Mechanism Of NAG-1 In Prevention Of Post-ERCP Pancreatitis By Diclofenac

Background Endoscopic Retrograde Cholangio-Pancreatography(ERCP) is an important method for the diagnosis and treatment of biliary and pancreas diseases. But because the invasiveness of ERCP, a series of related complications occured with increased pancreatic duct pressure resulted from the damage of duodenal papilla and Oddi sphincter by repeated intubation. The repeated developing and over filling of contrast agent, and the damage of pancreatic duct also have an important influence on the occurrence of complications. The main complications of ERCP were post-ERCP pancreatitis(PEP), gastrointestinal bleeding, perforation and biliary infection, and the most common and serious complication was PEP. The incidence of PEP was 1%-10%, and high-risk patient was up to 30%. Therefore, it is very important to prevent PEP. Extensive research on preventive effect of somatostatin and its analogs, calcium antagonists, nitroglycerin, IL-10, heparin, protease inhibitors, and steroidal anti-inflammatory drugs for PEP, but there was still have no uniform conclusion. Many evidence point to the effect of non-steroidal anti-inflammatory drugs(NSAIDs) in the prevention of PEP. NAG-1 has attracted more attention gradually during the study on NSAIDs prevention of PEP. As NAG-1 has certain anti-inflammatory effect, our study intends to investigate the effect of diclofenac on the prevention of PEP and its possible mechanism.Objective To study the effect of diclofeac on PEP, and the change of NAG-1 levels in prevention of diclofenac in PEP.Methods From September 2012 through October 2013, 120 patients after ERCP were randomly assigned to diclofenac group and control group. Patients in diclofenac group were received a Olfen which contains diclofenac 75 mg by intramuscular injection immediately after ERCP. We observed the plasma amylase levels at pre-ERCP, post-ERCP 3h and post-ERCP 24 h and the occurrence of stomachache, and counted the incidences of PEP in two groups. Meanwhile, the plasma NAG-1 levels at pre-ERCP, post-ERCP 3h and post-ERCP 24 h in patients were detected randomly. Meanwhile, the plasma NAG-1 levels, NAG-1 m RNA and NAG-1 protein expression at pre-ERCP, post-ERCP 3h and post-ERCP 24 h in patients were detected by ELISA, RT-PCR and Western Blot assay respectively.Results There was a significant difference on PEP incidences after ERCP between diclofenac group(6.67%) and control group(20.00%)(P<0.05). The plasma amylase activities at post-ERCP 3h and post-ERCP 24 h in diclofenac group(202.70±120.44 and 209.13±157.14 U/L) were notable lower than those in control group(283.57±178.39 and 305.97±208.69U/L). The levels of NAG-1 at 3h in diclofenac group were higher than those at pre-ERCP and post-ERCP 3h in control group(P<0.05). The levels of NAG-1, NAG-1 m RNA and NAG-1 protein expression at post-ERCP 3h were higher than those at pre-ERCP and control group at post-ERCP 3h in diclofenac group(P<0.01).Conclusions Diclofenac can prevent the occurrence of PEP, which may associate with the increased plasma NAG-1 levels.Background PEP was one of the most common and serious complication of ERCP. At present, massive studies have proved that NSAIDs can prevent PEP which may by inhibiting the synthesis of prostaglandin and inflammation cascade in pancreatitis. Diclofenac, one of NSAIDs, is an inhibitor of COX and PLA2, and our previous study have comfirmed that diclofenac play a role in the prevention of PEP. The levels of NAG-1, NAG-1m RNA and protein in plasma of post-ERCP patients with diclofenac intramuscular injection, which infer that diclofenac may decrease the incidence of PEP by up-regulation NAG-1 level. In order to investigate and expound the possible mechanism of diclofenac prevention of PEP, we prepare the mice model of acute pancreatitis and hope to provide experimental and theory evidence for diclofenac on prevention of PEP.Objective To investigate the protective effect of diclofenac sodium on the acute pancreatitis in mice induced by caerulein.Methods Male C57/B6 mice were randomly divided into the control group, AP group and AP + diclofenac sodium group. The AP model of mice was established by intraperitoneal injection of caerulein with a dose of 50μg/kg, one time per hour for seven times of injection. Diclofenac sodium were intraperitoneal injection with a dose of 15mg/kg one hour before AP was induced by caerulein. Meanwhile, the mice in control group received injection of the same dose of 0.9%(w/v) saline. At the end of the expriment all the mice were killed. The levels of amylase, lipase, TNF-α, IL-1?, IL-6, IL-8 and NAG-1 in serum were measured by colorimetric method and Enzyme-Linked Immunosorbent Assay respectively and NAG-1 m RNA and protein expression were measured by RT- PCR and Western Blot assay respectively. Prepare the pancreas and lung homogenate, and measure levels of IL-1?、IL-6、IL-8、TNF-ɑ、NAG-1 by ELISA, MPO level by colorimetric method, NAG-1 m RNA by PT-PCR and NAG-1 protein expression by Western Blot. Simultaneously, the weight ratio of pancreas and lung tissue to mice weight were detected, and pancreas and lung tissue were underwent pathological staining.Results The activities of amylase, lipase, the ratio of pancreatic and lung tissues wet weight with mice weight n AP group were higher than those in control group(P<0.05). AP+ diclofenac group were lower than those in AP group in different degree(P<0.05).Pancreatic pathology of AP group showed hyperemia and edema of pancreatic mesenchyme, widened lobular gap and infiltration of inflammatory cells as well as an acinar swelling. Widened lung mesenchyme, extravasation of red blood cells in alveoli, infiltration of inflammatory cells and odd alveolar wall damage were also found in AP group. The inflammation degree of pancreas and lung in AP+ diclofenac group were significantly lower than those in the AP group(P<0.01). The levels of IL-1?, IL-6, IL-8, TNF-ɑ in plasma and the levels of IL-1?, IL-6, IL-8, TNF-ɑ and MPO activities in pancreas and lung homogenates in AP group were higher than those in control group in different degree(P<0.05). Simultaneously, AP+ diclofenac group were lower than those in AP group in different degree(P<0.05). While, the plasma levels of NAG-1 among control, AP and AP+diclofenac group have not reach the difference(P>0.05). In pancreas and lung homogenates, the levels of NAG-1 in AP group were higher than those in control group(P<0.05) and AP+diclofenac group were higher than those in AP gorup(P<0.05). The levels NAG-1 m RNA and protein expression in plasma, pancreas and lung homogenates in AP group were higher than those in control group(P<0.05). Simultaneously, AP+ diclofenac group were higher than those in AP group(P<0.05).Conclusion Diclofenac can alleviate inflammatory reaction and lung injury in mice with acute pancreatitis and its mechanism may associate with upregulating the NAG-1 level and inhibiting the inflammatory mediators synthesis.