| Part1. Dose-effect relationship of intravenous administration of different doses of zerumbone treatment on severe acute pancreatitis.Objective:This study is to investigate the optimal Dose-effect relationship of zerumbone, a natural plant extract, treatment on severe acute pancreatitis in order to provide the optimal dose of zerumbone in protection against severe acute pancreatitis-associated liver injury in rats.Methods:Forty-eight male Wistar rats, were randomly divided into six groups (N=8):Control group (CON group), Severe acute pancreatitis group (SAP group), Zerumbone pretreatment group5mg/kg (ZER5mg/kg), Zerumbone pretreatment group10mg/kg (ZER10mg/kg), Zerumbone pretreatment group20mg/kg (ZER20mg/kg), Zerumbone pretreatment group40mg/kg (ZER40mg/kg). Severe acute pancreatitis model was induced by retrograde injection of5%sodium taurocholate (0.1ml/100g)in biliopancreatic duct. Various doses (5,10,20and40mg/kg) of zerumbone solution were administered via femoral vein half an hour prior to infusion5%sodium taurocholate. All rats were sacrificed at12h time point after the induction of severe acute pancreatitis. The dry cotton ball was used to absorb the ascites of rats;the blood sample underwent centrifuging to get serum; Lung wet/dry weight ratio was calculated from the middle lobe of right lung; the pancreatic tissue was fixed in4%paraformaldehyde. We measured the ascites, serum amylase (AMY), alanine transarninase (ALT), creatinine (Cr), Lung wet/dry weight ratio, and pathological examination of pancreas.Results:The levels of ascites, serum AMY, ALT, Cr, Lung wet/dry weight ratio, and pancreatic pathological score in SAP group were significantly (P<0.05) higher than in CON group. There was no difference between SAP group and ZER5mg/kg group. The levels of ascites, serum AMY, Lung wet/dry weight ratio, and pancreatic pathological score in ZER lOmg/kg, ZER20mg/kg, ZER40mg/kg group was significantly (P<0.05) lower than in SAP group. There was no differenc of serum ALT, Cr between ZER20mg/kg, ZER40mg/kg group and SAP group, but significantly (P<0.05) higher than in ZER10mg/kg group.Conclusion:5mg/kg zerumbone can not effectively alleviate pancreatitis.10,20, 40mg/kg zerumbone can alleviate the severity of severity of pancreatitis:decreasing ascites, serum amylase and pancreatic pathological score. But there is a greater liver and kidney toxicity in20,40mg/kg zerumbone group. Therefore, the10mg/kg zerumbone administered via femoral vein is the optimal, effective dosage. Part2. Effect of zerumbone on severe acute pancreatitis-associated liver injury in ratsObjective:This study is to investigate the effect of zerumbone, a natural plant extract, treatment on liver injury in severe acute pancreatitis rats in order to provide a theoretical basis for liver protection in severe acute pancreatitis.Method:Seventy male Wistar rats, were randomly divided into four groups: Control group (CON group)(N=8), Severe acute pancreatitis group (SAP group)(N=40), Zerumbone pretreatment group (ZER group)(N=10), Zerumbone drug control group (Drug-CON group)(N=10). Severe acute pancreatitis group (SAP group) was divided into four time points of1h,3h,6h and12h (N=10each time point). Severe acute pancreatitis model was induced by retrograde injection of5%sodium taurocholate (0.1ml/100g) in biliopancreatic duct in SAP group and ZER group. Rats were injected isotonic saline solution instead of taurocholate as a control in CON group and Drug-CON group.10mg/kg zerumbone solution was administered via femoral vein half an hour prior to establishing models in ZER group and Drug-CON group. All rats except in SAP group were sacrificed at12h time point after the induction of severe acute pancreatitis. The rats in SAP group were sacrificed at1h,1h,3h,6h and12h. The dry cotton ball was used to absorb the ascites of rats; the blood sample underwent centrifuging to get serum; the head of the pancreatic tissue and the right lobe of hepatic tissue were harvested and fixed in4%paraformaldehyde. We measured the mortality, ascites, serum amylase (AMY), alanine transarninase (ALT), aspartate Aminotransferase (AST), phospholipase, and pathological examination of pancreas and liver.Results:There were no difference of the levels of mortality, ascites, serum AMY, ALT, AST, phospholipase, and pathological examination of pancreas and liver between in Drug-CON group and in CON group. However, those in ZER group were significantly lower than in SAP group, and higher than in CON group (P<0.05). Conclusion:Zerumbone can reduce the mortality and ascites, effectively alleviate the enzyme and pathological injury in pancreatic and liver tissue in severe acute pancreatitis. But there was no evident adverse effect of zerumbone on rats. Part3. Effect mechanism of zerumbone on severe acute pancreatitis associated liver injury in ratsObjective:It was proved that zerumbone could protect the severe acute pancreatitis associated liver injury. The aim of this study is to investigate the possible mechanism.Method:Seventy male Wistar rats, were randomly divided into four groups: Control group (CON group)(N=8), Severe acute pancreatitis group (SAP group)(N=40), Zerumbone pretreatment group (ZER group)(N=10), Zerumbone drug control group (Drug-CON group)(N=10). Severe acute pancreatitis group(SAP group) was divided into four time points of lh,3h,6h and12h (N=10each time point). Severe acute pancreatitis model was induced by retrograde injection of5%sodium taurocholate (0.1ml/100g) in biliopancreatic duct in SAP group and ZER group. Rats were injected isotonic saline solution instead of taurocholate as a control in CON group and Drug-CON group.10mg/kg zerumbone solution was administered via femoral vein half an hour prior to establishing models in ZER group and Drug-CON group. All rats except in SAP group were sacrificed at12h time point after the induction of severe acute pancreatitis. The rats in SAP group were sacrificed at1h,1h,3h,6h and12h. The head of the pancreatic tissue and the right lobe of liver tissue were harvested and fixed in4%paraformaldehyde. The remaining part of pancreatic and the right lobe of liver tissue were stored at-80℃. The expression of nuclear factor-kappa B (NF-κB) P65in pancreatic and liver tissue was evaluated by immunohistochemistry assay. The nuclear NF-κB protein in pancreatic and liver tissue was evaluated by western blot. Inhibitor κB (I-κB) in cytoplasm was evaluated by western blot. Intercellular adhesion molecule (ICAM-1) mRNA and Interleukin (IL-1) mRNA in pancreatic and liver tissue were examined by reverse transcription-reverse transcription (RT-PCR).Results:The Expression of nuclear NF-κB p65protein in pancreas was gradually elevated from at3h, duration to6h, decreased at12h in the SAP group, which was higher than in the CON group. In the ZER group, the NF-κB p65expression declined to a much lower level than in the SAP group (P<0.05). The pancreatic NF-κB immunohistochemical assay highly expressed in nucleus in the SAP group, but lowly expressed in the cytoplasm in the CON group. Hepatic nuclear NF-κB expression increased gradually in the SAP group and the peak expression was the12h time point. In the ZER group, the NF-κB p65expression declined to a much lower level than in the SAP group (P<0.05). The NF-κB immunohistochemical assay in liver tissue highly expressed in nucleus in the SAP group, but lowly expressed in the cytoplasm in the CON group. ICAM-1and IL-1mRNA expression in ZER group were significantly lower than in SAP group (P<0.05).Conclusion:that treatment of zerumbone on rat inhibited NF-κB activation in pancreatic and liver tissue, and then down regulated the expression of ICAM-1and IL-1. It suggested that zerumbone attenuated the severity of severe acute pancreatitis and pancreatitis-induced liver injury... |
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