Protective Effect And Mechanism Of Abdominal Paracentesis Drainage On Liver Injury In Rats With Severe Acute Pancreatitis

Acute pancreatitis(AP)refers to an acute abdominal disease in which the pancreatic enzymes are activated by a variety of etiologies,and characterized by local inflammation which may eventually causes the systemic inflammatory response syndrome and multiple organs dysfunction.Clinically,about 20% of AP patients evolve into severe acute pancreatitis(SAP).Patients with SAP often manifest systemic inflammatory response syndrome and extrapancreatic organs failure which were the main causes of death,besides the local inflammation and necrosis of pancreatic tissue.Liver,as the main source of cytokines,is often firstly affected after SAP.Then,the injured liver secretes more inflammatory factors,resulting in the amplification of inflammation leading to the aggravation of SAP.Therefore,relieving SAPassociated liver injury(SALI)may be an effective way to treat SAP.In SAP,severe inflammatory response changes vascular permeability and cause fluid accumulation in the abdominal cavity named pancreatitis associated ascitic fluid(PAAF).Our group found that the mortality of SAP patients could be significantly reduced by receiving drainage of ascites.And a series of clinical or basic studies have confirmed that the abdominal paracentesis drainage(APD)is one of the most important treatments in SAP.Based on the above,we speculated that APD could improve SAP symptoms by alleviating SALI.It has been confirmed that the interplays between inflammation and oxidative stress lead to the deterioration of SAP.In the early stages of SAP,inflammatory mediators and toxic substances secreted by damaged acinar cells pass through the pancreatic bile duct or circulation to the liver.Then,damaged hepatocytes will release a large number of oxygen free radicals and rapidly consume intracellular antioxidant substances,resulting in the disruption of the oxidation-antioxidant balance.At the same time,the imbalance of oxidant-antioxidant balance can also trigger the activation of cytokine-secreting cells in liver,leading to the amplification of the inflammatory response cascade.Heme oxygenase-1(HO-1)is a rate-limiting enzyme that decomposes heme into carbon monoxide,iron and biliverdin,which is activated by nuclear factor erythroid-2-related factor2(Nrf-2).Nrf-2/HO-1 signaling pathway plays an important role in anti-inflammatory,antioxidant or autophagy regulation,and is one of the most important intracellular protective axes.It has been confirmed that Nrf-2 /HO-1 pathway plays an important role in many hepatopancreatic diseases,such as pancreatitis,hepatitis,and hepatic ischemia-reperfusion injury.Therefore,we speculated that APD may alleviate SALI through Nrf-2 /HO-1 pathway.Part 1: Explore the effect of APD on SALI and its mechanismObjective: Explore the therapeutic effect of APD on SALI in SAP rats and find out its possibility mechanism.Methods: Twenty-four male adult SD rats were randomly divided into sham operation group(SO group),SAP group and APD group.SAP was induced by retrograde injecting 4%sodium taurocholate into biliopancreatic duct.In APD group,a drainage tube with vacuum bulb was implanted in its right lower abdomen immediately after SAP modeling.All rats were sacrificed 12 h after modeling.The serum,pancreas and liver tissues were collected.HE staining was used to evaluate the pathological changes of pancreas and liver;ELISA was used to detect the levels of IL-6,IL-1β and TNF-α in serum;Serum amylase,lipase,AST and ALT levels were detected;Colorimetric methods were used to detected the levels of SOD,GSH and MDA in liver tissue;The structural changes of sub-organelles in hepatocytes were detected by electron microscopy;RT-PCR,Western Blotting and Immunohistochemistry were used to detect the level of mRNA and protein expression of Nrf-2/HO-1 signaling pathway in liver.Results: 1.APD significantly reduced the severity of SAP,manifested as the lower pancreatic pathology score,lower serum amylase,lipase and inflammatory factors levels in APD group(P<0.05);2.APD significantly relived SALI,which is manifested as rats in APD group showed lower histopathological score,lower serum AST and ALT levels,and lower oxidative stress in liver tissue(P<0.05).The swelling of mitochondria and endoplasmic reticulum was significantly decreased under electron microscope in APD group;3.APD active Nrf-2/HO-1 pathway in liver tissue.The expression level of Nrf-2 and HO-1 in APD group was significantly upregulated compared with SAP group,while keap1 expression was significantly decreased(P<0.05).Conclusion: APD can significantly reduce SALI,systemic inflammatory response and oxidative stress level in liver tissue,which may be related to the up-regulation of Nrf-2 /HO-1 pathway.Part Ⅱ: Study on the protective effect of HO-1 on SALI by APDObjective: To investigate the role of HO-1 in APD treatmentMethods: Thirty male adult SD rats were randomly divided into SO group,SAP group,SAP+Zn PP(HO-1 inhibitor)group,APD group and APD+Zn PP group.Zn PP(30mg/kg)was intraperitoneal injected 12 h prior to surgery.All rats were sacrificed 12 h after modeling.HE staining was used to evaluate the pathological changes of liver.ELISA was used to detect the levels of IL-6,IL-1β and TNF-α in serum;Serum AST and ALT levels were detected;Colorimetric methods were used to detected the levels of SOD,GSH and MDA in liver tissue;Western Blotting was used to detect the expression of HO-1 in liver tissues to confirm the effectiveness of Zn PP.Results: In SAP+Zn PP or APD+Zn PP groups,the HO-1 expression in liver was significantly lower than that in SAP or APD groups.After inhibiting HO-1 expression,the pathological score of liver was significantly higher than group without inhibitor.Serum AST,ALT and inflammatory factors TNF-α,IL-1β and IL-6 were significantly increased.The oxidative stress in liver tissue was also significantly increased(P<0.05).Conclusion: Zn PP could reverse the therapeutic effect of APD,demonstrating that Nrf-2 /HO-1 pathway plays a key role in the treatment of APDPart Ⅲ: Study on the role of PAAF in SALIObjective: To further explore the role of PAAF in promoting SALIMethods: Twelve adult male SD rats were induced to SAP and PAAF was collected 3h after surgery.In vivo experiment: Eighteen adult male SD rats were randomly divided into SO group,MAP group and intraperitoneal injection(PI)group.MAP was induced by six consecutive intraperitoneal injection of caerulein(20μg/kg at 1-h intervals)while saline was used in SO group.In PI group,4ml PAAF was intraperitoneal injected after MAP modeling.All rats were sacrificed 12 h after modeling.The serum and liver tissues were collected.HE staining was used to evaluate the pathological changes of liver;ELISA was used to detect the levels of IL-6,IL-1β and TNF-α in serum;Serum AST and ALT levels were detected;Colorimetric methods were used to detected the levels of SOD,GSH and MDA in liver tissue;Western Blotting was used to detect the expression of Nrf-2/HO-1 signaling pathway in liver.In vitro experiment: BRL-3A cells were planted in 6-well plates and divided into CON,LPS,1.25%PAAF,2.5%PAAF,5%PAAF and 10%PAAF groups.CON group was not treated;In LPS group,LPS was added into cell culture medium to make its concentration reach10μg/ml.In each PAAF group,based on the treatment of LPS group,different PAAF were added into the medium to make the concentration reach 1.25%,2.5%,5% and 10%,respectively.Twelve hours after processing,Western Blotting and Immunofluorescence were used to detect the expression of Nrf-2 / HO-1 signaling pathway.Results: 1.Compared with the MAP group,the pathological damage of liver tissue in PI group was significantly aggravated and the level of AST,ALT and inflammatory factors were increased;Also,oxidative stress was increased and the expression of Nrf-2 /HO-1 pathway was down-regulated in liver tissue in PI group(P<0.05);2.Expression of Nrf-2/HO-1pathway were decreased in BRL-3A cells in each PAAF groups compared with LPS group(P<0.05).Conclusion: PAAF can aggravate oxidative stress in liver by inhibiting the activation of Nrf-2/HO-1 signaling pathway,thus aggravating systemic inflammatory response and the severity of pancreatitis.