LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease

Abstract

Background & aims: Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr(-/-) mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time.

Methods: APOE2ki and Ldlr(-/-) mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control.

Results: Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr(-/-) mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr(-/-) mice. Finally, bone-marrow-derived-macrophages of Ldlr(-/-) mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice.

Conclusion: Ldlr(-/-) mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr(-/-) mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis.

Figure 1. Parameters of hepatic inflammation.

(A) Liver sections were stained for infiltrated macrophages and neutrophils (Mac-1), neutrophils (NIMP) and T cells (CD3) and counted as cells/mm². (B–D) Representative pictures of Mac-1, NIMP and CD3 stained liver sections of control C57Bl6, APOE2ki and Ldlr^−/− mice after 3 months of HFC diet, respectively (×200 magnification). (E) Hepatic gene expression of tumor necrosis factor (Tnf), monocyte chemotactic protein 1 (Mcp-1) and Cd68. Dotted bars indicate the earlier time point of 7 days. *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively.

Figure 2. Parameters of hepatic steatotis.

(A+B) Liver cholesterol and triglycerides after chow and 3 months of HFC diet in C57Bl6, APOE2ki and Ldlr^−/− mice. (C–E) Representative pictures (×200 magnification) of the Oil red O staining after the chow diet and 3 months of the HFC diet in C57Bl6, APOE2ki and Ldlr^−/− mice.

Figure 3. Parameters of hepatic fibrosis.

(A–C) Representative pictures of (A) Sirius Red (magnification×100) and (B+C) ERTR7 (fibroblast marker) (magnification ×400, ×100, respectively) positive sections after 3 months on the HFC diet in C57Bl6, APOE2ki and Ldlr^−/− mice, respectively. (D) Hepatic hydroxyproline content of C57Bl6, APOE2ki and Ldlr^−/− mice after 3 months of chow and HFC diet. (E–I) Gene expression analysis of tumor growth factor beta (Tgf-β), Collagen 1a1 (Col1a1), metalloproteinase (Mmp-9), tissue inhibitor of metalloproteinase 1 (Timp1) and alpha smooth muscle actin (αSMA). Data were set relative to the C57Bl6 mice on the chow diet. * Significantly different from chow group. *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively.

Figure 4. Parameters of apoptosis.

(A) Scoring of TUNEL stained liver sections for apoptosis after chow and 3 months of HFC diet in C57Bl6, APOE2ki and Ldlr^−/− mice. (B) Caspase 3/7 activity in liver homogenates. (C–G) Gene expression analysis of the apoptotic genes Bax, Bcl-2, Traf1, Bfl1 and Chop. (H) Aminotransferase (ALT) levels in plasma. Data were set relative to the C57Bl6 mice on the chow diet. * Significantly different from chow group. *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively.

Figure 5. Cholesterol-related hepatic gene expression.

Gene expression analysis of Cd36, scavenger receptor A (Sr-a), lipoprotein lipase (Lpl), ATP binding cassette G1 (Abcg1) and A1 (Abca1) and peroxisome proliferator activated receptor (Ppar-γ) after chow and 3 months of HFC diet in C57Bl6, APOE2ki and Ldlr^−/− mice. Data were set relative to the C57Bl6 mice on the chow diet. * Significantly different from chow group. *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively.

Figure 6. Loading of bone marrow-derived macrophages with oxLDL.

(A) Bone marrow-derived macrophages (BMM) of C57Bl6, APOE2ki and Ldlr^−/− mice were loaded with oxidized low-density lipoprotein (oxLDL) and gene expression of tumor necrosis factor (Tnf), interleukin 6 (Il6), monocyte chemotactic protein 1 (Mcp-1), Cd68, Cd36 and scavenger receptor A (Sr-a) was analysed. Data were set relative to the basal levels of C57Bl6 bone marrow. (B) Percentage of positive BMM at basal levels and after oxLDL loading. (C) Positivity of BMM at basal levels and after oxLDL loading. * indicate p<0.05.