The Protective Effect Of PS341 In Severe Acute Pancreatitis In Mouse

Severe acute pancreatitis(SAP) is a serious disease with high morbidity in clinic. For the sudden occurring and rapid progressing, it always leads to significant mortality which is up to 20%~30%.Up to now, the complete pathogenesis of severe acute pancreatitis remains poorly understood and the treatment still remains nonspecific and primarily supportive. Finding a cheaper and effective drug to reduce the high mortality of SAP is a serious task for us.Now several theories have been proposed to explain the pathogenesis: the most common cause of pancreatitis is the obstruction of bole duct by gallstone or something else. The bile duct and the pancreatic duct have the common opening which leads to the duodenum. Once the common duct is obstructed, bile will be perfused into the pancreas and activate pancreatic protein enzymes, which leads to autolysis and destruction of pancreas. This is also the most classic hypothesis of pancreatitis. And the theory also considers the invasion of pancreatic enzymes into the blood as the chief cause of SAP. But death is often the result of multiple organ dysfunction. Drugs which inhibit pancreatic enzymes don't work well. What is the problem? Many new hypothesis appears, such as "over-activation of leukocyte", "ischemia of pancreas and obstacle of microcirculation", "translocation of bacteria and followed infection". In 1988, Rindernecht suggested that over-activation of leukocyte was the chief cause of SAP, autolysis of pancreas caused by pancreatic enzymes maybe acted in part. Many researches have indicated that the inflammatory response is initiated by injured pancreatic acinar cells which produces inflammatorymediators, such as cytokines (e.g.,TNF-a) and adhesion molecules (e.g.,ICAM-l), ultimately leading to systemic complications.Blocking the activation of these mediators by using various approaches ameliated pancreatitis in different experimental models.Considering the central importance of the inflammatory response in pancreatitis, theraperutic strategies should be aimed at the key steps leading to this response. Because of the multitude of cytokines, chemokines, and other inflammatory molecules involved in this disease process, one can argue that the most effective strategy should target upstream "master regulation" of these molecules. Recent data suggests that one such key regulator is NF-kB. NF-kB comprises a family of transcription factors regulating the inflammatory, immune and cell death response. In unstimulated cells, NF-kB is kept in inactive in the cytoplasm by association with the inhibitory (IkB) proteins. When the inhibitory (IkB) is degradated by proteasome, NF-kB is activated, then it translocates into the nucleus and activates the expression of a multitude of genes that have KB-binding sites in their promoters or enhancers. NF-kB activation,at least in part, regulates the expression of cytokines(TNF-a,IL-6), chemokines(IL-8,KC), and other inflammatory molecules such as ICAM-1 and the inducible nitric oxide synthase, all of which are upregulated and play an important role in pancreatitis. Protein degration by proteasome mediated is one of the pathes of protein degration inside of eukaryocyte. As state above, its degration contributes the activation of NF-kB, which plays an important role in pancreatitis activation. Its specific inhibitor named PS341, can make the NF-kB unactivate by inhibiting proteasome, then block the generation of inflammatory factor and pancreatitis. It had been confirmed by animal models that inhibitor of proteasome can repress the proceed of pancreatitis.In this study, we made severe acute pancreatitis in mouse by intraperitoneal injection of caerulein and lipopolysacchoride, and treated mouse with PS341. 8hafter operation, we harvested serum for amylasex CRP and LDH test. The pancreas was also harvested for pathological examination. The differences of amylase^ CRPn LDH and pathological change between groups were estimated to find whether PS341 is effective for in vivo application in caerulein and lipopolysacchoride-induced pancreatitis.Materials and methodsAnimals: twenty-four ICR female mouse were used. The animals, weigh 20.0±1.5g, were provided by the Animal Center of college of medical science, Zhejiang university, China.Groups: The mouse were randomly divided into normal saline group(n=8), DMSO treated group(n=8) and PS341 treated group(n=8). Animal model:normal saline group: The animals were injected intraperitoneally with normal saline (10 ml/kg) seven times at a 1 h interval. But twice normal saline were injected at the six times. The intraperitoneal infusion of DMSO(10ml/kg) was treated 4.5h after the first injection of caerulein.DMSO treated group: DMSO treated group: The animals were injected intraperitoneally with caerulein(5mg/l, lOml/kg) seven times at a lh interval. The intraperitoneal infusion of LPS(0. 1%, lOml/kg) followed the six times injection. The intraperitoneal infusion of DMSO(10ml/kg) was treated 4.5h after the first injection of caerulein.PS341 treated group: The animals were injected intraperitoneally with caerulein(5mg/l, lOml/kg) seven times at a 1 h interval. The intraperitoneal infusion of LPS (0.1%, lOml/kg) followed the six times injection. The intraperitoneal infusion of PS341(0. 05g/l, lOml/kg) was treated 4.5h after the first injection of caerulein. Preparation of samples: 8h after operation, all animals were sacrificed by gatheringarteria carotis blood under light anesthesia. The serum was collected and stored in-20 "C refrigeratory. The tissue samples of pancreas was taken and fixed with 10%formalin solution.Biochemical measurements: The Amylase of serum, the CRP level and the LDHlevel were all tested by standard clinical methods for automated analysis.HE staining: Sections of the specimens were stained with HE, and then observedunder a light microscope.ResultLevels of amylase > CRP and LDH:Amylase in serum increased significantly in DMSO treated group as compared with normal saline group(P<0.01);Amylase in serum increased significantly in PS341 treated group as compared with normal saline group(P<0.01);Amylase^ CRP level and LDH level in serum decreased in PS341 treated group as compared with DMSO treated group(P<0.05, P<0.05, P<0.05);CRP level and LDH level in serum decreased in PS341 treated group as compared with normal saline group ( P<0.05, P<0.05). Findings under light microscope:normal saline group: a mild degree of interstitial edema, pancreatitic acinus have no hemorrhage and inflammatory;DMSO treated group: mucosal edema obviously, hemorrhage and inflammatory cell infiltration were revealed in mucosa or submucosa;PS341 treated group: mucosal edema, hemorrhage and inflammatory cell infiltration all decreased compared with the DMSO treated group.Conclusions1. PS341 inhibited the increases in serum amylase, inhibited the increases in serum CRP and serum LDH;2. PS341 inhibited the increases in tissue bleed and infiltrating inflammatory cells, and resulted in less cellular damage;3. PS341 succeed to ameliorate SAP in this mouse model.