The male C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China), they were housed in a temperature-controlled animal facility with a 12-h light–dark cycle and allowed to obtain rodent chow and water ad libitum. All experimental procedures of animals in this study were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine.

The animal model was established according to Gao’s group previously described with appropriate modifications (Chang et al., 2015; Ding et al., 2010). In detail, the animals were fed a HFD (60% kcal% fat; Cat# GD60, Guangdong Medical Laboratory Animal Center, Foshan, China; Table 1) or a HFD Control diet (10% kcal% fat; Cat# GD450B, Guangdong Medical Laboratory Animal Center, Foshan, China; Table 1) for 3 days, followed by a single gavage of ethanol (as a 31.25% solution in water) at a dose of 5 g/kg body weight (as 0.20 mL per 10 g body weight) or the same volume of distilled water (Chang et al., 2015). The food was not taken away after gavage. After 9 h of ethanol gavage, the animals were deeply anesthetized before blood collection from the orbital sinus for collecting serum and euthanized via cervical dislocation (Mackowiak et al., 2022) (Figure 1A). Then, the liver samples were harvested, and either frozen in −80°C or put in 4% Paraformaldehyde Fix Solution (Cat# G1101, Servicebio, Wuhan, China) before further analysis.

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglycerides (TG) were examined by an automatic biochemical analyzer as we had previously descried (Xu et al., 2024). Hepatic TG, malondialdehyde (MDA), myeloperoxidase (MPO) and the reduced glutathione (GSH) were examined by the commercial assay kits (Cat# A110-1-1, Cat# A003-1-2, Cat# A044-1-1, and Cat# A006-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) (Xu et al., 2024).

The fixed liver samples were embedded by the Tissue-Tek^® optimum cutting temperature (O.C.T.) compound (Cat# 4583, Sakura, Japan) to perform frozen liver sections for Oil Red O (Cat# G1015, Servicebio, Wuhan, China) staining, or embedded in paraffin to perform sections for staining with hematoxylin (Cat# Ba4097, BaSo, Zhuhai, China) and eosin (Cat# Ba4099, BaSo, Zhuhai, China) (H&E) as we previously described (Zhang et al., 2020; Xu et al., 2024). H&E staining was used to evaluate the morphological change of the liver tissue and lipid accumulation, and Oil Red O staining was used to visualize fat content in the liver samples.

The total proteins were extracted from the liver samples with lysis buffer, and immunoblot assay was performed according to the standardized processes (Zhang et al., 2020). The antibodies used in this study were described in Table 2. The gray value of protein bands was quantified by the ImageJ software (National Institutes of Health, Bethesda, MD, United States).

Immunofluorescence staining was performed to detect 3-nitrotyrosine (3-NT) in the liver. The frozen sections were subjected to pre-washing treatments as follows: washing with 1 × PBS for 30 min, 1 × TBS for 10 min, and 1 × TBST for 20 min. The liver sections were sealed with goat serum blocking solution (Cat# ZLI-9056, ZSGB-BIO, Beijing, China) at room temperature for 2 h. Then, the 3-NT antibody (Abcam, Cat# ab110282, United Kingdom) was incubated overnight with the liver sections at 4°C, followed by washing with 1 × TBS for 10 min and 1 × TBST for 20 min. The liver sections were incubated with Goat Anti-Mouse IgG (H + L) Fluor 594-conjugated antibody (Cat# S0005, Affinity Biosciences, Beijing, China) under dark conditions for 2 h. The sections were washed with 1 × TBS for 10 min followed by 1 × TBST for 20 min. Finally, the fluorescence intensity of 3-NT was observed under a fluorescence microscope after sealing the sections with anti-fluorescence attenuation sealer (containing DAPI) (Cat# S2110, Solarbio, Beijing, China).

The statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analysis for data with normal distribution (by Shapiro-Wilk test) and satisfying homogeneity of variance (by Brown-Forsythe test), performed by Brown-Forsythe and Welch ANOVA tests followed by Dunnett T 3 post hoc analysis for data with normal distribution and heteroscedasticity, and performed by Kruskal–Wallis test followed by Dunn’s post hoc analysis for data with skewed distributions (by Shapiro-Wilk test). All data were expressed as the mean ± SD or median ± interquartile range, a value of P < 0.05 was considered as significantly different. All histograms were performed using GraphPad Prism 10.0 (GraphPad Software Inc., San Diego, CA, United States).

It has been reported that 3 days of HFD feeding plus acute ethanol binge can induce liver damage in mice (Chang et al., 2015; Babuta et al., 2024c). Here, we established the same animal models (Figure 1A), and like the previous reports (Chang et al., 2015; Babuta et al., 2024c), our data also indicated that HFD feeding for 3 days plus one time of acute ethanol binge on the third day synergistically induced liver injury as indicated by increasing serum levels of ALT and AST (Figures 1B,C) and increasing hepatic fat deposition in mice as indicated by Oil Red O staining (Figure 1D).

In order to further investigate the mechanisms of short-term HFD feeding plus acute ethanol binge on acute liver injury, the other animals were randomly divided into three groups: Control group, HFD group, and short-term HFD feeding plus acute ethanol binge group. Similar to the previous reports that short-term HFD feeding, e.g., for 3–4 days, is sufficient to induce liver steatosis and impair glucose tolerance and hepatic insulin sensitivity (Lee et al., 2011; Ji et al., 2012; Wiedemann et al., 2013; Chang et al., 2015), our data of hepatic H&E staining showed that a small amount of fat vacuoles accumulated in the liver of mice fed a HFD (Figure 2A), hepatic Oil red O staining and hepatic TG levels showed that HFD feeding induced fat deposition in the liver (Figures 2B,C). HFD feeding also increased serum TG levels, and only slightly increase serum ALT and AST levels with no statistical significance (Figures 2D–F). Compared with HFD group, hepatic fat deposition, serum TG, ALT and AST levels were further enhanced in HFD + Al group (Figure 2). Therefore, short term HFD feeding plus ethanol binge synergistically induces acute liver injury and hepatic steatosis in mice.

HFD or excessive ethanol intake has been shown to promote the production of reactive oxygen species (ROS), thus, increase oxidative stress in the liver (Ma et al., 2022; Zhu et al., 2022; Park et al., 2023). The high levels of ROS can induce lipid peroxidation. 3-nitrotyrosine (3-NT), a product of reactive-nitrogen species (RNS) with the activated aromatic ring of tyrosine, is another classical biomarker of oxidative stress (Bandookwala and Sengupta, 2020). The immunofluorescence results indicated that hepatic 3-NT levels in HFD group and HFD + Al group were increased when compared with Control group, and hepatic 3-NT levels in HFD + Al group were higher than those in HFD group (Figures 3A,B). MDA, which is a biomarker of lipid peroxidation, has been examined to reflect oxidative stress (Ayala et al., 2014). HFD, and HFD plus ethanol binge increased MDA levels in the liver, and HFD plus ethanol binge upregulated more MDA levels in the liver (Figure 3C). MPO, which is the member of heme peroxidase family in immune cells, can contribute to ROS production (Hawkins and Davies, 2021), and thus, MPO levels also reflects the degree of oxidative stress and inflammation in the organ or tissue. Our data indicated that hepatic MPO levels were increased (Figure 3D), in contrast, GSH (an indicator of antioxidant capacity) levels were decreased in HFD + Al group compared with Control group (Figure 3E). Therefore, HFD plus ethanol binge synergistically contributed to hepatic oxidative stress in mice.

The increased ROS can further cause acute liver injury after short-term ethanol feeding by activating innate immune signals and inducing sterile inflammation (Tsutsumi et al., 2003; Gao et al., 2024). Here, the phosphorylation of MAPK and NF-κB p65 in the liver were evaluated by Western blotting. Our results showed that the phosphorylation of hepatic ERK, JNK, and NF-κB p65 were elevated in HFD group when compared with Control group, and the phosphorylated hepatic MAPK (p38 and JNK) were further increased in the mice subjected to HFD plus ethanol binge, and ERK and NF-κB p65 also showed increasing trends, however, it was not statistically significant (Figure 4). Therefore, HFD plus ethanol binge significantly increased the phosphorylation levels of MAPK and NF-κB in the liver.

The phosphorylation of MAPK and NF-κB p65 in the liver can induce the expression of inflammatory cytokines, and thus amplifying inflammation (Zhang et al., 2019; Zhang et al., 2023; Lan et al., 2024). Therefore, TNF-α, IL-1β, and IL-18 levels in the liver were determined by Western blotting. Our results showed that the hepatic protein levels of TNF-α, pro-IL-1β and the cleaved-IL-1β (the maturation form of IL-1β), and pro-IL-18 and the cleaved-IL-18 (the maturation form of IL-18) were increased in HFD + Al group compared with HFD group (IL-18 levels only show upward trends but has no statistical significance) (Figure 5). Therefore, HFD plus ethanol binge induced and amplified hepatic inflammation by aggravating both the expression and maturation of inflammatory cytokines in mice.

The pro-IL-1β and pro-IL-18 need to be processed and cleaved by the activated Caspase-1 (the cleaved Caspase-1) to convert into their mature forms (He et al., 2016). Therefore, to investigate whether the effect of HFD plus ethanol binge on the maturation of IL-1β and IL-18 was related to the activation of Caspase-1, we examined the protein levels of Caspase-1. Our results showed that both pro-Caspase-1 and cleaved-Caspase-1 were upregulated in HFD group when compared with Control group, and HFD plus ethanol binge further increased the expression and the activation of Caspase-1 (Figure 6A). Besides the cleavage of IL-1β and IL-18, Caspase-1 can also specifically cleave the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in gasdermin D (GSDMD), which is required and sufficient for pyroptosis (Shi et al., 2015). To further investigate whether HFD plus ethanol binge-induced acute liver injury is involved in pyroptosis, we examined hepatic GSDMD levels in the liver. Our results showed that both pro-GSDMD and cleaved-GSDMD levels were increased in HFD group and HFD + Al group, and HFD plus ethanol binge facilitated more expression and maturation of GSDMD than HFD alone (Figure 6B).

In addition to the Caspase-1 to GSDMD canonical pyroptosis signaling, Caspase-8 and Caspase-11 can also induce the non-canonical pyroptosis signaling by cleaving GSDMD, and Caspase-3 can induce the non-canonical pyroptosis signaling by cleaving gasdermin E (GSDME) (Shi et al., 2015; Wang Y. et al., 2017). Therefore, we further examined the non-canonical pyroptosis signals. Our results showed that both HFD and HFD plus ethanol binge can increased the hepatic levels of pro-Caspase-11 and cleaved-Caspase-11 (Figure 7A), pro-Caspase-8 and cleaved-Caspase-8 (Figure 7B), pro-Caspase-3 and cleaved-Caspase-3 (Figure 7C), and pro-GSDME and cleaved-GSDME (Figure 7D). Except for the levels of pro-Caspase-8, which has no statistical significance, other non-canonical pyroptosis signals in HFD + Al group were higher than these in HFD group (Figure 7). Therefore, HFD plus ethanol binge synergistically induced hepatic pyroptosis by excessively activating pyroptosis signals.