Intest Res.  2014 Jul;12(3):205-213. 10.5217/ir.2014.12.3.205.

Expression of Heat Shock Proteins and Cytokines in Response to Ethanol Induced Damage in the Small Intestine of ICR Mice

• Lee SW
• Choi DW
• Park SC
• Kim HJ
• Nam YH
• Choi DH
• Kang CD
• Lee SJ
• Chun WJ
• Ryu YJ

• KMID: 2174379
• DOI: http://doi.org/10.5217/ir.2014.12.3.205

Abstract

BACKGROUND/AIMS
Ethanol administration causes intestinal epithelial cell damage by increasing intestinal permeability and the translocation of endotoxins from intestinal bacterial flora. Heat shock proteins (HSPs) are associated with recovery and protection from cell damage. The aim of the current study was to investigate differences in the expression of HSPs in the small intestine and the biochemical changes attributable to ethanol-induced intestinal damage.
METHODS
Ethanol (20%) was injected intraperitoneally (2.75 g/kg, 5.5 g/kg, 8.25 g/kg) in ICR mice and the same volume of saline was administered to controls. After 1 hour, the proximal, middle, and distal segments were taken from the small intestine and the degree of damage was analyzed. In each segment, the expression of HSPs was analyzed by western blotting. The expression of inflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), and antioxidant enzyme such as glutathione-S-transferase were compared using real-time polymerase chain reaction assays.
RESULTS
In the control group, HSP70 increased in all segments of small intestine. Additionally, increases in the expression of HSP40 and HSP90 in the distal regions and an increase in HSP32 in the middle regions were observed. After ethanol treatment, greater histological damage was observed in the distal small intestine and significant decreases in HSPs were observed generally. Increased expression of IL-1beta, TNF-alpha, and COX-2 was observed in small intestinal tissues exposed to ethanol-induced damage. However, there was no significant difference in the expression of an antioxidant enzyme.
CONCLUSIONS
Significant differences in the expression of HSPs in different intestinal regions were observed. These differences may have been attributable to the distribution of intestinal bacteria.

Keyword

Intestine, small; Heat shock protein; Ethanol

MeSH Terms

Animals
Bacteria
Blotting, Western
Cyclooxygenase 2
Cytokines*
Endotoxins
Epithelial Cells
Ethanol*
Heat-Shock Proteins*
Interleukin-1beta
Intestine, Small*
Mice
Mice, Inbred ICR*
Permeability
Real-Time Polymerase Chain Reaction
Tumor Necrosis Factor-alpha
Cyclooxygenase 2
Cytokines
Endotoxins
Ethanol
Heat-Shock Proteins
Interleukin-1beta
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Pathology of the small intestinal mucosa and muscle induced by treatment with saline or ethanol (2.75 g/kg, 5.5 g/kg, 8.25 g/kg) (H&E, ×100). In saline-treated mice, villi were intact and normal lymphoid follicles were observed without infiltration of inflammatory cells. In mice treated with 2.75 g/kg of ethanol, focal falling off of villi and an edematous glandular layer were observed. These changes were more severe in the distal part with villi damage. In the group treated with 5.5 g/kg of ethanol, mulfifocal villi elimination, glandular layer destruction, erosions, and ulcers were observed with a severe aspect in the distal part. In mice treated with 8.25 g/kg of ethanol, diffuse damage extended to serosa, severe ulcers, and irregular expansion of lumen due to ischemic changes.

• 

  Fig. 2 Comparison of the differences in expression of heat shock proteins (HSPs) in the small intestine. (A) HSP70, (B) HSP90, (C) HSP40, and (D) HSP32. The upper part of each figure shows the representative bands indicative of changes in the expression of HSPs by western blot analysis and the lower part shows a quantitatively obtained graph from the western blot analysis that was repeated 3 times. All results were obtained with 3 independent experiments and values are displayed as mean±SD. * and † indicate P<0.05. ‡ and § indicate P<0.01. P, proximal small intestine; M, middle small intestine; D, distal small intestine.

• 

  Fig. 3 Comparison of the differences in the protein expression of (A) glutathione-S-transferase (GST) and (B) mRNA expression of interleukin-1β (IL-1β), (C) tumor necrosis factor-α (TNF-α), and (D) cyclooxygenase-2 (COX-2). Figure 3A shows the representative band indicative of changes in the expression of GST by western blot analysis. Figure 3B, C, and D show results obtained by extracting mRNA via reverse transcription-PCR assay to measure the expression of each gene. All results were obtained with 3 independent experiments and values are displayed as mean±SD. * and † indicate P<0.05. ‡ and § indicate P<0.01. P, proximal small intestine; M, middle small intestine; D, distal small intestine.

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