Root bark of <i>Ulmus davidiana var</i>. <i>japonica</i> restrains acute alcohol-induced hepatic steatosis onset in mice by inhibiting ROS accumulation

<div><p>Alcohol-induced hepatic steatosis and inflammation are key drivers of alcohol-induced liver injury, mainly caused by oxidative stress. The roots bark of <i>Ulmus davidiana var</i>. <i>japonica</i> is well known for its substantial antioxidative and antitumorigenic potency. In this study, we examined whether this plant can ameliorate alcohol-induced liver injuries characterized by hepatic steatosis and inflammation through its antioxidative activity. C57BL/6J mice were treated with the root bark extract of <i>Ulmus davidiana var</i>. <i>japonica</i> (RUE; 100 mg of extract/kg bodyweight; oral gavage) and alcohol (1 g/kg of bodyweight; oral gavage) for 5 days. Markers of acute alcohol-induced hepatic steatosis were determined and putative molecular mechanisms responsible for the protection of RUE were investigated. RUE noticeably protected against alcohol-induced hepatic steatosis and inflammation. Reactive oxygen species (ROS), over-produced by alcohol, negatively orchestrated various signaling pathways involved in the lipid metabolism and inflammation. These pathways were restored through the ROS scavenging activity of RUE in the liver. In particular, the expression of lipogenic genes (e.g., SREBP-1, ACC, and FAS) and inflammatory cytokines (e.g., IL-1β, and NF-κB p65) significantly decreased with RUE treatment. Conversely, the expression of fatty acid oxidation-related genes (e.g., SIRT1, AMPKα, and PGC1α) were increased in mice treated with RUE. Thus, the results indicate that RUE counteracts and thus attenuates alcoholic hepatic steatosis onset in mice, possibly by suppressing ROS-mediated steatosis and inflammation.</p></div

RUE decreases expression of inflammatory cytokines through MAPK-NF-κB pathway.

<p>(A) Inflammatory cytokines levels in serum were analyzed using mouse cytokine antibody array. A graph depicting the mean pixel density of dot blots; (B) Immunoblot analyses of inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-18) in the liver tissue with the loading control CyPB. All data are presented as the mean ± SEM. (*<i>P</i> < 0.05 vs. control); (C) Immunoblot analyses of p38 and JNK MAPK-NF-κB inflammatory pathway. Actin, non-phosphorylated p65, p38, and JNK were examined as the loading controls, and a graph depicting the quantification of the relative abundance of the genes is shown. All data are presented as the mean ± SEM. (*<i>P</i> < 0.05; **<i>P</i><0.01; ***<i>P</i><0.001 vs. control). Not detected bands were indicated as ND in the quantifications; (D) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in the liver tissue (200× magnification). TUNEL-positive cells were detected by immunofluorescence analysis. Histograms represent the quantification of fluorescein isothiocyanate-labeled nucleus over the total number of nucleus. Data are presented as the mean ± SEM. **<i>P</i><0.01 vs. control); (E) Immunoblot analysis of p53 with the loading control CyPB. Data are presented as the mean ± SEM. (*<i>P</i> < 0.05 vs. control). ¹RQ, relative quantity.</p

RUE decreases the level of alcohol-induced liver damage markers.

<p>(A) Representative images of hematoxylin & eosin (H&E) staining of liver (200× magnification); (B) Levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum. All data are presented as the mean ± SEM. (**<i>P</i> < 0.01 vs. control).</p

RUE attenuates alcohol-induced hepatic steatosis.

<p>(A) Representative images of boron-dipyrromethene (BODIPY) staining of lipid droplets in the liver tissue (200× magnification); (B) Levels of triglyceride (TG) accumulation in serum and liver tissue. All data are presented as the mean ± SEM. (*<i>P</i> < 0.05 vs. control); (C) Immunoblot analyses of lipogenic genes (SREBP-1, ACC, and FAS) in liver tissue with the loading control CyPB. All data are presented as the mean ± SEM. (*<i>P</i> < 0.05 vs. control); (D) Immunoblot analyses of fatty acid oxidative genes (SIRT1, AMPK-α, PGC1-α, and CPT1) with the loading control CyPB. All data are presented as the mean ± SEM. (*<i>P</i> < 0.05 vs. control) ¹RQ, relative quantity.</p

RUE attenuates alcohol-induced oxidative damages through its antioxidant activity and proposed molecular pathway in liver.

<p>(A) Representative images of 3,3’-diaminobenzidine (DAB) and nitro-tyrosine in the liver tissue (40× magnification). DAB and nitro-tyrosine were used to measure the level of reactive oxygen species (ROS) and reactive nitrogen species, respectively; (B) Representative images of DNA damages measured by Avidin-TRITC staining in the liver tissue (250× magnification). Avidin-TRITC-stained nucleus can be recognized as 8-OH-dG and nucleus were stained with 4’,6-diamino-2-phenylindole (DAPI). Histograms represent the quantification of avidin stained nucleus over the total number of nucleus. Data are presented as the mean ± SEM. (*<i>P</i> < 0.05; ***<i>P</i><0.001 vs. control); (C, D) Representative images of immunohistochemical and immunoblot analyses of lipid peroxidation adducts, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) (40× magnification). A graph depicting the quantification of the relative abundance of the genes is shown. Data are presented as the mean ± SEM. (*<i>P</i> < 0.05; **<i>P</i><0.01 vs. control); (E) Immunoblot analysis of glutathione disulfide (GSSG); (F) Proposed molecular pathways for the preventive effect of RUE against alcohol-induced liver injury through ROS scavenging.</p