Ellagic acid (98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Nanjing, China). Formalin, sodium carboxymethyl cellulose (CMC-Na), and ethanol were procured from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), free fatty acid (FFA), and gamma glutamyl transferase (γGT) kits were procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GSH-Px) kits were procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kits (IL-6, IL-1β, and TNF-α) were purchased from Beijing Sinouk institute of biological technology (Beijing, China). TRIzol reagent and FastQuant RT kit were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). All other chemicals used in the present study were analytical grade.

Forty male ICR mice, aged 7–9 weeks and weighing 22–24 g, were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) (Certificate SCXK (Beijing) 2012-0001). The mice were kept in a well-ventilated animal room for a 1-week acclimation period at 25°C, humidity (60–80%), under 12 h light/12 h dark cycle. The animal experiment project was approved by the Ethics Committee of the Beijing Key Laboratory of Functional Food from Plant Resources (Permit number: A330-19). All animal experimental procedures in this research also followed the National Institutes of Health's guidelines for the treatment and use of laboratory animals.

After 1-week acclimation period, four groups of mice were formed (n = 10 in each group); control group (C), model group (M), low-dose of ellagic acid group (EL), and high-dose of ellagic acid group (EH); and were fed with normal pellet diet ad libitum and water. The C group was given CMC-Na (0.5%) via oral way whereas M group was administered with 50% (v/v) alcohol (10 mL/kg bw daily) after 1 h of 0.5% CMC-Na by oral route for 4 weeks. The mice in EL and EH were administered orally with low (50 mg/kg bw) or high (100 mg/kg bw) dose of ellagic acid (suspended in 0.5% CMC-Na) after 1 h of receiving 50% alcohol for 4 weeks. Every 3 days, the mice were weighted, and the amount of gastric infusion received was adjusted based on the weight. The total animal experiment lasted for 4 weeks. After 4 weeks, all mice were fasted for 12 h before being weighed and killed. Using a capillary tube, blood samples were taken from each mouse retroorbital venous plexus. Liver, kidney, and spleen tissues were dissected out, rinsed, washed with ice-cold PBS, and weighed to measured organ index according to the following formula:

Following that, one section of the liver tissues was immersed in a 10% formaldehyde solution for histopathological examination whereas the remaining sections were kept for biochemical study, ELISA determination, and qPCR measurement.

The blood samples were clotted at 4°C for 6 h and then centrifugated at 4,000 g for 15 min to obtain the serum. After that, the serum was further subjected for the determination of various biochemical parameters such as ALT, AST, TC, TG, HDL, LDL, FFA, and γGT by using commercially available kits according to the instruction manuals.

Liver tissue homogenates were prepared, and the lipids were separated according to the previously described method (13). Bicinchoninic acid protein assay kit was used to assess the total protein concentrations in the liver homogenate. The hepatic lipid profiles such as TG, TC, and FFA were measured by using the same protocol described for serum FFA, TC, and TG levels. The antioxidant activities such as MDA, SOD, CAT, GSH, and GSH-Px levels in liver tissue homogenates were examined by using commercially available kits following the protocol of the manufacturer.

The IL-6, IL-1β, and TNF-α (proinflammatory cytokines) concentrations in the liver were determined by using commercial ELISA kit, following the instruction manual.

Samples of liver were separated from each mouse and fixed in formalin solution (10%) for 24 h, and after that dehydrated using graded alcohol and xylene, and implanted in paraffin. Paraffin implanted segments were further cut into the thickness of 5 μm, stained with Masson's trichrome (MAS) and hematoxylin and eosin (H&E) for histological investigation. The degree of histological damage to the liver sections was further determined by using a light microscope (BA-9000L, Osaka, Japan).

The total RNA was isolated from hepatic tissue by using TRIzol reagent according to the method reported by Mehmood et al. (33). RNA was reverse transcribed to cDNA using a FastQuant RT kit, and mRNA expression was quantified using real time polymerase chain reaction (qRT-PCR). The details of primers used in this study are presented in Table 1. To normalize mRNA expression, the expression of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was measured.

Colonic contents in mice were collected in individual sterile cryotube and stored in liquid nitrogen (34). The total DNA was extracted via the EZNA soil DNA extraction kit (Omega Bio-tek, Norcross, USA) according to instruction manuals. The V3–V4 region of the bacterial 16S ribosomal RNA gene was subjected to PCR amplification using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′ -GGACTACHVGGGTWTCTAAT-3′).

The Illumina MiSeq sequencing technology along with multivariate statistical methods were conducted to detect the diversity of the V3-V4 region of the bacterial 16S rRNA gene. Related library construction and the Miseq high-throughput sequencing process were performed by Majorbio (Shanghai, China). Operational taxonomic units (OTUs) were clustered at 97% similarity, and Venn diagram, analyses of community composition, and Spearsman correlation analysis were performed.

The data of the animal experiment was evaluated by using SPPSS version 22.0 (SPSS Inc., Chicago, IL, USA) and Graph Pad Prism version 8 (La Jolla, CA, USA). One-way analysis of variance (ANOVA) was used to compare variations between groups, followed by Duncan's multiple range test. Differences between groups were found statistically significant at p < 0.01 or p < 0.05 and the data was expressed as mean ± SD.

The food intake by different groups of mice was measured in g/day. The normal group (C) utilized a healthy amount of food throughout the period of the experiment (Table 2). Compared with the normal group, food utilization in the model group (M) after alcohol administration was prominently decreased (p < 0.05), resulting in significant loss of body weight and activities (p < 0.05). However, the feed intake and weight loss were significantly improved in the two ellagic acid groups (EL and EH, p < 0.05), which was close to the C group. As shown in Table 2, the liver index of the M group was prominently raised compared with mice in the C group (p < 0.05). However, the administration of different doses of ellagic acid (EL and EH) attenuated the liver swelling compared with the M group. In addition, there were no significant changes in organ indices of kidney and spleen among mice in C, M, EL, and EH groups (p > 0.05).

As displayed in Figures 1A–D, a significant elevation (p < 0.05) in the serum AST (Figure 1A), ALT (Figure 1B), γGT (Figure 1C), and ALP (Figure 1D) activities occurred in the M group when compared with the C group. The treatment with ellagic acid (EL and EH) markedly (p < 0.05) downregulated the activities of the enzymes; AST, ALT, γGT, and ALP; when compared with the M group.

The results regarding serum lipid profiles such as TG, TC, HDL-C, LDL-C, and FFA are presented in Figures 2A–E. Compared with the C group, a significant rise in the serum TC, TG, LDL-C, and FFA levels and a downregulation of HDL-C level were observed in the M group (p < 0.05). Interestingly, the serum TG, TC, HDL-C, LDL-C, and FFA levels were significantly downregulated (p < 0.05), and HDL-C level was upregulated by ellagic acid treatment (EL and EH) compared with the M group.

As shown in the Figures 3A–C, hepatic lipid indicators such as TC (Figure 3A), TG (Figure 3B), and FFA (Figure 3C) were markedly upregulated in M group compared with the C group (p < 0.05). Ellagic acid treatment (EL and EH) induced a significant decline in the hepatic lipid indicators, e.g., TC, TG, and FFA (p < 0.05). However, EL failed to restore FFA level compared to the M group.

One of the major pathological events during the development of alcoholic fatty liver disease is oxidative stress since it connects lipid metabolism dysfunction to downstream inflammation and apoptosis (2, 4). Antioxidant enzymes, e.g., GSH-Px, CAT, MDA, SOD, and GSH, play significant roles in protecting alcohol-induced hepatic damage and oxidative stress. Figures 4A–E showed the effect of ellagic acid on hepatic antioxidant enzymes (GSH-Px, CAT, MDA, SOD, and GSH). Hepatic MDA level significantly elevated in the M group compared with C group (p < 0.05). The ellagic acid-treated groups (EL and EH) markedly decreased the MDA content compared with the M group (p < 0.05). In addition, hepatic GSH-Px, CAT, SOD, and GSH levels were significantly decreased in M group compared with C group (p < 0.05), which was restored by ellagic acid administration (EL and EH groups). The results clearly indicated that ellagic acid showed strong potential to alleviate hepatic damage in alcohol-induced mice via downregulating MDA content and increasing the antioxidant enzymes (GSH-Px, CAT, MDA, SOD, and GSH).

To determine the protective effect of ellagic acid against ALD, we investigated the histopathological changes in the hepatic tissue by using H&E and MAS staining assay. As presented in the Figure 5, the structure of liver lobule was complete and clear with no evident inflammatory cell infiltration, and hepatocytes were in an ordered arrangement with centrally and round located nuclei in C group mice. However, after alcohol exposure (M group), a significant inflammatory cell infiltration, fibrosis, and enlargement of hepatocytes around the central vein was observed. The ellagic acid-treated groups (EL and EH) exhibited less edema, fibrosis, and inflammatory cell infiltration compared with M group.

The results regarding proinflammatory cytokines such as IL-6, IL-1β, and TNF-α in the hepatic tissues are presented in the Figures 6A–C. A significant (p < 0.05) elevation in the hepatic IL-6, IL-1β, and TNF-α were observed in M group mice compared with C group. Interestingly, treatment of ellagic acid (EL and EH) was able to markedly decrease the levels of TNF-α, IL-1β, and IL-6 compared with the M group (p < 0.05). These results suggested that ellagic acid treatment was an effective approach to attenuate alcohol-induced hepatic inflammation.

According to the results, hepatic mRNA expression of TLR4, a trigger of inflammation in ALD was significantly increased in M group mice (p < 0.05), which further caused the upregulation of MyD88 and downstream genes compared with the C group (Figures 7A–F). However, it has been observed that EL and EH significantly reduced the expression of TLR4 (Tlr4) and downstream MyD88 genes (Myd88) in a dose-dependent manner resulting in reduced inflammatory changes (p < 0.05). Expression of inflammatory mediators (NF-κB and COX-2) and a cluster of differentiation (CD14) was significantly higher in the liver of M group of mice (p < 0.05). However, treatment with ellagic acid considerably reversed the inflammatory necrosis by downregulating the NF-κB (Nfkb1), COX-2 (Cox2), and CD14 (Cd14) genes expression (p < 0.05). Furthermore, a high dose of ellagic acid (100 mg/kg bw) was more effective in inhibiting the overexpression of TLR4, COX-2, CD14, and NF-κB genes than the low dose of ellagic acid (50 mg/kg bw, p < 0.05). It was also noticed that both doses of ellagic acid (EL and EH) reduced the hepatic inflammation by suppressing the expression of iNOS gene (Nos2) compared with the M group.

In the Figure 8A, Venn chart showed the number and overlap of sample OTUs in each and among the group. The results revealed that a total of 454 OTUs were shared by all the samples. At the phylum level (Figure 8B), ethanol administration elevated the relative abundance of Firmicutes, Verrucomicrobia, Actinobacteria, and decreased the relative abundance of Bacteroidetes and Proteobacteria compared with the C group. The administration of ellagic acid markedly modulated gut microbiota to some extent such as decreasing the relative abundance of Actinobacteria and Verrucomicrobia compared with the model group. At the genus level (Figure 8C), the norank_f__Muribaculaceae, Lactobacillus, Kurthia, Akkermansia, Bacteroides, and Lachnospiraceae_NK4A136_group were found to be the most prevalent six genera in colonic samples of tested mice. Alcohol administration decreased the relative abundance of norank_f__Muribaculaceae and Bacteroides, which were modulated to some extent by the treatment of EL and EH. In addition, the relative abundance of Bacteroides increased and Lachnospiraceae_NK4A136_group decreased in the EH group compared with the M group. The EL also improved the relative abundance of Bifidobacterium compared with the M group. Collectively, our results indicated that ellagic acid could significantly modulate gut microbiota composition.

Correlations between the microbiome and several biochemical indicators were tested by Spearman's correlation analysis. As shown in Figure 8D, the relative abundance of Bifidobacterium was significantly correlated with hepatic FFA, γGT, LDL, IL-6, and GSH. The relative abundance of Lactobacillus was significantly correlated with SOD. The relative abundance of Akkermansia was significantly correlated with IL-1β. The relative abundance of Parasutterella was significantly correlated with serum TG. The relative abundance of Candidatus_Arthromitus was significantly correlated with γGT and HDL. The relative abundance of (Eubacterium)_coprostanoligenes_group was significantly correlated with SOD. The relative abundance of Alistipes was significantly correlated with MDA.