Abdominal paracentesis drainage ameliorates severe acute pancreatitis in rats by regulating the polarization of peritoneal macrophages

Abstract

Aim: To investigate the role of peritoneal macrophage (PM) polarization in the therapeutic effect of abdominal paracentesis drainage (APD) on severe acute pancreatitis (SAP).

Methods: SAP was induced by 5% Na-taurocholate retrograde injection in Sprague-Dawley rats. APD was performed by inserting a drainage tube with a vacuum ball into the lower right abdomen of the rats immediately after the induction of SAP. To verify the effect of APD on macrophages, PMs were isolated and cultured in an environment, with the peritoneal inflammatory environment simulated by the addition of peritoneal lavage in complete RPMI 1640 medium. Hematoxylin and eosin staining was performed. The levels of pancreatitis biomarkers amylase and lipase as well as the levels of inflammatory mediators in the blood and peritoneal lavage were determined. The polarization phenotypes of the PMs were identified by detecting the marker expression of M1/M2 macrophages via flow cytometry, qPCR and immunohistochemical staining. The protein expression in macrophages that had infiltrated the pancreas was determined by Western blot.

Results: APD treatment significantly reduced the histopathological scores and levels of amylase, lipase, tumor necrosis factor-α and interleukin (IL)-1β, indicating that APD ameliorates the severity of SAP. Importantly, we found that APD treatment polarized PMs towards the M2 phenotype, as evidenced by the reduced number of M1 macrophages and the reduced levels of pro-inflammatory mediators, such as IL-1β and L-selectin, as well as the increased number of M2 macrophages and increased levels of anti-inflammatory mediators, such as IL-4 and IL-10. Furthermore, in an in vitro study wherein peritoneal lavage from the APD group was added to the cultured PMs to simulate the peritoneal inflammatory environment, PMs also exhibited a dominant M2 phenotype, resulting in a significantly lower level of inflammation. Finally, APD treatment increased the proportion of M2 macrophages and upregulated the expression of the anti-inflammatory protein Arg-1 in the pancreas of SAP model rats.

Conclusion: These findings suggest that APD treatment exerts anti-inflammatory effects by regulating the M2 polarization of PMs, providing novel insights into the mechanism underlying its therapeutic effect.

Keywords: Abdominal paracentesis drainage; Peritoneal macrophages; Polarization; Severe acute pancreatitis.

Conflict of interest statement

Conflict-of-interest statement: The authors declare that there are no conflicts of interest related to this study.

Figures

Figure 1

Abdominal paracentesis drainage ameliorates severe acute pancreatitis in a rat model. A: Model establishment. Retrograde injection of Na-taurocholate (left) and a rat after abdominal paracentesis drainage (APD) treatment (right); B and C: Histopathological analysis of the pancreas. Comprehensive disruption of the pancreatic structure with widespread infiltration of leukocytes, acinar cell vacuolization and necrosis was observed in severe acute pancreatitis (SAP) rats; localized leukocyte infiltration and relatively intact acinar structure were observed in APD rats; D-I: Plasma levels of amylase, lipase, tumor necrosis factor-α, interleukin (IL)-1β, IL-10 and transforming growth factor-β, respectively. Data indicate the mean ± SD of six mice (C-I). aP < 0.05 vs sham, bP < 0.05 vs SAP.

Figure 2

Different polarized phenotypes of peritoneal macrophages in each group. A: Gating strategy for the peritoneal macrophage population; B-D: Representative dot plot (B) and the percentages (C) and M1/M2 ratio (D) of CD68+CD86+ (M1) cells and CD68+CD163+ (M2) cells in each group; E: Relative expression levels of CD206 and iNOS gene in peritoneal cells measured by real-time PCR and normalized to GAPDH mRNA. The data represent at least three independent experiments (A-B) or indicate the mean ± SD of six mice (E). aP < 0.05 vs abdominal paracentesis drainage.

Figure 3

Abdominal paracentesis drainage alters the inflammatory environment in the peritoneal cavity. Protein levels in peritoneal lavage were measured by Luminex. The results reflect the mean ± SD obtained from six animals in each group. aP < 0.05 vs abdominal paracentesis drainage.

Figure 4

In vitro simulated peritoneal inflammatory environment of abdominal paracentesis drainage rats changes the polarized phenotype of peritoneal macrophages. Primary peritoneal macrophages were cultured in medium simulating different inflammatory environments. The percentages of CD86+ and CD163+ cells were measured by flow cytometry. The data are representative of at least three independent experiments. aP < 0.05 vs CON, bP < 0.05 vs abdominal paracentesis drainage.

Figure 5

Number of M2 macrophages increases in the pancreas of abdominal paracentesis drainage rats and exerts anti-inflammatory effects. A: Pancreatic tissues from each group were stained with DAPI (blue), CD163 (green in upper two panels), CD86 (green in lower two panels) and CD68 (infrared represented by carmine). Representative images are shown; B and C: Protein levels of Arg-1, CD163, CD86 and iNOS in the pancreatic tissues of each group were measured by Western blot, and the relative expression of these proteins was normalized to GAPDH. Data are representative of at least three independent experiments. aP < 0.05 vs severe acute pancreatitis.

Figure 6

Possible mechanisms responsible for the beneficial effects of abdominal paracentesis drainage on severe acute pancreatitis. Once severe acute pancreatitis (SAP) occurs, the inflammatory cells are activated following acinar cell injuries and exudate full of pro-inflammatory mediators collects in the peritoneal cavity, which can polarize the peritoneal macrophages (PMs) towards the M1 phenotype and lead to the overexpression of pro-inflammatory mediators by PMs. By removing the pancreatitis-associated ascitic fluids, abdominal paracentesis drainage (APD) could improve the inflammatory environment of the peritoneal cavity, thus promoting M2 polarization of PMs in the peritoneal cavity. Meanwhile, APD could also promote M2 polarization of macrophages in pancreatic tissues. These events could upregulate the expression of anti-inflammatory cytokines, which ultimately ameliorate pancreatic injury. APD: Abdominal paracentesis drainage; SAP: Severe acute pancreatitis.