Dissertation/Thesis Abstract

The Effect of Fish-Oils on the Hepatic Activity of BAMBI Following LPS-Induced Inflammation
by Schaller, Megan Lynn, M.S., Southern Illinois University at Edwardsville, 2018, 67; 10792198
Abstract (Summary)

Non-alcoholic steatohepatitis, defined as excess hepatic lipid and chronic inflammation, provides an environment prone for the development of hepatic fibrosis. Recent evidence suggests that the anti-fibrotic protein BAMBI is downregulated in the presence of inflammation due to an increased concentration of circulating endotoxins and may be central to the development of fibrosis. Diets rich in fish-oil based omega-3 fatty acids (ω-3 FA), eicosapentaenoic acid and docosahexaenoic acid, are known to provide anti-inflammatory effects; however, the effects of fish-oil based ω-3 FA on hepatic fibrosis are not well-established. Furthermore, the interaction between fish-oils and BAMBI activity during high-fat feeding remains to be elucidated. Therefore, the purpose of this study was to determine the effects of type and amount of dietary fat for 32 weeks on the hepatic expression of BAMBI and markers of hepatic inflammation using an LPS challenge model. We hypothesized that a fish-oil dietary intervention would reduce hepatic pro-inflammatory cytokine release and increase the hepatic expression of BAMBI following simulated chronic inflammation. To evaluate the hypotheses for this study, male C57BL/6 mice were randomly assigned to one of four diets (n = 9/group) for 32 weeks: low-fat lard based (LFL, 10% kcal fat), low-fat fish-oil based (LFFO, 10% kcal fat), high-fat lard based (HFL, 41% kcal fat), or high-fat fish-oil based (HFFO, 41% kcal fat). Following 32 weeks, hepatic tissue (100 mg) from each diet was incubated for 20 hours with and without LPS (25 µg/mL media) to mimic an in vivo hepatic inflammatory response to LPS. Hepatic mRNA expression was identified in control and LPS stimulated tissue using quantitative RT-PCR for genes associated with inflammation and fibrotic activity: CD14, TLR4, MyD88, BAMBI, TGF-β1, STAT3, IL-6, IL-1β0, TNF-α, IFN-γ, IL-1ββ, and MCP-1. In addition, the concentration of cytokines IL-1β, IL-6, IL-1β0, TNF-α, MCP-1, and IFN-γ in media were determined using a multiplex assay system. Protein abundance of BAMBI and its mediator, NF-κB p50, were measured using Western blot analysis. Following LPS stimulation, CD14 was 2.5-fold lower in HFFO when compared to HFL (p = 0.01). Despite the decrease in CD14, TLR4 showed no difference between groups in control and LPS stimulated tissues. In contrast, MyD88 expression was 4- and 3-fold greater in control and LPS stimulated HFFO, respectively, when compared to HFL (p = 0.002). In control condition, BAMBI mRNA expression was 1.8-fold higher in HFFO (p = 0.01) than all other groups. However, expression of TGF-β1 in control and LPS stimulated HFFO was significantly greater than HFL (p = 0.02). While both extracellular CD14 and intracellular MyD88 were influenced by HFFO feeding, no differences were observed in TLR4 expression between groups. BAMBI mRNA expression was elevated with fish-oil supplementation but only in the control condition. Protein abundance of BAMBI showed no differences among diet groups, despite lower abundance of NF-κB p50 in HFFO when compared to HFL (p = 0.029). In samples without LPS, IL-6 and MCP-1 expression was significantly lower in the HFFO diet group when compared to HFL (p = 0.036, 0.006). Control cytokine concentration in media of IL-6 and MCP-1 showed no differences between high-fat groups. When comparing fat type in samples without LPS, IL-1β0 and IFN-γ expression were 3.8-fold (p = 0.001) and 5.6-fold higher (p = 0.001), respectively, in HFFO with no difference and 1.5-fold (p = 0.009) lower media concentrations compared to the HFL diet. Following LPS stimulation, IL-6 mRNA expression was lower in HFFO when compared to HFL and was 5.4-fold lower (p = 0.014) in HFFO media concentration. In contrast to samples without LPS, LPS stimulation showed no differences in mRNA expression or media concentration of IL-1β0, IFN-γ, and MCP-1 between high-fat diet groups. When comparing fat amount following LPS, secretion of IFN-γ and MCP-1 were lower (p < 0.05) in the high-fat diet groups when compared to LFFO, despite no differences in gene expression of these cytokines. The higher basal (untreated LPS samples) cytokine gene expression and secretion of IL-6 in HFFO alone showed that IL-6 response is dependent to both fat amount and fat type. In contrast, IFN-γ and MCP-1 expression were more sensitive to fat amount versus type of fat consumed, exemplified by lower cytokine activities post-LPS stimulation. In the control condition, we observed a disconnect between tissue expression and media concentration for IL-1β and TNF-α, whereas fish-oils had lower expression and media of IL-6 and MCP-1. Fish-oils protected against LPS stimulation as observed by lower media concentration of IL-1β, IL-6, and MCP-1. Overall, the consumption of fish-oils partially restricted LPS inflammatory signal progression across the cell-membrane into the hepatocyte. (Abstract shortened by ProQuest.)

Indexing (document details)
Advisor: Wooten, Joshua S.
Commitee: Guilford, Brianne L., Witt, Ken A.
School: Southern Illinois University at Edwardsville
Department: Kinesiology and Health Education
School Location: United States -- Illinois
Source: MAI 57/06M(E), Masters Abstracts International
Source Type: DISSERTATION
Subjects: Pathology, Kinesiology, Physiology
Keywords: BAMBI, Fish-oils, Lipopolysaccharide, NAFLD
Publication Number: 10792198
ISBN: 9780438009370
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