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, 10, 1743-51
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Sulfasalazine Inhibits Inflammation and Fibrogenesis in Pancreas via NF-κB Signaling Pathway in Rats With Oxidative Stress-Induced Pancreatic Injury

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Sulfasalazine Inhibits Inflammation and Fibrogenesis in Pancreas via NF-κB Signaling Pathway in Rats With Oxidative Stress-Induced Pancreatic Injury

Ya-Ru Wang et al. Drug Des Devel Ther.

Abstract

Background: Pathogenesis and effective therapeutics of chronic pancreatic inflammation and fibrosis remain uncertain.

Purpose: To investigate the effects of sulfasalazine (SF) on pancreatic inflammation and fibrogenesis.

Methods: Chronic pancreatic injury in rats was induced by diethyldithiocarbamate (DDC) and interfered by SF through intraperitoneal injection. The rats were divided into five groups: group N, normal control group, rats were treated with dilated water only; group DS1, rats received SF (10 mg/kg) 2 hours before DDC treatment; group DS2, rats were treated with DDC and then SF (100 mg/kg, twice a week); group DS3, rats were treated with DDC, then SF (100 mg/kg, thrice a week); and group DDC, rats were treated with DDC only. Pancreatic inflammation and fibrosis were determined by hematoxylin and eosin staining and Sirius red staining. The genes and proteins related to NF-κB pathway and fibrogenesis including NF-κB/p65, TNF-α, ICAM-1, α-SMA, and Con 1 were detected by immunohistochemical staining, reverse transcription polymerase chain reaction, and Western blotting.

Results: Rats in the DDC and DS1 groups showed the highest histological scores after DDC treatment, but the scores of DS2 and DS3 groups decreased significantly when compared with the DDC group. Sirius red staining showed collagen formation clearly in DDC and DS1 rats rather than in DS2 and DS3 rats. NF-κB/p65, ICAM-1, and α-SMA were strongly expressed in DDC and DS1 rats, while DS2 and DS3 rats showed mild to moderate expression by immunohistochemistry. Reverse transcription polymerase chain reaction showed increased levels of NF-κB/p65, ICAM-1, TNF-α, α-SMA, and Con 1 mRNA in DDC and DS1 rats in comparison to normal controls. The mRNA levels of these molecules in DS2 and DS3 rats were significantly lower than those in DS1 and DDC rats. Western blotting demonstrated that the NF-κB/p65, ICAM-1, and α-SMA expressions in pancreatic tissues of the rats of the DDC group were more clear than those of the normal control, DS2, and DS3 rats.

Conclusion: SF inhibits pancreatic inflammation and fibrogenesis via NF-κB signaling pathway.

Keywords: NF-κB; fibrogenesis; inflammation; pancreatic injury; sulfasalazine.

Figures

Figure 1
Figure 1
Pancreatic histological alterations (A) and histological scores (B). Notes: (A, H&E staining, original magnification 200×) Representative histological changes in pancreas in normal group (a), group DS1 (b), DS2 (c), DS3 (d), and DDC (e). Quantitative analysis (B) demonstrates different pancreatic histological alterations in rats with different sulfasalazine (SF) treatments in different groups. Group N, normal control group, rats were treated with dilated water only; DS1, rats received SF (10 mg/kg) 2 hours before DDC treatment; group DS2, rats were treated with DDC and then SF (100 mg/kg, twice a week); group DS3, rats were treated with DDC, then SF (100 mg/kg, thrice a week); and group DDC, rats were treated with DDC only. Abbreviations: H&E, hematoxylin and eosin; DDC, diethyldithiocarbamate.
Figure 2
Figure 2
Fibrogenesis in pancreas and the corresponding statistical analysis. Notes: Collagen deposition was demonstrated by Sirus red staining and observed under common light (A, original magnification 200×) and polarized microscopes (B, original magnification 200×) in normal group (a), group DS1 (b), DS2 (c), DS3 (d), and DDC (e). Under polarization microscope, collagen appears bright orange-red and/or bright green. Quantitative analysis (C) demonstrates different levels of pancreatic fibrogenesis in rats with different sulfasalazine (SF) treatments in different groups. Group N, normal control group, rats were treated with dilated water only; DS1, rats received SF (10 mg/kg) 2 hours before DDC treatment; group DS2, rats were treated with DDC and then SF (100 mg/kg, twice a week); group DS3, rats were treated with DDC, then SF (100 mg/kg, thrice a week); and group DDC, rats were treated with DDC only. Abbreviation: DDC, diethyldithiocarbamate.
Figure 3
Figure 3
Immunohistochemistry staining for NF-κB/p65, ICAM-1, and α-SMA and the corresponding statistical analysis. Notes: NF-κB/p65 (A, original magnification 200×), ICAM-1 (B, original magnification 200×), and α-SMA (C, original magnification 200×) expression in normal group (a), group DS1 (b), DS2 (c), DS3 (d), and DDC (e). Quantitative analysis (D) demonstrates different levels of pancreatic fibrogenesis in rats with different sulfasalazine (SF) treatment in different groups. Group N, normal control group, rats were treated with dilated water only; DS1, rats received SF (10 mg/kg) 2 hours before DDC treatment; group DS2, rats were treated with DDC and then SF (100 mg/kg, twice a week); group DS3, rats were treated with DDC, then SF (100 mg/kg, thrice a week); and group DDC, rats were treated with DDC only. Abbreviations: DDC, diethyldithiocarbamate; IOD, integrated optical density.
Figure 4
Figure 4
Agarose gel electrophoresis for PCR products and the corresponding statistical analysis. Notes: Genes (A) NF-κB/p65 (mark: 100, 250–2,000 bp); (B) ICAM-1 (mark: 100–600 bp); (C) TNF-α (mark: 100–600 bp); (D) α-SMA (mark: 100–600 bp); (E), Con 1 (mark: 100, 250–2,000 bp); (F) actin (mark: 100–600 bp); groups 1, N; 2, DS1; 3, DS2; 4, DS3; 5, DDC; and (G) the statistical analyses of mRNA levels between different groups with different sulfasalazine (SF) treatments. Group N, normal control group, rats were treated with dilated water only; DS1, rats received SF (10 mg/kg) 2 hours before DDC treatment; group DS2, rats were treated with DDC and then SF (100 mg/kg, twice a week); group DS3, rats were treated with DDC, then SF (100 mg/kg, thrice a week); and group DDC, rats were treated with DDC only. Abbreviations: PCR, polymerase chain reaction; DDC, diethyldithiocarbamate; OD, optical density; mark, marker.
Figure 5
Figure 5
Western Blotting for detection of protein expression and the corresponding statistical analyses. Notes: Proteins (A) NF-κB/p65 (65 kDa); (B) ICAM-1 (85 kDa); (C) α-SMA (42 kDa); group 1, DS3; 2, DS2; 3, DS1; 4, N; 5, DDC. (D) Statistical analyses show differences in the expression of NF-κB/p65, ICAM-1, and α-SMA in different groups with different sulfasalazine (SF) treatments. Group N, normal control group, rats were treated with dilated water only; DS1, rats received SF (10 mg/kg) 2 hours before DDC treatment; group DS2, rats were treated with DDC and then SF (100 mg/kg, twice a week); group DS3, rats were treated with DDC, then SF (100 mg/kg, thrice a week); and group DDC, rats were treated with DDC only. Abbreviations: DDC, diethyldithiocarbamate; OD, optical density.

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