Protection of Inonotus hispidus (Bull.) P. Karst. against Chronic Alcohol-Induced Liver Injury in Mice via Its Relieving Inflammation Response

Abstract

Alcoholic liver disease (ALD) can be induced by excessive alcohol consumption, and has a worldwide age-standardized incidence rate (ASIR) of approximately 5.243%. Inonotus hispidus (Bull.) P. Karst. (IH) is a mushroom with pharmacological effects. In ALD mice, the hepatoprotective effects of IH were investigated. IH strongly ameliorated alcohol-induced pathological changes in the liver, including liver structures and its function-related indices. Intestinal microbiota and serum metabolomics analysis showed that IH altered the associated anti-inflammatory microbiota and metabolites. According to results obtained from Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA), IH downregulated the levels of pro-inflammation factors interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α), enhanced the expressions of peroxisome proliferator-activated receptor alpha (PPARα) and 15-hydroxprostaglandin dehydrogenase (15-PGDH), and inhibited the phosphorylated activation of Janus kinase (JAK) 1 and signal transducer and activator of transcription (STAT) 3, confirming the hepatoprotection of IH against alcohol damage via anti-inflammation. This study provides the experimental evidence for the hepatoprotective effects of IH in chronic ALD.

Keywords: Inonotus hispidus; chronic alcohol damage; hepatoprotection; inflammation.

Figure 1

Inonotus hispidus (Bull.) P. Karst. (IH) alleviated chronic liver damage caused by alcohol. (A) Animal model establishment and agent administration process. (B) Body weight of the mice was measured (n = 12). (C) Hematoxylin and Eosin (H&E) staining was used to analyze the histopathological changes of the liver in mice with chronic alcoholic liver injury (n = 3) (200×, scale bar: 100 μm) (a. large number of fatty vacuoles, b. hepatocellular swelling, c. inflammatory cell infiltrate, d, e. few fatty vacuoles). Liver-related indicators (D) aspartate aminotransferase (AST), (E) alanine aminotransferase (ALT), (F) triglyceride (TG), (G) total cholesterol (TC), (H) low-density lipoprotein cholesterol (LDL-C), (I) high-density lipoprotein cholesterol (HDL-C), (J) alcohol dehydrogenase (ADH) were analyzed to verify the alleviating effect of IH on liver injury (n = 8). The data are shown as the mean ± S.E.M. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control (CTRL) mice; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Alcohol mice.

Figure 2

IH regulated intestinal microbiota in chronic alcoholic liver disease (ALD) mice (n = 3). (A) Venn diagram representation of shared/ unique operable taxonomic units (OTUs) in the intestinal microbiota of CTRL, Alcohol and Alcohol + IH mice. (B) Based on the Bray–Curtis hierarchical clustering analysis in the β-diversity clustering analysis, the similarity among the three groups was presented as a hierarchical tree. (C) Principal coordinate analysis (PCoA) of Bray–Curtis distance derived from β-diversity analysis was used to characterize the community differences between the three groups. (D) α-diversity indices of three different groups. # p < 0.05 vs. CTRL mice. (E) Histograms of the distribution of the linear discriminant analysis (LDA) values of significantly different species based on the results of linear discriminant analysis of effect size (LEfSe) analysis, also showing the significantly enriched species within each group. (F) Bacterial taxonomic profiling of intestinal microbiota at the genus level (top 10). (G) The heat map shows significant changes in the 20 genera with the highest abundance.

Figure 3

IH regulated serum metabolites in chronic ALD mice (n = 3). (A) Differential abundance scores of enriched metabolic pathways among the groups. A score of 1 indicates that all identified metabolites are upregulated, and −1 indicates that all identified metabolites in the pathway are downregulated. (B) The heat map of different serum metabolites. (C) The correlation analysis of altered metabolites in serum. * p < 0.05. (D) The correlation analysis of intestinal microbiota with differential metabolites. * p < 0.05, ** p < 0.01. Color keys represents the correlation coefficients.

Figure 4

IH alleviated the inflammatory response in chronic ALD mice. IH decreased the expression levels of (A) interleukin (IL)-1β, (B) IL-6, and (C) tumor necrosis factor-α (TNF-α) in the liver, according to the enzyme-linked immunosorbent assay (ELISA) results (n = 8). (D) IH decreased the protein expression levels of IL-6, TNF-α and IL-1β in the liver of alcohol mice, according to the Western blot results. (E) Quantification of protein expression normalized to β-actin and expressed as the percentage of CTRL mice. The data are shown as the mean ± S.E.M. ## p < 0.01, ### p < 0.001 vs. CTRL mice; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Alcohol mice.

Figure 5

IH altered the inflammation-related factors in chronic ALD mice. (A) IH administration increased the levels of 15-hydroxprostaglandin dehydrogenase (15-PGDH) and peroxisome proliferator-activated receptor alpha (PPARα), and decreased the levels of phospho- (P-) Signal transducer and activator of transcription (STAT) 3 and P- Janus kinase (JAK) 1 in the liver of chronic ALD mice. (B) Quantification of protein expression normalized to β-actin or their related total proteins, and expressed as the percentage of CTRL mice. In immunohistochemical staining in mouse liver tissue, IH administration (C) decreased the level of P-STAT3 and (D) increased the level of PPARα (n = 3) (200×, scale bar: 100 μm). The data are shown as the mean ± S.E.M. # p < 0.05, ### p < 0.001 vs. CTRL mice; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Alcohol mice.