The cholesterol derivative 27-hydroxycholesterol reduces steatohepatitis in mice.

<p>BACKGROUND & AIMS: Non-alcoholic steatohepatitis is characterized by hepatic steatosis with inflammation. Although steatosis is benign and reversible, inflammation can increase liver damage. Hepatic inflammation has been associated with accumulation of cholesterol in lysosomes of Kupffer cells. 27-Hydroxycholesterol (27HC), a derivative of cholesterol formed by CYP27A1, can mobilize cholesterol from the lysosomes to the cytoplasm. We investigated whether 27HC can change the intracellular distribution cholesterol and reduce hepatic inflammation in mice. METHODS: We transplanted bone marrow from irradiated wild-type or Cyp27a1(-/-) mice to mice that do not express the low density lipoprotein receptor (Ldlr(-/-)), which are hyperlipidemic; 9 weeks later, mice were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 3 months. In a separate experiment, Ldlr(-/-) mice were given subcutaneous injections of 27HC and placed on regular chow or HFC diets for 3 weeks. Blood and liver tissues samples were collected and analyzed for intracellular cholesterol distribution and inflammation. RESULTS: In Ldlr(-/-) mice that received bone marrow transplants from Cyp27a1(-/-) mice, lysosomes of Kupfer cells had a greater accumulation of cholesterol than those of mice that received bone marrow from wild-type mice, after the HFC diet. Liver histology and gene expression analyses showed increased inflammation and liver damage in mice given bone marrow transplants from Cyp27a1(-/-) mice and placed on the HFC diet. Administration of 27HC to Ldlr(-/-) mice, following the HFC diet, reduced the accumulation of lysosomal cholesterol and hepatic inflammation, compared with mice that were not given 27HC. CONCLUSIONS: Accumulation of cholesterol in lysosomes of Kupfer cells promotes hepatic inflammation in mice. The cholesterol derivative 27HC reduces accumulation of cholesterol in lysosomes and might be used to treat non-alcoholic steatohepatitis.</p>

Protective Role of Plant Sterol and Stanol Esters in Liver Inflammation: Insights from Mice and Humans

<div><p>The inflammatory component of non–alcoholic steatohepatitis (NASH) can lead to irreversible liver damage. Therefore there is an urgent need to identify novel interventions to combat hepatic inflammation. In mice, omitting cholesterol from the diet reduced hepatic inflammation. Considering the effects of plant sterol/stanol esters on cholesterol metabolism, we hypothesized that plant sterol/stanol esters reduces hepatic inflammation. Indeed, adding plant sterol/stanol esters to a high-fat-diet reduced hepatic inflammation as indicated by immunohistochemical stainings and gene expression for inflammatory markers. Finally, adding sterol/stanol esters lowered hepatic concentrations of cholesterol precursors lathosterol and desmosterol in mice, which were highly elevated in the HFD group similarly as observed in severely obese patients with NASH. In vitro, in isolated LPS stimulated bone marrow derived macrophages desmosterol activated cholesterol efflux whereas sitostanol reduced inflammation. This highly interesting observation that plant sterol/stanol ester consumption leads to complete inhibition of HFD-induced liver inflammation opens new venues in the treatment and prevention of hepatic inflammation.</p></div

Effect of plant stanols on macrophages <i>in vitro</i>.

<p>Changes in Tnf-α concentrations in supernatant and LXR target gene expression of bone marrow derived macrophages after incubation with sitostanol (0.6 and 1.2 µm) or desmosterol (0.25, 0.5 and 1.0 µm) and 4 h LPS stimulation. (A) Tnf-α concentrations, (B) <i>LXRα</i> mRNA, (C) <i>Abca1</i> mRNA, and (D) <i>Abcg1</i> mRNA expression after sitostanol exposure, (E) Tnf-α mRNA, (F) <i>LXRα</i> mRNA, (G) <i>Abca1</i> mRNA, and (H) <i>Abcg1</i> mRNA expression after desmosterol exposure. Data were set relative to cells incubated with cyclodextrin (carrier control). *P<0.05; **P<0.01; ***P<0.001.</p

Hepatic non-cholesterol sterol concentrations.

<p>Hepatic concentrations of (A) campesterol, (B) sitosterol, (C) campestanol and (D) sitostanol were measured. To analyze endogenous cholesterol synthesis, hepatic (E) lathosterol and (F) desmosterol were measured. All values are shown as absolute concentrations (ng/mg tissue). n = 10 per group. *P<0.05, ***P<0.001.</p

Lipid Measurements.

<p>(A, B) Plasma and liver cholesterol measurements. (C, D) Plasma and liver triacylglycerol levels. (E) Scoring of liver slides for the accumulation of fat (steatosis) using HE staining. A score ranging from 0–3 (3 = highest steatosis) was given by an experienced pathologist. *P<0.05, **P<0.01, and ***P<0.001, respectively.</p

Plasma cholesterol precursor levels in severely obese patients.

<p>Serum levels of (A) desmosterol, (B) lathosterol and (C) cholestanol were measured in control (n = 20), NAFLD (n = 8) and NASH (n = 25) patients. All values are shown as absolute levels (mg/dl serum). *P<0.05.</p

Schematic representation of direct effects of sitostanol vs desmosterol.

<p>Schematic representation of direct effects of sitostanol vs desmosterol.</p

Hepatic gene expression.

<p>(A–E) Gene expression analysis of the macrophage marker <i>Cd68</i>, monocyte chemoattractant protein 1 (<i>Mcp-1</i>), interleukin 1β (<i>IL-1β</i>), tumor necrosis factor α (<i>Tnf-α</i>) and intercellular adhesion molecule 1 (<i>Icam</i>). Relative expression was normalized to endogenous control gene Cyclophilin A. Data were set relative to group on chow diet. n = 10 per group. *P<0.05, **P<0.01, and ***P<0.001, respectively.</p

Population characteristics.

<p>Data are represented as mean ± SEM.</p><p>*Significantly different from Normal (p<0.05).</p><p>Population characteristics.</p

Parameters of hepatic inflammation.

<p>(A, B) Liver sections were stained for infiltrating macrophages and neutrophils (Mac-1) and neutrophils (NIMP). From each liver, 6 random pictures were taken at 200x magnification to cover the whole slide. Positive cells for the specific staining were then counted being indicative for inflammation (C) Result of scoring for inflammation by an experienced pathologist using the HE staining in all groups. (D) Liver sections were stained for T-cells (CD3) and positive cells counted. (E, F) Representative pictures of Mac-1 staining and NIMP staining in the four experimental groups (200x magnification). *P<0.05, **P<0.01, and ***P<0.001, respectively.</p