Effect Of Abdominal Paracentesis Drainage On The Polarization Of Peritoneal Macrophages In Rats With Severe Acute Pancreatitis

BackgroundDue to lacking of specific treatment,non-surgical treatment remains the therapeutic strategy for early stage severe acute pancreatitis(SAP)for decades.The overall mortality rate of acute pancreatitis is about 2%,and the mortality rate of severe acute pancreatitis is as high as 30%.Pancreatitis associated ascitic fluids(PAAF)is a common complication of acute pancreatitis patients.Previous studies of PAAF have confirmed that substances such as the pro-inflammatory mediators,digestive enzymes,hemoglobin,free fatty acid contained in PAAF play a critical role in promoting the progress of SAP.Our previous studies have demonstrated that by removing PAAF,abdominal paracentesis drainage(APD)could decrease the concentrate of pro-inflammatory mediators in serum and ascites,reduce systemic inflammatory response and improve the condition of SAP patients.However,the mechanism by which APD improves the condition of SAP has not been fully elucidated.Acute pancreatitis is an inflammatory disease characterized by systemic inflammatory response syndrome(SIRS),inflammatory-related immunopathological responses play an important role in the progression of acute pancreatitis.After abnormal activation of zymogens,damaged acinar cells are rapidly recognized by the innate immune system and initiate inflammation cascade and further leads to SIRS.Macrophages are an important member of the innate immune system,and studies have shown that macrophages are the main source of inflammatory factors in the SAP inflammatory storm.Especially,the production of PAAF directly affects the microenvironment of peritoneal macrophages.It has been demonstrated that macrophages can initiate different transcriptional programs under the stimulation of different microenvironments which is called polarization and can be can be roughly divided into pro-inflammatory classical polarization(M1 polarization)and anti-inflammatory alternative polarization(M2 polarization).PAAF induces the M1 polarization and the production of pro-inflammatory mediators in peritoneal macrophage.The polarization state of macrophages is highly plasticized.In recent years,researches on the effect of inducing macrophage to change their polarization phenotype in the treatment of inflammatory diseases have been conducted in-depth,these researched revealed that by using cytokine intervention,plasmid transfection,adoptive transfer and other methods affect the microenvironment of macrophages or directly change their own gene expression,increase the proportion of M2 macrophages,plays a therapeutic role in various diseases such as pancreatitis,sepsis,colitis,rheumatoid arthritis and so on.In previous studies,we found that APD can continuously discharge PAAF,reduce the concentration of pro-inflammatory cytokines such as TNF-?,IL-1? and IL-6 in the peritoneal cavity,and increase the concentration of anti-inflammatory cytokines such as IL-4 and IL-10.however,the mechanism by which APD improves the peritoneal inflammatory environment and reduces systemic inflammatory responses,particularly the mechanism by which APD increase the concentrate of anti-inflammatory cytokines,is unclear.Based on existing studies,we hypothesized that APD may play a role in increasing the concentration of anti-inflammatory cytokines and reducing the degree of systemic inflammatory response by inducing M2 polarization in peritoneal macrophages.Therefore,this aim of study is to explore the polarized phenotype of peritoneal macrophage during the course of SAP and the effects of APD on the polarization of peritoneal macrophages,and provide an experimental basis for better elucidating the mechanism of APD exerting anti-inflammatory effects in SAP treatment.Part I: Effect of APD on peritoneal macrophage polarization in rats with severe acute pancreatitisObjective: To observe the effects of APD on the polarization phenotype of peritoneal macrophages in SAP rats by establishing rat SAP and APD models.Methods: Rats were randomly divided into three groups: SAP group,APD group,and sham operation group(SHAM group),with 6 rats in each group.The SAP rat model was established by retrograde injection of 5% sodium taurocholate into the pancreatic duct.On the basis of this model,the abdominal drainage tube was implanted in the right lower abdomen to establish the APD model.The SHAM group turned the pancreas and closed the abdomen after laparotomy.Rats were sacrificed 12 h after the model was established,samples were collected.The extent of pathological damage of pancreatic tissue was observed by H&E staining and was scored.The level of serum amylase,lipase,and the secretion of pro-inflammatory factor and anti-inflammatory factor was determined by ELISA.These results were used to observed the severity of pancreatitis and pancreatic tissue damage as well as the degree of systemic inflammatory response.To observe the effect of APD on the polarization phenotype of peritoneal macrophages in SAP rats,flow cytometry was used to detect the phenotypic polarization of peritoneal macrophages,qPCR was used to detect the expression of iNOS and CD206 mRNA in total free cells in peritoneal cavity.Results: Compared with the SAP group,pancreatic injury was significantly reduced in the APD group,and the pathological score was significantly decreased,which was implied by: H&E staining showed that the acinar structure integrity of pancreatic tissue in APD group was better than that of SAP rats,acinar edema,necrosis,red blood cell exudation,inflammatory cell infiltration,etc.were significantly lighter than SAP group rats.In addition,the levels of amylase,lipase,pro-inflammatory factors TNF-? and IL-1? in the APD group were significantly lower than those in the SAP group,and the concentrations of the anti-inflammatory factors IL-10 and TGF-? were increased.The results showed that the degree of pancreatic tissue damage and systemic inflammatory response in the APD group was significantly less than that in the SAP group.According to the phenotypic analysis of peritoneal macrophages,compared with the SAP group,the M2 macrophages(CD68+CD163+ cells)in the APD group were significantly increased,and the mRNA expression of the M2 macrophage-associated protein CD206 gene was significantly up-regulated.The M1 macrophage(CD68+CD86+ cells)and mRNA expression of its related protein iNOS gene was down-regulated.Simultaneously,the proportion of M1/M2 which is an indicator of the balance of pro-inflammatory/anti-inflammatory response,was also significantly decreased,indicating that the immune balance in the peritoneal cavity of APD group has a tendency to move toward anti-inflammatory.Part II: Mechanism of M2 polarization of peritoneal macrophages induced by APD in rats with SAP and its application valueObjective: To analyze the mechanism of APD affecting phenotypic polarization of peritoneal macrophages and its role in SAP therapy.Methods: Animal grouping and model establishment methods are the same as part I.The concentration of inflammatory factors in the peritoneal lavage fluid of each group was measured,and the difference of inflammatory environment was analyzed.Secondly,the primary peritoneal macrophages were isolated and cultured,and the peritoneal lavage fluid of each group was added to RPMI1640 medium in proportion to simulate different peritoneal inflammatory environment,and the environment was used to intervene the primary cultured peritoneal macrophages.Flow cytometry was used to detect the polarization phenotype of primary peritoneal macrophages in different simulated environments.Subsequently,immunofluorescence staining of pancreatic tissue was performed.CD68+CD86+ and CD68+CD163+ were used as surface markers of M1 and M2 macrophages,respectively,and the distribution of the two types of cells in pancreatic tissue was observed.Finally,Western blot was used to detect the expression of Arg-1 protein in pancreatic tissue to analyze the role of M2 macrophages in pancreatic tissue.Results: APD significantly reduced the concentrations of pro-inflammatory mediators IL-1? and L-select in the peritoneal cavity of rats and increased the concentrations of the anti-inflammatory mediators IL-4 and IL-10.Cellular experiments showed that in vitro simulated APD abdominal inflammatory environment can still increase the M2 polarization of primary peritoneal macrophages,indicating that APD can affect the polarization phenotype of peritoneal macrophages by changing the peritoneal inflammatory environment.Immunofluorescence staining showed that both M1 and M2 macrophages were distributed in pancreatic tissue.In the pancreas of APD group,the number of M2 macrophages was higher than that in SAP group,and this result was consistent with that of the expression levels of CD163 and CD86 detected by western blot.In addition,we also found that the expression of Arg-1 protein in pancreatic tissue of APD group was significantly higher than that of SAP group,indicating that M2 macrophages in the pancreatic tissue of APD group may exert its effect on immunoregulation and tissue repairing by up-regulating the expression of Arg-1.Conclusion: The drainage of PAAF in the peritoneal cavity of SAP rats by APD can reduce the concentration of pro-inflammatory mediators in the peritoneal cavity and improve its inflammatory environment,thereby increasing the M2 polarization of the peritoneal macrophages as well as the distribution of M2 macrophages in the pancreas tissues.Meanwhile,it up-regulates the expression of Arg-1 and other proteins with anti-inflammatory and tissue repair effects,consequently reducing the degree of systemic inflammation and pancreatic tissue damage in SAP rats,and increasing the concentration of anti-inflammatory cytokines in ascites and serum,ultimately alleviate SAP patients' condition.These findings provide new insights into the mechanisms underlying APD as the treatment of severe acute pancreatitis.