| Objective: Severe acute pancreatitis ( SAP ) is characterized by hemodynamic alterations and a systemic inflammatory response, leading to a high mortality rate. The pathogenesy of SAP has not been interpreted yet and there is not effective therapeutic measures clinically. The release of cytokine contributes to systemic response such as systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), adult respiratory distress syndrome (ARDS), and sepsis. Moreover, cytokines have been shown to play a pivotal role in multiple organ dysfunction, a major cause of death in SAP. The two-hit hypothesis of SIRS induced by cytokine explains the variable individual response to SAP. Ginkgolide B (BN52021) is a very important and effective ingredient of extract from Ginkgo Biloba Leaf. It played an critical role in the treatment of SAP and in the pharmacologic action of anti-inflammatory, anti-thrombus or antishock and so on, and was a powerful antagon of Platelet activating factor (PAF), however, the mechanism of which is not clear. In vivo, PAF transfers singles and regulate the activity of cells mainly by binding to platelet activating factor receptor (PAF-R). PAF-R a member of G-protein-coupled recepters family chracterized by seven membrane spanning regions. After activated, PAF-R couples with special heterotrimeric G-proteins to activate corresponding signal transduction pathway.The purpose of the research is to investegte the effect of BN52021 on rats' severe acute pancreatitis , by establishing rat severe pancreatitis model, treating the model with BN52021, detecting the area percentage of collagen fibers and the activity of blood serum amylase, studying the optimal dosage of BN52021, and evaluating the effect of BN52021 on phospholipase C-β2 (PLC-β2) in pancreatic tissue.Methods: Firstly, 180 Wistar rats (180g-220g) were randomly divided into three groups (NC, n=60; SAP, n=60; BN, n=60), and every group was randomly divided into 6 subgroups (1h, 2h, 3h, 6h, 12h, 24h), 10 rats in each. The sodium aurocholatet was injected through pancreaticobiliary duct in retrograde direction to establish SAP model. The animals were sacrificed respectively at 1h, 2h, 3h, 6h, 12h and 24h after the injection of BN52021 or physiological saline. Blood and pancreatic tissue were collected immediately. Serum amylase was detected with Hitachi Clinical Analyzer 7180. A part of pancreas tissue were deposited in refrigeratory at -80℃after being stayed Liquid nitrogen over night;the rest were fixed by neutral formaldehyde buffer (40g/L) and then were made into olefin slice, stained by HE to observe pathology change of pancreas tissue.Then, 70 Wistar rats were randomly divided into seven groups (sham operation group, SO, n=10; SAP model group, SAP, n=10; 0.5% DMSO contrast group, DMSO, n=10; 2.5 mg/kg BN52021 treating group, BN1, n=10; 5.0 mg/kg BN52021 treating group, BN2, n=10; 10.0 mg·kg-1 BN52021 treating group, BN3, n=10; 20.0μg/kg Sandostatin treating group, SS, n=10). The area percentage of collagen fibers was detected, the activity of blood serum amylase was observed and the effect of BN52021 on PAF-R mRNA in pancreatic tussue was examined with RT-PCR.Finally, The activity of PLC-β2 in pancreatic tissue stored in refrigeratory at -80℃was examined with western blot, the data of which was analyzed with statistic software.Results: Firstly, compared with NC group, there is a significant increase of serum amylase in SAP group compared with NC group (P<0.05) and in BN group (P <0.05). However, compared with SAP group, there was a significant decrease in BN group at 1h, 2h, 3h, 6h and 24h (P<0.05). Pathologic result showed that the area of edema, hemorrhage and necrosis enlarged gradually in SAP group. However, the area was smaller and the degree of destruction was lighter in BN group compared with SAP group.Then, the results of the serum amylase, phospholipase A2 (PLA2), ascites, pathologic score and PAF-R mRNA expression showed that, compared with 2.5 mg/kg and 10.0 mg/kg, 5.0 mg/kg of BN52021 by vein injection was the optimal dosage in treating rats with SAP (P<0.05). Finally, from 1 hour to 3 hour, PLC-β2 in all of the three groups reached to the lowest level. From 6 hour to 24 hour, PLC-β2 in NC group went up to the highest level, and cut down subsequently. However, PLC-β2 in SAP group increased gradually. After treated with BN52021, compared with SAP group, PLC-β2 in BN group decreased, and there was a significantly difference between two groups in 12 hour (P<0.01) and 24 hour (P<0.01).Conclusion: Firstly, SAP animal model was established successfully by retrograde infusion of 5% sodium taurocholate into the pancreatic duct in wistar rats and BN52021 had therapeutic effect on SAP.Then, 5mg/kg of BN52021 by vein injection may be the optimal dosage in treating Wistar rats with SAP。Finally, treatment with BN52021 could partly inhibit the activity of PLC-β2. BN52021 had effective treatment on SAP, the mechanism of which might correlate with the decreased level of PLC-β2 induced by BN52021. |
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