A medium-chain fatty acid analogue prevents endotoxin liver injury in a murine model

Abstract

Parenteral nutrition (PN) is lifesaving for patients with short bowel syndrome and other gastrointestinal disorders, however long-term use may lead to complications including hepatosteatosis and sepsis. We have previously demonstrated the anti-steatotic, -fibrotic, and -inflammatory properties of SEFA-6179, an engineered medium-chain fatty acid analogue. We hypothesized that SEFA-6179 treatment would protect against endotoxin-induced liver injury in a murine model of PN-induced hepatosteatosis. C57Bl/6J mice were administered a high-carbohydrate liquid diet plus intravenous lipid emulsion (Intralipid, 4 g fat/kg/d) or intravenous saline for 19 days to induce hepatosteatosis. SEFA-6179 (100 mg/kg) or vehicle (MCT/medium-chain triglyceride) was administered via oral gavage for four days leading up to intraperitoneal challenge with lipopolysaccharide (15 mg/kg) or saline on day 19. Age-matched, chow-fed controls received the same treatments. The primary outcome was liver biomarkers: alanine aminotransferase and aspartate aminotransferase. Pro-inflammatory cytokines, IL-6, TNF-alpha, and monocyte chemoattractant protein (MCP1), were analyzed. Liver immunofluorescence staining was performed to evaluate macrophage phenotypes. In endotoxin-challenged mice, pre-treatment with SEFA-6179 lowered liver enzymes and pro-inflammatory cytokine levels compared to vehicle. On liver histology, SEFA-6179 pre-treatment led to greater polarization of M1/pro-inflammatory macrophages to an M2/anti-inflammatory phenotype compared to vehicle. SEFA-6179 is currently in Phase II clinical trials. These findings support the potential application of SEFA-6179 in high-risk, PN-dependent patients.

Keywords: Fatty acid; Inflammation; Liver steatosis; Parenteral nutrition.

Fig. 1

Experimental design investigating an engineered medium-chain fatty acid analogue, SEFA-6179, in a murine model of PN-associated liver disease and endotoxin challenge. Mice were randomized to parenteral nutrition-equivalent intake or standard rodent chow. Mice received various intravenous (saline or soybean oil-based lipid emulsion i.e. Intralipid), oral gavage (MCT or SEFA-6179), and intraperitoneal (saline or LPS) treatments. A total of n = 70 C57Bl/6J mice were utilized (n = 5 per group, total of 14 groups). MCT, medium-chain triglyceride; SEFA-6179, structurally engineered fatty acid-6179; LPS, lipopolysaccharide (E. coli).

Fig. 2

(A) Body weights, (B) Parenteral nutrition consumption, and (C) Normalized organ weights of liver, spleen, and kidney by treatment group over the course of a 19-day experiment involving a murine model of parenteral nutrition-associated liver disease. For (A) and (B), treatment groups are named in the following order: ad libitum oral intake (PN vs. chow), intravenous injection (saline vs. Intralipid), oral gavage (MCT vs. SEFA), and intraperitoneal treatment (saline vs. LPS). Mean ± Standard Error of Mean. PN, parenteral nutrition; SEFA, structurally engineered fatty acid (i.e. SEFA-6179); MCT, medium-chain triglyceride; LPS, lipopolysaccharide.

Fig. 3

Representative liver histology in a murine model of PN-associated liver disease. (A) Standard H&E stain demonstrates evidence of steatosis such as hepatocyte ballooning. (B) Oil Red O stain demonstrates areas of fat and triglyceride deposition as orange-red in color. 40X magnification, Bar = 60uM. MCT, medium-chain triglyceride; PN, parenteral nutrition; SEFA, structurally engineered fatty acid (i.e. SEFA-6179).

Fig. 4

Plasma liver enzyme concentrations including (A) alanine aminotransferase and (B) aspartate aminotransferase in a murine model of parenteral nutrition-associated liver disease for PN + Saline, PN + Intralipid, and Chow-Fed Groups. Values expressed are as the mean ± standard error of the mean. ALT, alanine aminotransferase; AST, aspartate aminotransferase; PN, parenteral nutrition; MCT, medium-chain triglyceride; LPS, lipopolysaccharide; SEFA, structurally engineered fatty acid (i.e. SEFA-6179); OG, oral gavage; IP, intraperitoneal; whereby * indicates P < 0.05; ** indicates P < 0.01, *** indicates P < 0.001 on unpaired Student t-test.

Fig. 5

Pro-inflammatory cytokine levels in a murine model of parenteral nutrition-associated liver disease for PN + Saline, PN + Intralipid, and Chow-Fed Groups. (A) Serum IL-6 Levels, (B) Liver Lysate TNF-alpha Levels, (C) Liver Lysate MCP1 Levels. Mean ± Standard Error of Mean. PN, parenteral nutrition; ALT, alanine aminotransferase; AST, aspartate aminotransferase; MCT, medium-chain triglyceride; LPS, lipopolysaccharide; SEFA, structurally engineered fatty acid (i.e. SEFA-6179); OG, oral gavage; IP, intraperitoneal; IL-6, interleukin-6; TNF-alpha, tumor necrosis factor-alpha, MCP1, monocyte chemoattractant protein-1, whereby * indicates P < 0.05; ** indicates P < 0.01, *** indicates P < 0.001 on unpaired Student t-test.

Fig. 6

(A) Representative immunofluorescence stains of liver specimens in a murine model of PN-induced hepatosteatosis and endotoxin-driven inflammation. M1/pro-inflammatory macrophages were stained with anti-CD80 (pink, 1:100), M2/anti-inflammatory macrophages were stained with anti-CD206 (green, 1:100), nuclear material was stained with DAPI as a control (blue). Bright field microscopy at 40X magnification, Bar = 100 uM. (B) Ratio of M1 to M2 macrophages quantified using ImageJ software according to published protocol. MCT, medium-chain triglyceride; LPS, lipopolysaccharide; SEFA, structurally engineered fatty acid (i.e. SEFA-6179); OG, oral gavage; IP, intraperitoneal, whereby * indicates P < 0.05 on unpaired Student t-test.