The Effect Of Chondroitin-Sulfate On Limiting The Damage Of Pancreatic Cells During Experimental Acute Necrotizing Pancreatitis

ObjectiveSeveral authors provided evidence that during the initiation and manifestation of experimental AP, large amounts of ROS are produced. Depletion of pancreatic antioxidants may be attributable in part to inflammation and/or to the activation of proenzymes because it is known that activated proteases such as carboxypeptidase or metalloproteinases can cleave GSH, SOD and so on. The imbalance in the regulation of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may determine membrane lipid peroxidation and cellular destruction in pancreatic tissue.There are tow prominent cellular features of early acinar cell events during AP: rapid disruption of the actin cytoskeleton with reduction or loss of cell polarity, enlarged acinar lumina, and loss of the actin network surrounding the lumina; the interstitial space between the polarized epithelial (acinar) cells of the pancreas undergoes an important structural changes. Exreavasation of protein-rich fluid from increasingly permeable pancreatic blood vessels enter and distend the interstitial space. This phenomenon is referred to as pancreatic edema. The E-cadherin/catenin/actin complex that regulates cell-cell contacts in a number of epithelial cell systems is also operative in the exocrine pancreas. The biological function that can be inferred from related tissues would be the formation of adherens junctions, organization of the actin cytoskeleton, and the polarized distribution of organelles and cellular proteins.HSP27 which modulates actin dynamics following its phosphorylation is induced by stress. And overexpression of HSP27 is known to confer resistance to heat and other stresses. A previous study suggested that regulation of actin and preservation of glutathione together accounted for the protective effects of HSP27. Not only was HSP27 protein expression in the pancreas increased but also its phosphorylation which has been shown to protect the cell architecture against supramaximal concentrations of CCK in Chinese hamster ovary cells stably transfected with the CCK-A receptor.Glycosaminoglycans (GAGs), a large family of heterogeneous polysaccharides, are linear sulfate-substituted polymers composed of alternating hexuronic acid and hexosamine units that play important roles in all living organisms. GAGs are present in blood, and the main GAG of nomal human and animals plasma is Chondroitin-Sulfate (CS), which is markedly increased in several diseases including AP in human and experimental rat models, possess antioxidant activity capable of inhibiting lipid peroxidation. In fact, recently, several papers have reported antioxidant activity exerted by GAGs, mainly for CS, in a variety of in vitro and in vivo experimental models. The use in therapy is still understudy.To help understand the relevance of cytoskeletal changes, HSP27 and CS in acute necrotizing pancreatitis (ANP), we studied the effection of CS on sodium-taurocholate induced experimental ANP in vivo and in vitro. Our work includes three parts listed below.Paper oneMethodsMale Wistar rats were divided randomly into three groups: group A,group B and group C. Group A was injected 5% sodium taurocholate. Group B received chondroitin-sulfate therapy, and both groups served as experimental groups, while group C as control group. Rats in three groups were killed at30',1h,3h,6h,12h.The levels of malonyl dialdehyde (MDA),total superoxide dismutase (SOD),glutathione (GSH),serum amylase (SAM) and ATP were measured . Histopathology was observed .ResultsLevels of SAM increased in group A and group B, with related pathological changes of ANP .Levels of GSH,SOD in group A decreased markely during pancreatitis (P<0.05) ,however MDA increased significantly (P<0.05) .The levels of GSH,SOD,ATP in group B were higher than those in group A (P<0.05) .The level of MDA was lower than that in group A (P<0.05) . And the histopathologic damage was attenuated to a certain extent.ConclusionRetrograde infusion of sodium taurocholate via the pancreatic duct produced pancreatic necrosis and a marked increase in serum amylase activity; induced a severs depletion of reduced glutathione (GSH), superoxide dismutase (SOD) levels; primed lipid peroxidation. Intraperitoneal pretreatment of rats with chondroitin-sulfate ameliorated pancreatic cell conditions; restored the endogenous antioxidants (GSH, SOD, ATP) reduced; limited cell membrane peroxidation. Our data confirm the antioxidant activity of this glycosaminoglycan.Paper TowMethodsMale Wistar rats were divided randomly into three groups: group A,group B,and group C. Group A was injected 5% sodium taurocholate. Group B received chondroitin-sulfate therapy,and both groups served as experimental group, while group C as control group. Rats in three groups were killed at 30', 1h, 3h, 6h, 12h respectively. The levels of pancreatic indexes (pancreatic wet/body weight) were measured. Changes of adhesion junctions were examined by using a electron microscope. E-cadherin, one of adhesion junction protein, was localized by immunocytochemistry for confocal fluorescence microscopy. Its expression was studied by western-blot and RT-PCR . Immunostained F-actin with rhodamine-phalloidin was analyzed using confocal laser scanning system.. HSP27 and its phosphorylation were measured by Western-blotting using anti-HSP27 antibody and anti-phospho-HSP27 antibody respectively. The expression of HSP27 mRNA was determined by RT-PCR.ResultsIn group A, there was a rapidly and sustainly increasing in levels of pancreatic indexes, and a serious interetitial edema during ANP (P<0.05). Damages of adhesion junctions were observed. E-cadherin was localized at the basolateral cell membrane from where it rapidly dissociated early in pancreatitis and localized into cytoplasm. During an increase of E-cadherin mRNA, E-cadherin protein markly declined (P<0.05). In group B, there were moderate pancreatic edema and disruption of adhesion junctions. Although dissociated also, yet the level of E-cadherin protein was marked higher than that in group A (P<0.05). However, the levels of E-cadherin mRNA were not significant between group A and group B (P<0.05).ANP resulted in early disruption of the cytoskeleton with dramatic changes with a loss of F-actin. Administration of CS moderated the damage of actin-cytoskeleton. Moreover, administration of CS induced a much larger HSP27 response and a higher level of HSP27 phosphorylation and mRNA expression.ConclusionIn this study, we observed a serious damage of adhesion junction, which was responsible for the formation of pancreatic edema during.taurocholate-induced ANP in rate. Treatment of CS, a member of GAGs family, can protect adhesion junction by increasing E-cadherin protein concentration and attenaut pancreatic edema.We demonstrated that the cytoskeleton alterations are a common feature of AP, and administration of CS increases HSP27, its mRNA and its phosphorylation levels. All of these can protect actin cytoskeleton during ANP.Paper ThreeMethodsAfter intraductular injection with HBSS-DVC containing 2mg/mL collagenase I ,the rat pancreas was subjected to digestion in HBSS-DVC containing 2mg/mL collagenase I and filtered and centrifugalized until most of the pancreatic acini were completely isolated from the pancreas.Immunostained F-actin with rhodamine-phalloidin was analyzed using confocal laser scanning system. Content of F-actin protein was determined by Flow cytometry. HSP27 and its phosphorylation were measured by Western-blotting using anti-HSP27 antibody and anti-phospho-HSP27 (Ser15, 86) antibody respectively. The expression of HSP27 mRNA was determined by RT-PCR.ResultAbout 4×10~6-5×10~6 pancreatic acini cells were obtained in 1 rat pancreas, with an average purity over 70%.ANP resulted in early disruption of the cytoskeleton with dramatic changes with a loss of F-actin. Administration of CS moderated the damage of actin-cytoskeleton. Moreover, administration of CS induced a much larger HSP27 response and a higher level of HSP27 phosphorylation and mRNA expression.ConclusionIsolation of the rat pancreatic acini cells with this method is relatively simple and with less collagenase consumption.We demonstrated that the cytoskeleton alterations are a common feature of AP, and administration of CS increases HSP27, its mRNA and its phosphorylation levels. All of these can protect actin cytoskeleton during ANP.