Ethanol and dietary unsaturated fat (corn oil/linoleic acid enriched) cause intestinal inflammation and impaired intestinal barrier defense in mice chronically fed alcohol

Abstract

Alcohol and dietary fat both play an important role in alcohol-mediated multi-organ pathology, including gut and liver. In the present study we hypothesized that the combination of alcohol and dietary unsaturated fat (USF) would result in intestinal inflammatory stress and mucus layer alterations, thus contributing to disruption of intestinal barrier integrity. C57BL/6N mice were fed Lieber-DeCarli liquid diets containing EtOH and enriched in USF (corn oil/linoleic acid) or SF (medium chain triglycerides: beef tallow) for 8 weeks. Intestinal histology, morphometry, markers of inflammation, as well as levels of mucus protective factors were evaluated. Alcohol and dietary USF triggered an intestinal pro-inflammatory response, characterized by increase in Tnf-α, MCP1, and MPO activity. Further, alcohol and dietary USF, but not SF, resulted in alterations of the intestinal mucus layer, characterized by decreased expression of Muc2 in the ileum. A strong correlation was observed between down-regulation of the antimicrobial factor Cramp and increased Tnf-α mRNA. Therefore, dietary unsaturated fat (corn oil/LA enriched) is a significant contributing factor to EtOH-mediated intestinal inflammatory response and mucus layer alterations in rodents.

Figure 1. Evaluation of the intestinal pathology in response to ethanol and saturated fat (SF) or unsaturated fat (USF) diets

(A-B) Representative microphotographs of hematoxylin and eosin staining of the intestinal sections. Distal ileum from mice fed Lieber-DeCarli control and EtOH liquid diets for 8 weeks. Sections were analyzed using a light microscope (original magnification for A: x200, for B:x400). The mucosa appear normal in SF and USF groups (A-1, and A-3). Histological injury is noted in mice fed alcohol, characterized by shorten villi (A-2, and A-4). Inflammatory cell infiltrates (B-1) and dilation of blood vessels (B-2) were observed predominantly in the lamina propria of USF+EtOH group. Arrows indicate infiltration of lymphocytes and neutrophils, and dilated capillaries. (C-D) Morphometrical analysis of the ileal mucosa in response to ethanol and dietary fat. Ten complete villus-crypt junctions of the ileal segments of the individual mouse were measured, n=3 animals/per group. Values are mean+SEM. The value for the SF group was set at 1 as a control for comparison purposes. Statistical differences were analyzed by two-way ANOVA followed by the Tukey’s multiple comparison test. Means without a common letter differ at p<0.05.

Figure 2. Intestinal markers of inflammation in response to alcohol and saturated fat (SF) and unsaturated fat (USF) diets

(A and B) Tnf-α mRNA and protein levels. (C and D) Mcp1 mRNA and protein levels. The relative mRNA expression was measured by qRT-PCR. Values are mean+SEM, n=6 animals/per group. Cytokine protein levels were determined using MILLIPLEX Cytokine/Chemokine Panel kit (Millipore, Billerica, MA) on the Luminex100 IS System (Austin, TX). Values are mean+SEM, n=6–9 animals/per group. Statistical differences were analyzed by two-way ANOVA followed by the Tukey’s multiple comparison test. Means without a common letter differ at p<0.05.

Figure 3. Intestinal MPO activity in response to EtOH and SF and USF dietary fat

USF+EtOH significantly increased ileal MPO activity, a marker of inflammation and neutrophil infiltration, compared with SF+EtOH. Values are mean+SEM, n=5–6 animals/per group. Statistical differences were analyzed by two-way ANOVA followed by the Tukey’s multiple comparison test. Means without a common letter differ at p<0.05.

Figure 4. Effects of saturated fat (SF) or unsaturated fat (USF) on the intestinal mucus layer integrity in response to chronic alcohol feeding

(A) Quantitative analysis of intestinal Goblet cells. (B) Muc2 mRNA levels assessed by qRT-PCR. Values are mean+SEM, n=6 animals/per group. Statistical differences were analyzed by two-way ANOVA followed by the Tukey’s multiple comparison test. Means without a common letter differ at p<0.05.

Figure 5. Down-regulation of the intestinal mucosa antimicrobial peptide, Cramp, in response to ethanol and dietary unsaturated but not saturated fat

(A) Cramp mRNA levels assessed by qRT-PCR. Values are mean+SEM, n=6 animals/per group. Statistical differences were analyzed by two-way ANOVA followed by the Tukey’s multiple comparison test. Means without a common letter differ at p<0.05. (B) Negative correlation was observed between intestinal antimicrobial factor Cramp and intestinal Tnf-α mRNA levels.