Abdominal paracentesis drainage attenuates intestinal inflammation in rats with severe acute pancreatitis by inhibiting the HMGB1-mediated TLR4 signaling pathway

Figure 1 Effects of abdominal paracentesis drainage on pancreatic histopathology and proinflammatory cytokines. A: Representative images of rat pancreatic tissue from different groups following hematoxylin and eosin staining (bar = 100 μm); B: Histology severity score of pancreas tissue; C: Amylase; D: Lipase; E: Tumor necrosis factor-α; F: Interleukin-6; G: High mobility group box 1. All data are presented as mean ± SD (n = 6). ^aP < 0.05 vs sham group; ^bP < 0.05 vs severe acute pancreatitis group. SAP: Severe acute pancreatitis; APD: Abdominal paracentesis drainage; TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6; PAAF: Pancreatitis-associated ascitic fluid; HMGB1: High mobility group box 1.

Figure 2 Effects of abdominal paracentesis drainage on intestinal mucosa injury induced by severe acute pancreatitis. A: Representative images of rat distal ileal tissue from different groups following hematoxylin and eosin staining (bar = 100 μm); B: Histological severity score of intestinal tissues; C: D-lactate; D: Diamine oxidase; E: Endotoxin. All data are presented as mean ± SD (n = 6). ^aP < 0.05 vs sham group; ^bP < 0.05 vs severe acute pancreatitis group. SAP: Severe acute pancreatitis; APD: Abdominal paracentesis drainage; DAO; Diamine oxidase.

Figure 3 Effects of abdominal paracentesis drainage on intestinal inflammation and mucosal cell apoptosis induced by severe acute pancreatitis. A: Intestinal tumor necrosis factor-α; B: Intestinal interleukin-6; C: Intestinal myeloperoxidase activity; D: Representative images (400 × magnification) of terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay; E: Statistical results of the number of apoptotic intestinal mucosal cells in each group; F: Immunoblot of Bax, Bcl-2, pro-caspase-3 and cleaved-caspase-3 protein expression from intestinal samples; G-I: Densitometry analysis of the immunoblot data of apoptosis-related proteins in intestinal tissue. Data are expressed as means ± SD, n = 6 enzyme-linked immunosorbent assay and TUNEL assay results per group; means ± SD, n = 3 immunoblot results per group. ^aP < 0.05 vs sham group; ^bP < 0.05 vs severe acute pancreatitis group. TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6; MPO: Myeloperoxidase; SAP: Severe acute pancreatitis; APD: Abdominal paracentesis drainage; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 4 Effects of abdominal paracentesis drainage on intestinal toll-like receptor 4 signaling pathway in rats with severe acute pancreatitis. A: Representative immunohistochemistry images of toll-like receptor 4 (TLR4) in intestinal tissue (bar = 100 μm); B: TLR4 mRNA measurement by real-time polymerase chain reaction (PCR); C: Immunoblotting of TLR4, myeloid differentiation factor 88 (MyD88) and p65 protein expression and p65 phosphorylation level from intestinal samples; D-F: Densitometry analysis of TLR4, MyD88 and ratio of p65 to phosphorylated p65. Data are expressed as means ± SD, n = 6 real-time PCR results per group; means ± SD, n = 3 immunoblotting results per group. ^aP < 0.05 vs sham group; ^bP < 0.05 vs severe acute pancreatitis group. SAP: Severe acute pancreatitis; APD: Abdominal paracentesis drainage; MyD88: Myeloid differentiation factor 88; TLR4: Toll-like receptor 4; p-p65: Phosphorylated p65; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5 Effects of pancreatitis-associated ascitic fluid intraperitoneal injection with or without anti-high mobility group box protein 1 neutralizing antibody on intestinal toll-like receptor 4 signaling pathway in cerulein-treated rats. A: High mobility group box protein 1 in serum; B: Toll-like receptor 4 (TLR4) mRNA measurement by real-time polymerase chain reaction; C: Immunoblotting of TLR4, myeloid differentiation factor 88 (MyD88) and p65 protein expression and p65 phosphorylation level from intestine samples; D: Representative immunohistochemical images of TLR4 in intestinal tissue (bar = 100 μm); E-G: Densitometry analysis of TLR4, MyD88 and ratio of p65 to phosphorylated p65. Data are expressed as means ± SD, n = 6 enzyme-linked immunosorbent assay results per group; means ± SD, n = 3 immunoblotting results per group. ^aP < 0.05 vs control group; ^bP < 0.05 vs pancreatitis-associated ascitic fluid injection group. HMGB1: High mobility group box protein 1; CN: Control; PI: Pancreatitis-associated ascitic fluid (PAAF) injection; PIH: PAAF and 200 μg anti-HMGB1 neutralizing antibody; PIC: PAAF + control lgY; TLR4: Toll-like receptor 4; MyD88: Myeloid differentiation factor 88; p-p65: Phosphorylated p65; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 6 Effects of intraperitoneal pancreatitis-associated ascitic fluid injection with or without anti-high mobility group box protein 1 neutralizing antibody on intestinal inflammation and cell apoptosis in cerulein-treated rats. A: Intestinal tumor necrosis factor-α; B: Intestinal interleukin-6; C: Statistical results of the number of apoptotic intestinal mucosal cells in each group; D: Representative images (400 × magnification) of terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay; E: Immunoblotting of Bax, Bcl-2, pro-caspase-3 and cleaved-caspase-3 protein expression from intestinal samples; F-H: Densitometry analysis of the immunoblotting data of apoptosis-related proteins in intestinal tissue. Data are expressed as means ± SD, n = 6 enzyme-linked immunosorbent assay and TUNEL assay results per group; means ± SD, n = 3 immunoblotting results per group. ^aP < 0.05 vs control group; ^bP < 0.05 vs pancreatitis-associated ascitic fluid group. TNF-α: Tumor necrosis factor-α; CN: Control; PI: Pancreatitis-associated ascitic fluid (PAAF) injection; PIH: PAAF and 200 μg anti-high mobility group box protein 1 neutralizing antibody; PIC: PAAF + control lgY; IL-6: Interleukin-6; TUNEL: Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4',6-diamidino-2-phenylindole.

Figure 7 Possible mechanisms responsible for the protective effects of abdominal paracentesis drainage treatment on severe acute pancreatitis-induced intestinal inflammation and accompanying apoptosis. TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6; HMGB1: High mobility group box protein 1; IKK: IkappaB kinase; NF-kB: Nuclear factor kB; TLR4: Toll-like receptor 4.