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. 2012;7(9):e46201.
doi: 10.1371/journal.pone.0046201. Epub 2012 Sep 27.

Netrin-1 Protects Against L-Arginine-induced Acute Pancreatitis in Mice

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Free PMC article

Netrin-1 Protects Against L-Arginine-induced Acute Pancreatitis in Mice

Ji Chen et al. PLoS One. .
Free PMC article

Abstract

Acute pancreatitis (AP) is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with infiltration of leukocytes. The neuronal guidance protein, netrin-1, has been shown to control leukocyte trafficking and modulate inflammatory responses in several inflammation-based diseases. The present study was aimed toward investigating the effects of netrin-1 in an in vivo model of AP in mice. AP was induced in C57BL/6 mice by administration of two intraperitoneal injections of L-Arginine (4 g/kg). Mice were treated with recombinant mouse netrin-1 at a dose of 1 µg/mouse or vehicle (0.1% BSA) intravenously through the tail vein immediately after the second injection of L-Arginine, and every 24 h thereafter. Mice were sacrificed at several time intervals from 0 to 96 h after the induction of pancreatitis. Blood and tissue samples of pancreas and lung were collected and processed to determine the severity of pancreatitis biochemically and histologically. Immunohistochemical staining demonstrated that netrin-1 was mainly expressed in the islet cells of the normal pancreas and the AP model pancreas, and the pancreatic expression of netrin-1 was down-regulated at both the mRNA and protein levels during the course of AP. Exogenous netrin-1 administration significantly reduced plasma amylase levels, myeloperoxidase activity, pro-inflammatory cytokine production, and pancreas and lung tissue damages. Furthermore, netrin-1 administration did not cause significant inhibition of nuclear factor-kappa B activation in the pancreas of L-Arginine-induced AP. In conclusion, our novel data suggest that netrin-1 is capable of improving damage of pancreas and lung, and exerting anti-inflammatory effects in mice with severe acute pancreatitis. Thus, our results indicate that netrin-1 may constitute a novel target in the management of AP.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of netrin-1 in the pancreas.
RT-PCR (A) and Western blot analysis (B) of netrin-1 expression and immunohistochemical staining (C) of netrin-1 at the indicated times in mouse (C57BL/6) in normal and AP model pancreas. Acute pancreatitis was induced by two intraperitoneal injections of L-Arginine (4 g/kg, 1 h apart). Data are presented as mean±SD (n = 6 per group in normal mice, n = 8 per group in the AP model). * P<0.05.
Figure 2
Figure 2. Effect of netrin-1 on pancreatic inflammation.
A. Plasma amylase activity. B. Pancreatic myeloperoxidase (MPO) activity. C. Histological scores (sum of interstitial edema, inflammatory cell infiltration, hemorrhage, and acinar cell necrosis). D. Histology. Representative micrographs of H&E-stained pancreatic sections at the indicated times are shown. Acute pancreatitis was induced by 2-hourly two intraperitoneal injections of L-Arginine (4 g/kg, 1 h apart). Mice were treated with recombinant mouse netrin-1 at a dose of 1 µg/mouse or vehicle (0.1% BSA) immediately after the second injection of L-Arginine and every 24 h thereafter. Data are presented as mean±SD (n = 6 per group in the normal mice, n = 8 per group in both the AP model and netrin-1 treated AP model). * P<0.05.
Figure 3
Figure 3. Effect of netrin-1 on lung MPO and histology.
A. Lung myeloperoxidase (MPO) activity. B. Histological scores (sum of inflammatory cell infiltration, pulmonary edema, and hemorrhage). C. Histology. Representative micrographs of H&E-stained lung sections at the indicated times are shown. Acute pancreatitis was induced by two intraperitoneal injections of L-Arginine (4 g/kg, 1 h apart). Mice were treated with recombinant mouse netrin-1 at a dose of 1 µg/mouse or vehicle (0.1% BSA) immediately after the second injection of L-Arginine and every 24 h thereafter. Data are presented as mean±SD (n = 6 per group in the normal mice, n = 8 per group in both the AP model and netrin-1 treated AP model). *P<0.05.
Figure 4
Figure 4. Effect of Netrin-1 on plasma cytokines level.
IL-1β, IL-6, TNF-α, and IL-10 plasma concentrations at the indicated times are shown for the normal group and AP groups with and without netrin-1 treatment. Acute pancreatitis was induced by two intraperitoneal injections of L-Arginine (4 g/kg, 1 h apart). Mice were treated with recombinant mouse netrin-1 at a dose of 1 µg/mouse or vehicle (0.1% BSA) immediately after the second injection of L-Arginine and every 24 h thereafter. Data are presented as mean±SD (n = 6 per group in the normal mice, n = 8 per group both in the AP model and netrin-1 treated AP model). * P<0.05.
Figure 5
Figure 5. Effect of netrin-1 on pancreatic NF-κB activation.
RT-PCR (A) and Western blot analysis (B) of NF-κB p65 expression at the indicated times are shown for the normal group and AP groups with and without netrin-1 treatment. Acute pancreatitis was induced by two intraperitoneal injections of L-Arginine (4 g/kg, 1 h apart). Mice were treated with recombinant mouse netrin-1 at a dose of 1 µg/mouse or vehicle (0.1% BSA) immediately after the second injection of L-arginine and every 24 h thereafter. Data are presented as mean±SD (n = 6 per group in the normal mice, n = 8 per group both in the AP model and netrin-1 treated AP model). * P<0.05.

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Grant support

The study was funded by Shanghai Municipal Science and Technology commission (no. 10JC1412700) and the Natural Science Foundation of China (no. 30901770 and no. 81150110493). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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