Triterpenoids Extracted From Antrodia cinnamomea Mycelia Attenuate Acute Alcohol-Induced Liver Injury in C57BL/6 Mice via Suppression Inflammatory Response

Abstract

Excessive alcohol consumption causes liver injury-induced mortality. Here we systematically analyzed the structure of triterpenoids extracted from Antrodia cinnamomea mycelia (ACT) and investigated their protective effects against acute alcohol-induced liver injury in mice. Liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry were performed to determine the structures of ACT constituents. Alcohol-induced liver injury was generated in C57BL/6 mice by oral gavage of 13 g/kg white spirit (a wine at 56% ABV). Mice were treated with either silibinin or ACT for 2 weeks. Liver injury markers and pathological signaling were then quantified with enzyme-linked immunosorbent assays, antibody array assays, and Western blots, and pathological examinations were performed using hematoxylin-eosin staining and periodic acid-Schiff staining. Triterpenoids extracted from A. cinnamomea mycelia contain 25 types of triterpenoid compounds. A 2-weeks alcohol consumption treatment caused significant weight loss, liver dyslipidemia, and elevation of alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and alkaline phosphatase activities in the serum and/or liver. These effects were markedly reversed after 2-weeks ACT administration. Triterpenoids extracted from A. cinnamomea mycelia alleviated the organ structural changes and inflammatory infiltration of alcohol-damaged tissues. Triterpenoids extracted from A. cinnamomea mycelia inhibited proinflammatory cytokine levels and enhanced anti-inflammatory cytokine levels. Acute alcohol treatment promoted inflammation with significant correlations to hypoxia-inducible factor 1α (HIF-1α), which was reduced by ACT and was partially related to modulation of the protein kinase B (Akt)/70-kDa ribosomal protein S6 kinase phosphorylation (p70S6K) and Wnt/β-catenin signaling pathways. In conclusion, ACT protected against acute alcohol-induced liver damage in mice mainly through its suppression of the inflammatory response, which may be related to HIF-1α signaling.

Figures

Figure 1

Total ion chromatogram of ACT…

Figure 1

Total ion chromatogram of ACT as obtained by LC-MS. ACT, triterpenoids separated from…

Figure 1 Total ion chromatogram of ACT as obtained by LC-MS. ACT, triterpenoids separated from A. cinnamomea mycelia.

Figure 2

ACT treatment decreases liver injury…

Figure 2

ACT treatment decreases liver injury markers and lipid abnormalities. Mice were treated with…

Figure 2 ACT treatment decreases liver injury markers and lipid abnormalities. Mice were treated with ACT for 14 days, and their liver injury markers were quantified with enzyme-linked immunosorbent assay. (A) The experimental protocol and drug administration. (B–F) The levels of (B,C) alanine aminotransferase and (D,E) aspartate aminotransferase in both the serum and liver, respectively, and the levels of (F) γ-glutamyl transferase, (G) alkaline phosphatase, (H) triglyceride, (I) total cholesterol, and (J) low-density lipoprotein in the liver. Data are expressed as the means ± SD (n = 8) and were analyzed using a one-way analysis of variance with parametric tests. #P < 0.05 and ###P < 0.001 vs. the control group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the model group. ACT, triterpenoids separated from A. cinnamomea mycelia; Sil, silibinin.

Figure 3

ACT treatment alleviates structural organ…

Figure 3

ACT treatment alleviates structural organ changes and hepatocyte apoptosis. Histopathological analyses of the…

Figure 3 ACT treatment alleviates structural organ changes and hepatocyte apoptosis. Histopathological analyses of the (A) liver, (B) kidney, (D) spleen, and (E) heart by H&E staining, and of the (C) kidney by PAS staining (scale bar 100 μm; magnification × 400). ACT, triterpenoids separated from A. cinnamomea mycelia; Sil, silibinin; H&E, hematoxylin and eosin; PAS, periodic acid–Schiff.

Figure 4

Antibody array assay. The effects…

Figure 4

Antibody array assay. The effects of ACT and Sil on 111 cytokines in…

Figure 4 Antibody array assay. The effects of ACT and Sil on 111 cytokines in the livers of mice with acute alcohol-induced liver injury were detected using the Proteome Profiler Mouse XL Cytokine Array kit (n = 3). (A) The array graph represents the cytokine expressions. (B) Scatter diagram of the 111 cytokines. The relative density is the ratio of the absolute value and the reference spot value. The red dots indicate the proteins validated by enzyme-linked immunosorbent assay. ACT, triterpenoids separated from A. cinnamomea mycelia; Sil, silibinin. ① RANTES, regulated upon activation normal T cell expressed and secreted; ② YKL-40, human cartilage glycoprotein 39; ③ CXCL13, chemokine (C-X-C motif) ligand 13; ④ ICAM-1, intercellular cell adhesion molecule 1; ⑤ IFN-γ, interferon γ; ⑥ IL-1α, interleukin 1α; ⑦ IL-7, interleukin 7; ⑧ IL-22, interleukin 22; ⑨ IL-23, interleukin 23; ⑩ IL-33, interleukin 33; ⑪ NGAL, neutrophil gelatinase-associated lipocalin; ⑫ RBP4, retinol-binding protein 4; ⑬ P-selectin; ⑭ PAI-1, plasminogen activator inhibitor 1; ⑮ TPO, thrombopoietin; ⑯ TNF-α, tumor necrosis factor α; ⑰ VCAM-1, vascular cell adhesion molecule 1; ⑱ VEGF, vascular endothelial growth factor.

Figure 5

ACT treatment regulates HIF-1α via…

Figure 5

ACT treatment regulates HIF-1α via Akt/p70S6K and Wnt/β-catenin signaling in the liver and…

Figure 5 ACT treatment regulates HIF-1α via Akt/p70S6K and Wnt/β-catenin signaling in the liver and spleen. Fourteen-day ACT administration regulated the phosphorylation levels of Akt, mTOR, GSK-3β, p70S6K, and β-catenin and the expression levels of Wnt 1, Wnt3, and HIF-1α in the liver (A) and spleen (B) of mice with acute alcohol-induced injuries. Quantified protein expression was normalized by related total protein expression and/or GAPDH expression. Data are expressed as the means ± SD (n = 3) and were analyzed using a one-way analysis of variance with parametric tests. #P < 0.05 and ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the model group. ACT, Triterpenoids separated from A. cinnamomea mycelia; Sil, silibinin; Akt, protein kinase B; mTOR, mammalian target of rapamycin; GSK-3β, glycogen synthase kinase 3 beta; p70S6K, 70-kDa ribosomal protein S6 kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.