A Study On The Protective Effects And Possible Mechanism Of Lopoxin A₄ Analogue LXA₄-ME On Acute Mecrotishing Pancreatitis In Rats

Objective: To establish the model of acute necrotising pancreatitis in rats and evaluate its manifestation and complications.Methods: Eighty male SD rats were randomly divided into sham operation group (n=40)and acute necrotizing pancreatitis group (ANP group,n=40). The rats were anesthetized with 3.6% chloral hydrate (10mL/kg) by intraperitoneal injection. ANP model was established by retrograde pancreatic duct injection of 5% sodium taurocholate (1mL/kg) , and evaluated by the general condition ,the amount of ascites and seratologic analysis of amylase (AMY), total bilirubin (TBIL), alanine aminotransferase (ALT), creatinine (SCR), blood urea nitrogen (BUN) at 12h and 24h respectively (20 in each time point). The pathologic changes of pancreas, liver and lung were also observed at the same time point.Results:1. The amount of ascites in ANP group at 12h,24h time points was much more than that in the sham operation group at the same time point(P<0.01).2. The serum levels of AMY,TBIL,ALT,SCRand BUN were as follows①The level of AMY in ANP rats was higher than that in the sham operation group and there was significant difference at 12h,24h(P<0.01).②The level of TBIL in ANP rats was significantly higher than that in the sham operation group and there was significant difference at12h,24h(P<0.01). ③The level of ALT in ANP rats was significantly higher than that in the sham operation group and there was significant difference at 12h,24h(P<0.01).④The level of SCR in ANP rats was significantly higher than that in the sham operation group and there was significant difference at 12h,24h(P<0.01).⑤The level of BUN in ANP rats was higher than that in the sham operation group and there was significant difference at12h,24h(P<0.01).3. The pathological changes of pancreas, liver and lung tissue were as follows①The pathological damage of pancreatic tissue in ANP rats included tissue edema, extensive hemorrhage and necrosis. The pathological score of pancreas was significantly higher than that of sham operation group at 12h,24h(P<0.01).②The pathological damage of liver tissue in ANP rats included liver cell swelling,degeneration and other pathological changes.The pathological score of liver tissue in ANP group was significantly higher than that of sham operation group at 12h,24h(P<0.01).③The pathological damage of lung tissue in ANP rats included significant alveolar exudate,inflammatory cell infiltration and other pathological changes.The pathological score of lung was significantly higher than that of sham operation group at 12h,24h(P<0.01).Conclusion:The ANP model induced by 5% sodium taurocholate is well consistent with ANP in pathological changes of pancreas, liver and lungs, and in manifestations, so it quite mimics severe acute pancreatitis in human. Further more it is relatively stable and is done with a high rate of success.PartⅡThe protective effects and possible mechanism of LXA₄-ME on acute necrotizing pancreatitis in ratsObjective: To study the the protective effects and its possible mechanism of LXA₄-ME on acute necrotizing pancreatitis in rats.Methods: One hundred and twenty male SD rats were randomly divided into Sham operation group (n=40), ANP group(n=40) and LXA₄-ME group(n=40). Sham operation group was given normal saline after the sham operation. ANP was induced by retrograde infusion of 5% sodium taurocholate into the pancreatobiliary duct. ANP group was treated with normal saline after the esbablishment of ANP. To LXA₄-ME group LXA₄-ME was administed(87.5 ug/kg, i.v.) after the onset of ANP. The general conditions of all rats were observed and their sera were obtained respectively at 12h and 24h time points after operation, for biochemical analysis of serum AMY and for measurement of TNF-αand sE-selectin by ELISA. The histological score of pancreatic tissue was calculated respectively at the 12h and 24h time points. At the same time points, the MPO activity and MDA levels in pancreatic tissue were determined, and the mRNA expression of TNF-α, E-selectin and NF-κB p65 in pancreatic tissue was evaluated by RT-PCR. The expression of NF-κB p65 was also measured by immunehistochemistry.Results:1. Compared with ANP group the general condition of LXA₄-ME group was better, and its mortality was lower(P<0.05, respectively).2. The levels of AMY in LXA₄-ME group were significantly lower than those in ANP group respectively at 12h and 24h time points after operation(P <0.05,respectively).3.The levels of TNF-αand sE-selectin in ANP group rats were remarkably increased at 12h and 24h time points after operation, and significantly higher than those in LXA₄-ME group(P<0.05, respectively).4. Histological score of pancreas in LXA₄-ME group was significantly lower than that in ANP group (P<0.05, respectively).5.①The activity of MPO in ANP group rats was significantly increased at 12h and 24h after operation(P<0.01, respectively), the activity of MPO in LXA₄-ME group was significantly lower than that in ANP group(P<0.05, respectively).②The level of MDA in ANP group rats was significantly increased at 12h and 24h after operation(P<0.01, respectively), the level of MDA in LXA₄-ME group was significantly lower than that in ANP group(P<0.05, respectively).6. Expression of TNF-αmRNA,E-selectin mRNA and NF-κB p65 mRNA in pancreasThe expression of TNF-αmRNA,E-selectin mRNA and NF-κB p65 in pancreas of Sham operation group was very low, While, the expression of them in ANP group was significantly increased at both 12h and 24h(P<0.01, respectively), The expression of TNF-αmRNA,E-selectin mRNA and NF-κB p65 mRNA in pancreas of LXA₄-ME group at 12h and 24h was reduced compared with that in ANP group at the corresponding time point(P<0.05, respectively) .7. NF-κB p65 protein expression in pancreasThere was almost no NF-κB p65 protein expression in Pancreas of sham operation group, but the NF-κB p65 protein expression in ANP group was visible(P<0.01, respectively).There was no significant difference at 12h and 24h . Compared with that in ANP group,the expression of NF-κB p65 protein in pancreas of LXA₄-ME group decreased (P<0.05, respectively).Conclusion:1. The severity of neutrophil infiltration , level of oxygen free radical and the expression of TNF-α,E-selectin in pancreatic tissues of ANP group significantly elevated.The increased activation of NF-κB in them was also observed.2. LXA₄-ME can significantly relieve the pathological damage of pancreas in rats with ANP and reduce the amount of ascites and serum levels of AMY in them. It can also significantly decrease their mortality. These results show that the protective effect of LXA₄-ME on ANP is associated with the reduction of neutrophil infiltration,oxidative stress levels and expression of inflammatory cytokines and adhesion molecules,the mechanism of whicth may be due to the suppression of NF-κB activation by LXA₄-ME and the subsequent down-regulated expression of NF-κB-dependent cytokines and adhesion molecules.