| Severe acute pancreatitis(SAP) is a kind of sudden inflammatory diseases with high death rate, It has the tendency of development of persistent multiple organ failure and a wide range of infected necrosis. The pathological process of acute pancreatitis including edema and hemorrhage, even necrosis. The development of acute pancreatitis is a complex process, involving lifestyle and environmental factors and so on many aspects, such as alcoholism, pancreatic duct stenosis, hyperlipidemia, infection and so on. The pathogenesis is not yet fully understood.Hypoxia inducible factor-1(HIF-1 hypoxia inducible factor-1) was extremely sensitive on hypoxia, under hypoxic condition of HIF-1 showed and practice, as well as the degree of hypoxia dependent stable high expression. HIF-1 had a wide range of target genes, regulation of nearly a hundred kinds of downstream gene expression, including and the development of inflammation, tissue repair, cell differentiation and apoptosis, tumor growth and vascular relaxation and shrinkage control of physiological and pathological processes, which could produce a series ofstress reaction. The apoptosis and necrosis of pancreatic acinar cells in acute pancreatitis is caused by hypoxia, the hypoxia inducible factor-1 alpha(HIF-1 alpha) activation and cause a series of stress reaction.Aquaporinfamily are small molecule protein which are widely existed in mammalian epithelial and endothelial cell membrane, They are the channel which allow water molecules rapidly across the membrane, playing a key role in physiological and pathological processes in the different mammalian organs. AQP1 gene knockout or gene deficient mice increased impaired vascular permeability, promoting inflammatory cells leak. Recent studies show that in experimental models of Alzheimer's disease, ischemic stroke and subarachnoid hemorrhage, curcumin may limit many ion channels transport and limit inflammation and nerve injury, researchers speculated that curcumin may by reducing the expression of AQP1 and restricts the development of the brain edema, but the mechanism is not yet clear.Curcumin analogues of biphenyl(diphenyl difluoroketone, two fluorine ketone EF24) with stronger and better solubility and stability effect than curcumin. In the past, EF24 was mainly researched in cancer, but the study of inflammation was poorly understood, there was no researche on its role in pancreatic injury resulting in severe acute pancreatitis.Objective: The establishment of rat model with severe acute pancreatitis, histological changes in pancreatic during SAP was observed, the expression of HIF-1?, AQP1 protein and mRNA in the pancreas with the application of EF24 were studied from molecular level. We tried to elucidate the mechanism of EF24 in pancreatic tissue during SAP, providing new ideas for the clinical treatment of SAP.Methods:1 The 60 SD rats were randomly divided into control(Con) group, SAP model group(SAP) and EF24(EF24) of the group. Each group was further divided into sub groups(6 h group, 12 h group), 10 rats in each group.2 SAP group and EF24 group rats were injected intraperitoneal with 20% L- arginine(L-Arg) 3.0 g/kg. After an hour, they were injected with the same does of L-Arg. The SAP group rats were injected intraperitoneally with polyethylene glycol(PEG:NS =1:1) 100 ?l after an hour. In EF24 group, the rats were injected intraperitoneally with EF24(0.4 mg/kg) 1 h after induction of acute pancreatitis. The Con group rats were injected intraperitoneally with polyethylene glycol(PEG :NS =1:1) 100 ?l.3 Blood, pancreas tissues were taken 6 or12 hours after the modeling,blood was taken from the femoral artery,stayed at room temperature for 1 h to be solidified, centrifugate at the speed of 3000 rpm for 10 min, placed in-80 ?ultra low temperature refrigerator. The levels of serum IL-6, TNF-? at the two time points were measured using the ELISA method. The expression of pancreas tissue's HIF-1?, AQP1 mRNA and protein were evaluated by RT-PCR and Western-blot.4 Statistical Methods: Statistical analysis was performed by SPSS 17.0 for windows. All data were presented as mean SD. The significance was assumed at P<0.05.Results:1 Histopathological changes of pancreatic tissue: structural disorder, cell and interstitial hyperemia and edema obviously, glandular cell bleeding, necrosis, inflammatory cell infiltration is visible in pancreatic tissue in the model group and degree of pathological damage extended with time, pancreatic pathology was evaluated by Schmidt score, suggesting that severe acute pancreatitis could be successfully induced by L-arginine, and compared with Group SAP, pancreatic lesions degree abated at corresponding time points in the Group EF24.2 The expression of serum IL-6: serum IL-6 levels of SAP group(6 h 99.9 4.6; 12 h 131.1 8.9) increased significantly at each time point compared with Con group(6 h 38.8 5.4; 12 h 30.9 7.2)(P < 0.01). Serum IL-6 levels of Group EF24(6 h 7.46 7.0; 12 h 79.7 8.3) increased significantly compared with that of Con group(P < 0.01). EF24 group decreased significantly compared with SAP group(P < 0.01). Comparison between each two time points showed that 12 h group of SAP was significantly higher than 6 h group(P < 0.01).3 The expression of serum TNF-?: TNF-? content of SAP Group(6 h 106.6 10.0; 12 h 146.3 8.3) at each time point increased significantly compared with Con group(6 h 31.1 5.5; 12 h 28.7 4.0)(P < 0.01). Compared with Con group, serum TNF-? levels of EF24 group(6 h 75.1 5.0; 12 h 83.2 6.8) increased significantly(P < 0.01). EF24 group decreased significantly compared with SAP group(P < 0.01). Comparison between each two time points showed that 12 h group of SAP group was significantly higher than 6 h group(P < 0.01).4 The expression of pancreas HIF-1? mRNA with RT-PCR: compared with Con group, HIF-1? mRNA in SAP group and EF24 group increased significantly(P < 0.01). HIF-1? mRNA in EF24 group was lower than that in SAP group in each subgroup(P < 0.01). No significant difference was found between respective time point of group EF24 and Con(P > 0.05). Comparison between each two time points showed that 12 h group of SAP group was significantly higher than 6 h group(P < 0.01).5 The expression of pancreas AQP1 mRNA with RT-PCR: compared with Con group, AQP1 mRNA in SAP group and EF24 group decreased significantly(P < 0.01). AQP1 mRNA in EF24 group was lower than that in Con group in each subgroup(P < 0.01). EF24 group and Con group showed no significant difference at 6 h, 12 h time point in AQP1 mRNA(P > 0.05). Comparison between each two time points showed that 12 h group of SAP group was significantly lower than 6 h group(P < 0.01).6 The expression of pancreas HIF-1? protein: compared with Con group, HIF-1? protein in SAP group and EF24 group increased significantly(P < 0.01). HIF-1? protein in EF24 group was lower than that in SAP group in each subgroup(P < 0.01). No significant difference was found between EF24 group and Con group in HIF-1? protein at 6 h, 12 h time point(P > 0.05). Comparison between each two time points showed that 12 h group of SAP group was significantly higher than 6 h group(P < 0.01).7 The expression of pancreas AQP1 protein: compared with Con group, AQP1 protein in SAP group and EF24 group decreased significantly(P < 0.01). AQP1 protein in EF24 group was lower than that in Con group in each subgroup(P < 0.01). No significant difference was found between EF24 group and Con group in AQP1 protein at 6 h, 12 h time point(P > 0.05). Comparison between each two time points showed that 12 h group of SAP group was significantly lower than 6 h group(P < 0.01).Conclusion:1 The expression of HIF-1? proteins and mRNA were higher, and AQP1 proteins and mRNA were lower in SAP rats' pancreatic, suggesting that the two factors were involved in pancreatic tissue injury of the severe acute pancreatitis.2 EF24 could reduce the levels of IL-6, TNF-? in serum of SAP, and reduced pancreatic injury suggesting that EF24 played a protective role in the severe acute pancreatitis.3 EF24 could inhibit the expression of HIF-1? in SAP pancreatic tissue and up regulate the expression of AQP1, suggesting certain relationship between EF24 and HIF-1?, AQP1, EF24 reduced SAP pancreatic injury by inhibiting the expression of HIF-1? and up regulation of AQP1 expression. |
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