Construction of chronic-binge ethanol feeding mouse model using ApoH
−/− C57BL/6 and WT mice
The ApoH−/− mouse model was used to explore the potential function of ApoH in the liver. In this study, a mouse model of chronic-plus-single binge ethanol feeding was constructed using C57BL/6 ApoH−/− and WT mice (Fig. 1A). Liver function parameters were analyzed based on ALT and AST levels, which were significantly increased in ApoH−/− mice than in WT mice fed the control diet (P < 0.05; Fig. 1B and C). AST levels were remarkably elevated in ApoH−/− mice fed the ethanol diet than in knockout mice pair-fed the control diet (P < 0.05; Fig. 1B and C). Liver triglyceride content also increased significantly in ApoH−/− mice than in WT mice under control diet (P < 0.05), whereas it was significantly increased in WT and ApoH−/− mice when both were fed ethanol (P < 0.05; Fig. 1D). Further, the degree of steatosis was evaluated in the stained liver tissue (Fig. 1E). Hepatic steatosis was observed in normal diet-fed ApoH−/− mice, whereas severe steatosis was observed in alcohol-fed WT and ApoH−/− mice (Fig. 1F). These findings suggested that absence of the ApoH gene induces hepatic steatosis, which is exacerbated by ethanol consumption.
RNA-seq analysis of liver sections in the chronic-binge ApoH
−/− mouse model highlighted the role of metabolic pathways
To explore the underlying mechanism of alcohol induced liver injury using the alcoholic fatty liver mouse model (the NIAAA model), RNA-seq was performed on liver sections from Lieber-DeCarli alcohol diet-fed C57BL/6 WT and ApoH−/− mice and their respective controls. The ApoH mRNA expression in ApoH−/− mice and alcohol-fed WT mice are illustrated in Fig. 2A and B, respectively. The Venn diagram and UpSet plots illustrate the interactions between combinations of the four different groups while hiding those without interactions (Fig. 2C). Figure 2D shows the number of differential expression genes (DEGs) between the WT and ApoH−/− mice and their alcohol-fed groups. Volcano plots further indicated the expression of differential genes in the various groups (Fig. 2E and G). Metabolic pathways were indicated to be predominant in KEGG signaling pathways (Fig. 2F and H). Among them, the DEGs between WT mice fed an alcohol diet and those fed paired control diet were predominantly associated with MAPK signaling pathway, retinol metabolism, and steroid hormone biosynthesis (Top 3). In contrast, the DEGs between ApoH−/− mice fed an alcohol diet and the ones fed paired control diet were predominantly enriched in retinol metabolism, steroid hormone biosynthesis, and PPAR signaling pathway (Top 3).
Subsequently, the number of DEGs was summarized in the different groups (Fig. 3A). A total of 82 DEGs were identified between the control-fed WT and ApoH−/− mice and 47 DEGs were identified between alcohol-fed WT and ApoH−/− mice. A comparison of gene expression profiles across the alcohol-fed groups led to the identification of 19 DEGs (Fig. 3B). Further, KEGG pathway analysis indicated that oxidation–reduction and lipid metabolic processes are most enriched in the signaling pathways (Fig. 3C). The remaining 28 DEGs between alcohol-fed WT and ApoH−/− mice were analyzed, and Fig. 3D illustrates the detailed information using a heatmap. The functional genes were enriched in oxidation–reduction and lipid metabolic processes, based on KEGG pathway analysis (Fig. 3E). Some cytochrome P450 (CYP) enzyme-related genes were identified among the 47 DEGs.
CYP450 enzymes act as a terminal oxidase in the multi-function oxidase system to metabolize different endogenous substrates and xenobiotics. The isoenzymes CYP2E1 and CYP2A5 primarily participate in alcohol metabolism and alcohol-induced liver injury [24,25,26]. The changes in expression of certain CYP450 genes were examined, namely, Cyp1a1, Cyp1b1, Cyp2a5, Cyp2b10, Cyp2e1, Cyp11a1, and Cyp26a1 (Fig. 3F). The mRNA levels of Cyp2a5 and Cyp2b10 were significantly increased in alcohol-fed WT mice than in normal diet-fed WT controls (P < 0.05). However, no significant difference was observed between the two ApoH−/− groups. The expression of Cyp1b1, Cyp11a1, and Cyp26a1 was undetectable in these samples. Taken together, the results suggested that ApoH downregulation primarily impacts liver lipid metabolism, which is mainly associated with CYP450 enzyme-activated pathways.
Changes in community diversity and bacterial species abundance in the chronic-binge ApoH
−/− mouse model
Recently, the gut-liver axis has been deemed one of the main regulatory factors in ALD [3, 4, 27]. Therefore, to explore the gut community diversity and differences in bacterial species abundance between alcohol- or control diet-fed WT and ApoH−/− mice. The total bacterial composition and abundance were remarkably reduced in WT and ApoH−/− mice fed an alcohol diet. However, at the phylum level, the abundance of Bacteroidetes increased in WT and ApoH−/− mice after 10-day chronic ethanol feeding, whereas that of Firmicutes increased sharply after the whole chronic-binge ethanol feeding. Abundance of Actinobacteria was lower in ApoH−/− mice than in WT mice fed either alcohol or control diet (Fig. 4A). In the beginning of adaptive liquid diet feeding in ApoH−/− mice, the abundance of norank_f_Muribaculaceae, Lachnospiraceae, Clostridia, and Turicibacter increased, whereas that of Lactobacillus and Bifidobacterium decreased. However, at the end of chronic-binge control diet feeding, the abundance of Lachnoclostridium and Parabacteroides increased, whereas that of Romboutsia, Lactobacillus, and Bifidobacterium decreased in ApoH−/− mice than in WT mice (Fig. 4B).
Thereafter, alpha diversity was analyzed, and Shannon index for the OTU level was used as a metric/index (Fig. 4C–E). No significant difference was observed in alpha diversity between WT and ApoH−/− mice administered the alcohol and pair-fed control diets at three different time points, namely the first day, chronic 10-day feeding, and final binge intake (Shannon index: P > 0.05). No significant difference was observed in ApoH−/− mice at the three different time points (Fig. 4F). However, at the end of the construction of chronic-binge model, alpha diversity of enterobacteria from ApoH−/− mice was lower than that corresponding to mice on the first day and to mice after chronic 10-day ethanol feeding (P < 0.05; Fig. 4G). Additionally, beta diversity and PCA indicated the bacterial community structure to be segregated differently between the four groups at the genus level (Fig. 4H and I).
Alcohol feeding-dependent ApoH downregulation impacted metabolic functions of gut microbiota
The different microbial community profiles was explored to predict their function using KEGG enrichment analysis in PICRUSt software. The results indicated that the gut microbiota primarily performed metabolic functions in the different groups; however, no significant difference was observed between them (Fig. 5A). The sequencing data was further analyzed using STAMP software to predict potential metabolic differences. The principal components 1, 2, and 3 accounted for 90.4%, 6.3%, and 2.2% of the total variation in the predicted functions, respectively (Fig. 5B). It was proceeded to analyze the relative abundance and distribution of metabolism and lipid metabolism in the different groups. Significant differences were observed across the different groups, as illustrated in Fig. 5C and D (P < 0.05). The heatmap indicated abundance of the selected active KEGG pathways in WT mice fed either an alcohol or control diet (Fig. 5E). From the selected KEGG pathway, numerous active signaling pathways were identified, including metabolism, lipid metabolism, glycan biosynthesis and metabolism, and cell growth and death, in WT mice fed either alcohol or control diet. The carbohydrate metabolism pathway was more active in alcohol-fed ApoH−/− mice than in alcohol-fed WT mice (P = 0.032) (Fig. 5F). Moreover, “metabolism” and “lipid metabolism” signaling pathways were more highly activated in alcohol-fed ApoH−/− mice than in control diet-fed ApoH−/− mice (P = 0.017 and P = 0.030; Fig. 5G and H, respectively). In conclusion, ApoH downregulation could affect the metabolic regulation of gut microbiota in mice, leading to changes in the metabolic outcome/pathways.