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Docosahexaenoic Acid Inhibits Cerulein-Induced Acute Pancreatitis in Rats

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Docosahexaenoic Acid Inhibits Cerulein-Induced Acute Pancreatitis in Rats

Yoo Kyung Jeong et al. Nutrients.

Abstract

Oxidative stress is an important regulator in the pathogenesis of acute pancreatitis (AP). Reactive oxygen species induce activation of inflammatory cascades, inflammatory cell recruitment, and tissue damage. NF-κB regulates inflammatory cytokine gene expression, which induces an acute, edematous form of pancreatitis. Protein kinase C δ (PKCδ) activates NF-κB as shown in a mouse model of cerulein-induced AP. Docosahexaenoic acid (DHA), an ω-3 fatty acid, exerts anti-inflammatory and antioxidant effects in various cells and tissues. This study investigated whether DHA inhibits cerulein-induced AP in rats by assessing pancreatic edema, myeloperoxidase activity, levels of lipid peroxide and IL-6, activation of NF-κB and PKCδ, and by histologic observation. AP was induced by intraperitoneal injection (i.p.) of cerulein (50 μg/kg) every hour for 7 h. DHA (13 mg/kg) was administered i.p. for three days before AP induction. Pretreatment with DHA reduced cerulein-induced activation of NF-κB, PKCδ, and IL-6 in pancreatic tissues of rats. DHA suppressed pancreatic edema and decreased the abundance of lipid peroxide, myeloperoxidase activity, and inflammatory cell infiltration into the pancreatic tissues of cerulein-stimulated rats. Therefore, DHA may help prevent the development of pancreatitis by suppressing the activation of NF-κB and PKCδ, expression of IL-6, and oxidative damage to the pancreas.

Keywords: NF-κB; acute pancreatitis; docosahexaenoic acid; interleukin-6; protein kinase C δ.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effect of docosahexaenoic acid (DHA) on pancreatic edema in rats. The ratio of pancreas to body weight was used as an indicator of pancreatic edema. Values are mean ± S.E. for the 10 rats in each group. * p < 0.05 vs. untreated group (without cerulein), + p < 0.05 vs. cerulein group (cerulein alone).
Figure 2
Figure 2
Effect of DHA on the abundance of lipid peroxide (LPO) and activity of myeloperoxidase (MPO) in the pancreas. (A) The abundance of lipid peroxide is expressed as nmol/mg protein; (B) Myeloperoxidase activity is expressed as units/mg protein. Values are mean ± S.E. for the 10 rats in each group. * p < 0.05 vs. untreated group (without cerulein), + p < 0.05 vs. cerulein group (cerulein alone).
Figure 3
Figure 3
Effect of DHA on cerulein-induced histopathological changes in the pancreas. Images (AC) display representative examples of pancreatic tissues. (A) Normal pancreatic tissue is seen in the untreated group; (B) Abnormal architecture, including inflammatory cell infiltration (arrow) and edematous lesion, are observed in cerulein-treated group; (C) Reduced edematous lesions are observed in the group treated with cerulein and DHA. Hematoxylin & eosin (H&E) stain, magnification: 400×; Scale bar, 20 μm (magnification in each top right panel is 800×).
Figure 4
Figure 4
The effect of DHA on histological scores in pancreatic injury. H&E-stained sections were evaluated for edema, inflammatory cell infiltration, and necrosis of acinar cells. Values are mean ± S.E. for the 10 rats in each group. * p < 0.05 vs. untreated group (without cerulein); + p < 0.05 vs. cerulein group (cerulein alone).
Figure 5
Figure 5
The effect of DHA on the serum level of IL-6, and the levels of IL-6 mRNA and protein in the pancreas. (A) The level of IL-6 in the serum was determined using ELISA (B) RT-PCR was performed on reverse-transcribed RNA isolated from the pancreatic tissue. The mRNA level of IL-6 was normalized to that of GAPDH; (C) The protein level of IL-6 in the pancreas was determined using ELISA and expressed as pg/mg protein. Values are mean ± S.E. for the 10 rats in each group. * p < 0.05 vs. untreated group (without cerulein), + p < 0.05 vs. cerulein group (cerulein alone).
Figure 6
Figure 6
Effect of DHA on the levels of phospho-IκBα (p-IκBα), total IκBα, and NF-κB-DNA binding activity in the pancreas. (A) Western blotting was performed using antibodies against p-IκBα and total IκBα. Protein levels of p-IκBα and total IκBα were compared with that of the loading control actin and expressed as a percentage ratio of the band density; (B) Electrophoretic mobility shift assay (EMSA) for the activity of NF-κB was performed using nuclear extracts from pancreatic tissues. Each lane represents data for an individual animal. Values are mean ± S.E. for the 10 rats in each group. * p < 0.05 vs. untreated group (without cerulein), + p < 0.05 vs. cerulein group (cerulein alone).
Figure 6
Figure 6
Effect of DHA on the levels of phospho-IκBα (p-IκBα), total IκBα, and NF-κB-DNA binding activity in the pancreas. (A) Western blotting was performed using antibodies against p-IκBα and total IκBα. Protein levels of p-IκBα and total IκBα were compared with that of the loading control actin and expressed as a percentage ratio of the band density; (B) Electrophoretic mobility shift assay (EMSA) for the activity of NF-κB was performed using nuclear extracts from pancreatic tissues. Each lane represents data for an individual animal. Values are mean ± S.E. for the 10 rats in each group. * p < 0.05 vs. untreated group (without cerulein), + p < 0.05 vs. cerulein group (cerulein alone).
Figure 7
Figure 7
Effect of DHA on the level of PKCδ in the pancreas. Immunohistochemical analysis was performed using an anti-PKCδ antibody. (A) A scant signal for PKCδ was detected in the untreated group; (B) An intense signal for PKCδ was detected in the cerulein-treated group; (C) Few cells, positive for PKC-δ, were detected in the group treated with cerulein and DHA. Magnification: 200×.

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