Astaxanthin prevents ischemia-reperfusion injury of the steatotic liver in mice

<div><p>Steatosis has a low tolerance against ischemia-reperfusion injury (IRI). To prevent IRI in the steatotic liver, we attempted to elucidate the protective effect of astaxanthin (ASTX) in the steatotic liver model by giving mice a methionine and choline-deficient high fat (MCDHF) diet. Levels of lipid peroxidation and apoptosis, the expression of inflammatory cytokines and heme oxygenase (HO)-1, in the liver were assessed. Reactive oxygen species (ROS), inflammatory cytokines, apoptosis-related proteins and members of the signaling pathway were also examined in isolated Kupffer cells and/or hepatocytes from the steatotic liver. ASTX decreased serum ALT and AST levels, the amount of TUNEL, F4/80, or 4HNE-positive cells and the mRNA levels of inflammatory cytokines in MCDHF mice by IRI. Moreover, HO-1 and HIF-1α, phosphorylation of Akt and mTOR expressions were increased by ASTX. The inflammatory cytokines produced by Kupffer, which were subjected to hypoxia and reoxygenation (HR), were inhibited by ASTX. Expressions of Bcl-2, HO-1 and Nrf2 in hepatocytes by HR were increased, whereas Caspases activation, Bax and phosphorylation of ERK, MAPK, and JNK were suppressed by ASTX. Pretreatment with ASTX has a protective effect and is a safe therapeutic treatment for IRI, including for liver transplantation of the steatotic liver.</p></div

ASTX attenuated necrosis, infiltration of macrophages, oxidant stress and apoptosis as well as the biochemical parameters related to IR injury in the steatotic liver.

<p>(A) The necrotic cells, TUNEL-positive cells, F4/80-positive cells and 4-HNE-positive cells in MCDHF diet, IR and ASTX treatment group were indicated arrows. Scale bars represent 200μm, respectively. (B) The number of necrotic areas, TUNEL-positive cells, F4/80-positive cells and 4-HNE positive cells were counted. Data are expressed as the means ± SEM; *p < 0.05.</p

ASTX decreased the inflammatory cytokines expression and the biochemical parameters, but increased the HO-1 and HIF-1α expression in the steatotic liver under IR injury.

<p>(A) The serum ALT and AST levels of 15 minutes ischemia and 3 hours reperfusion. (B) The mRNA expression of IL-6, osteopontin (OPN), inducible nitric oxide synthase (iNOS), HO-1 and in HIF-1α in different groups. (Data are representative of 4~7 animals for each group). Data are expressed as the means ± SEM; *p < 0.05.</p

ASTX prevented steatotic primary hepatocyte apoptosis when hepatocytes subjected to HR.

<p>(A) Expressions of Bax, Bcl-2, cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3 of steatotic primary hepatocyte by hypoxia/reoxygenation injury were detected by western blot analysis. Representative blot (left) and quantified protein levels (right) are shown(n = 3). (B) Protein expression of p38 MAPK, p-p38 MAPK, ERK, p-ERK, JNK and p-JNK was detected by western blots(n = 3). α-tubulin was used as a loading control. Representative blot (up) and quantified protein levels (down) are shown. Data are expressed as the means ± SEM; *p< 0.05, **p< 0.01. Data are representative of three independent experiments and indicate the mean ratio of triplicate results from each experiment.</p

Sample of a porous titanium alloy rod and gross specimens at 3 months after treatment.

<p>Blue arrowheads indicate treatment sites.</p

Schematic illustration of experimental design.

<p>Schematic illustration of experimental design.</p

Quantitative analysis of the parameters for reconstructed trabeculae, as calculated by micro-CT.

<p>Quantitative analysis of the parameters for reconstructed trabeculae, as calculated by micro-CT.</p

Section design for histology.

<p>Sections A, B, and C were prepared for staining.</p

The 3-D reconstruction of talar specimens performed by micro-CT.

<p>Quantitative study showed that the trabecular reconstruction of the IM group was prior to that of the DC group at each time point.</p

Percentages of reconstructed trabeculae in the ROIs of the two groups by histological study (<i>n</i> = 6, x ± s).

<p>Percentages of reconstructed trabeculae in the ROIs of the two groups by histological study (<i>n</i> = 6, x ± s).</p