Experimental Study On Effects Of Omega-3 Polyunsaturated Fatty Acids On Severe Acute Pancreatitis And Its Mechanisms

Objective To investigate the anti-inflammatory effect and its mechanisms ofω3 polyunsaturated fatty acids (ω-3 PUFAs) in severe acute pancreatitis.Methods 60 rats were divided into three groups:the Experiment Group (EG), the Negative Control Group (NCG) and the Blank Control Group (BCG). Acute pancreatitis in rat was induced by low-pressure intraductal infusion of glycodeoxycholic acid (GDOC) combined with intravenous cerulein in the EG and the NCG. Rats in the BCG underwent only midline laparotomy. Six hours after pancreatitis induction, animals in the NCG and the EG were given saline or supplemented with long-chain omega-3 fatty acids (10% FO emulsion,0.20 g/kg per day).24hs later, all rats were sacrificed and peripheral blood, pancreas and lungs tissues were collected; Serum alanine transarninase (ALT), aspartate aminotransferase (AST), amylase, interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-a) were detected and histological evaluation was done; The CD4+/CD8+T cell ratio in Peripheral blood was detected by flow cytometry and mRNA expression of Foxp3, IL-2 in Peripheral blood mononuclear cells were analyzed using real time PCR; The expression of uncoupling protein-2 (UCP-2) gene in pancreas tissues was derermined by real time PCR and western blot; Nuclear factor-kappaB (NF-KB) activation was determined by immunohistochemistry.Results Serum IL-6,TNF-a were decreased and IL-10 was increased significantly in the EG than in the NCG. ALT, AST, amylase and degree of pancreatic damage indicated no difference between the pancreatitis groups. The CD4+/CD8+T cell ratio in Peripheral blood and the expression of Foxp3,IL-2 of peripheral blood mononuclear cells in the EG was lower significantly than in the NCG. UCP-2 gene expression was up-regulated in pancreas tissues in the NCG compared with in the EG and the BCG. NF-KB had more activation in pancreas/lung tissues of the NCG.Conclusionω-3 PUFAs down-regulated inflammation response on severe acute pancreatitis in rats, its underlying mechanism may involve modulating T cell function, inhibiting the activation of NF-KB and expression of UCP-2 gene.Objective To investigate the regulatory role of inflammation ofω-3 PUFAs and the mechanisms in vitro.Methods AR42J cells were divided into three groups:the group A cells did nothing as control, the group C cells were overstimulated with caerulein (10-8 mol/L), and the group B cells were treated with DHA (50μmol/L). The expression of TNF-a, IL-6, UCP-2 mRNA were determined with real time PCR. The NF-KBp65 subunits in nucleus protein was detected with western blot analysis. Necrosis and apoptosis of AR42J cells were detected with Hoechest33258 and propidium iodide staining and observed with fluorescence microscope.Results Cells in group B and group C which sitmulated with caerulein had upregulated TNF-a, IL-6, UCP-2 mRNA expression and the levels of mRNA expression in group B were lower than those in group C. In addition, the protein expression of UCP-2 gene and NF-KBp65 subunits in nucleus prote in group B were lower compared with those in group C. More necrosis took place in the cells in group C contrast with the cells in group B.Conclusion In vitro,ω-3 PUFAs could inhibited the cytokine mRNA transcription sitmulated with caerulein, down-regulated the UCP-2 gene expression and reduced the nuclear translocation of NF-KB in AR42J cell culture system.Objective To investigate the effect of UCP-2 gene expression on cell death modes of pancreatic acinar cells in an inflammatory condition, and explore the role of UCP2 as an important modifier of the severity of acute pancreatitis.Methods Three specific target sequences from UCP-2 mRNA sequencewere designed according to the principle of designing on siRNA. RNAi lentivirus vector targeting uncoupling protein 2 gene were constructed and identified. The best one was chosen for following study. AR42J cells were divided into three groups:the Experimental Group (infected with RNAi lentivirus vector), the Negative Control Group (infected with empty lentivirus vector), the Blank Control Group (not infected with lentivirus). Fluorescence microscope detected the efficiency of infecting and Real time PCR determined the effect of RNA interference. After stimulated with caerulein, apoptosis was analyzed by flow cytometry using PI (propidium iodide) staining at different time points. ATP, ROS, in cells were also detected.Results The construction of the siRNA plasmids were identificated to be successful by PCR and DNA sequencing. The best one was selected by western blot and the titer of virus was 5×108TU/ml. The expression level of UCP-2 gene in the Experiment Group was knocked-down. Compared with the Negative Control Group, the Experiment Group had increased ROS, ATP and apoptosis, but decreased necrosis.Conclusion Inhibiting the expression of UCP-2 gene could affect cell death modes of pancreatic acinar cells in an inflammatory condition, increase apoptosis and decrease necrosis.