxidase (MPO). Evans blue was used to observe pancreatic microcirculat- orypermeability index (PI),i.e.,Total RNA extraction from pancreatic tissue by Trizol? Reagent Kit; Semi-Quantitative RT-PCR were performed in combined one tube reaction kit; The primer for COX-2 and P -actin were as following: P -actin (sense)5'-GATGGTGGGTATGGGTCAG AA-3',(anti-sense)5'-CTAGGAGCCAGGGCAGTAATC-3';COX-2(sense)5t -CTGTATCCCGCCCTGCTGGT-3',COX-2(antisense)5'-GAGGCACTTGC GTTGATGGT-3'. The cytoprotein was isolated by Lowry method, and COX-2 protein was detected by specific anti-rat COX-2 antibody with western blotting technique and the results were analyzed by ultraviolet trans-illuminator. The activity of MPO, a marker of inflammation and an index of tissue leukocyte recruitment, was determined following the method described by Calkins. The permeability index of the pancreas and lungs was determined by intravenous injection of Evans blue via a tail vein following the method described by Marc S. et al. Results(D RT-PCR and Western blot detection of COX-2 in the pancreas: COX-2 transcription remains at low level in normal control. Whereas, the expression of COX-2 in AIP group was increased, with peaked at 8th hr, andkept at high level within 24th hr. Setting the level of COX-2 expression at2nd hr as baseline, COX-2 expression of 4th hr and 8th hr in AIP weresignificant high (PO.05.PO.01 respectively).(3) Immunohistochemistry (IHC) revealing COX-2 distribution in thepancreas: COX-2 mainly in the pancreatic acini and vascular endothelium inthe process of AIP, while the COX-2 protein signal was undetectable in NC.No significant changes were found in different sites during the AIP, but theexpression level of COX-2 at various time group bore statistic significance,with the peak at 8th hr and 16th hr and then dropped (PO.05).(3) The AMS and MPO for AP were omitted.?The microvascular permeability in the pancreas and lungs: PI fromcontrol was shown little change before and after injection of 0.9% sodiumchloride, while both pancreas and lungs in AIP were shown a significantincrease in microvascular permeability from 4th hr to 16th hr and the increasein the lungs was higher than that in the pancreas (Lungs, 8th hr and 16th hr,PO.001; pancreas, 8th hr and 16th hr, PO.01).?The status of the PGE2 response on exposure to NS398 was furtherconfirmed the COX-2 levels in isolated pancreatic acinar cells afterinhibition with varying concentrations of NS-398.ConclusionsIncreased COX-2 expression is one of the predictor for capillarypermeability, edema formation and the severity of AIP. Based onpharmacologic inhibition of COX-2 can improves the disturbance in AIP,future studies on the clinical application of COX-2 inhibiting substances inAP seems to be promising. |
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