Cholesterol-Induced Non-Alcoholic Fatty Liver Disease and Atherosclerosis Aggravated by Systemic Inflammation

<div><p>Although triglyceride accumulation in the liver causes non-alcoholic fatty liver disease (NAFLD), hypercholesterolemia is also a main cause of NAFLD as well as atherosclerosis. However, NAFLD and atherosclerosis have not been investigated simultaneously in animal models fed a high-cholesterol diet. Moreover, it is unclear whether systemic inflammation can exacerbate both pathologies in the same model. Accordingly, this study investigated the effect of additional systemic inflammation on NAFLD and atherosclerosis induced by cholesterol overload in wild animals. New Zealand white rabbits were divided into 4 groups: groups I (control) and II received normal chow, and groups III and IV received a 1% cholesterol diet. To induce inflammation via toll-like receptor (TLR)-4 signaling, groups II and IV received subcutaneous injections of 0.5 mL of 1% carrageenan every 3 weeks. After 3 months, total cholesterol markedly increased in groups III and IV, and the serum expressions of systemic inflammatory markers were elevated in the groups II–IV. Early NAFLD lesions (e.g., mild fatty changes in the liver with sporadic fibrosis) and atherosclerosis (e.g., intimal hyperplasia composed of foam cells) were observed in both the liver and aorta specimens from group III, and advanced lesions were observed in group IV. The expressions of inflammatory cellular receptors, TLR-2 and TLR-4, in the aorta gradually increased from group I to IV but were similar in the liver in groups II–IV. Cholesteryl ester (CE) levels were higher in group IV than in group III, although the difference was not significant. CE levels in the aorta were similar between groups III and IV. Systemic inflammation can simultaneously exacerbate existing early lesions due to cholesterol overload in both the liver and aorta of rabbits. However, the cellular response of inflammatory receptors and expression of cholesterol metabolites differ between these organs.</p></div

Levels of serum inflammatory cytokines and reactants, and white blood cell count in peripheral blood.

<p><i>p</i>-values represent overall differences among groups as determined by the Kruskal–Wallis test.</p>a–c<p>The same letters indicate no statistical significance according to the Mann–Whitney <i>U</i>-test.</p><p>CGN, carrageenan; CHO, cholesterol; IL, interleukin; TNF, tumor necrosis factor; CRP, C-reactive protein, WBC, white blood cells.</p

Levels of serum lipids and biochemical parameters in the 4 groups.

<p><i>p</i>-values represent overall differences among groups as determined by the Kruskal–Wallis test.</p>a,b<p>The same letters indicate no statistical significance according to the Mann–Whitney <i>U</i>-test.</p><p>CGN, carrageenan; CHO, cholesterol; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase.</p

Representative histopathologic features of rabbit liver sections.

<p>(A, E, I, M) Hematoxylin–eosin stain, ×40 and (B, F, J, N) ×100′. (C, G, K, O) Masson trichrome stain (×40). (D, H, L, P) Sirius red stain (×40). (A–D) Group I (control) exhibited no fatty changes or fibrosis. (E, F) Liver sections from group II (carrageenan injection) showed no remarkable fatty changes, but mild portal expansion and collagen deposition were observed in sections stained with (G) Masson's trichrome and (H) Sirius red. (I–L) In group III (cholesterol diet), macro- and microvesicular fatty changes were widely observed; the portal areas were widened with sporadic portal–portal fibrosis and portal–central fibrosis. (M–P) Group IV (carrageenan injection and cholesterol diet) exhibited marked macro- and microvesicular fatty changes with frequent portal–portal and portal–central fibrosis. Collagen deposition around fibrotic areas is visualized as red color by Sirius red staining.</p

Correlations among log CRP and cytokines.

<p>Data are Spearman's correlation coefficients, <i>r</i>, with (<i>P</i>-values).</p><p>CRP, C-reactive protein; IL, interleukin; TNF, tumor necrosis factor.</p

Cholesterol levels in serum, the liver, and the aorta.

<p>Concentrations of cholesterol, cholesteryl esters, and phytosterols are expressed in µg/mL or µg/g; concentrations of cholesterol precursors and hydroxycholesterols are expressed in ng/mL or ng/g.</p><p>I, Group I; II, Group II; III, Group III; IV, Group IV; I–II, Group I versus Group II; I–III, Group I versus Group III; I–IV, Group I versus Group IV; II–IIV, Group II versus Group IV; III–IV, Group III versus Group IV; NS^a, not significant; ND^b, not detected; NC^c, not comparable; CGN, carrageenan; CHO, cholesterol; CON, control.</p

Examples of lipoprotein electrophoresis gels from each group.

<p>Groups I (control) and II (carrageenan injection) exhibited normal patterns, and groups III (cholesterol diet) and IV (carrageenan injection and cholesterol diet) exhibited type IV phenotype according to the Fredrickson-Levy-Lees classification. The result shows that inflammation did not affect the lipoprotein phenotype in blood.</p