XZTZ consists of six traditional Chinese medicines, including Shan zha as Crataegus pinnatifida Bunge [Rosaceae; Crataegi fructus], He ye as Nelumbo nucifera Gaertn. [Nelumbonaceae; Nelumbinis folium], Zhi qiao as Citrus × aurantium Engl [Rutaceae; Aurantii fructus], Bai zhu as Atractylodes macrocephala Koidz. [Compositae; Atractylodis macrocephalae rhizoma], Fu ling as Poria cocos (Schw) Wolf [Polyporaceae; Poria], Ze xie as Alisma orientale (Sam.) Juzep. [Alismataceae; Alismatis rhozoma]. The original botanical drugs were purchased from Traditional Chinese Medicine Pharmacy of Zhejiang Provincial Hospital of Traditional Chinese Medicine. The details of the specific metabolites and contents of the XZTZ dosage are shown in Table 1. The herbs in the XZTZ were soaked in 10 times the amount of distilled water for 1 h, and then extracted by reflux for 3 times. The extract was concentrated to the relative density of 1.05, dried under 60°C, lyophilized into dry powder, and the lyophilized powder was used for subsequent experiments (1 g of lyophilized powder is equal to 12.05 g original herb). Then, it was diluted with normal saline to achieve two concentrations, including 291.2 mg/kg (low dose, XZTZ-L) and 582.4 mg/kg (high dose, XZTZ-H), based on the body surface area of adults (Standard weight 70 kg). Commencing in the second week of the modeling process, the mice were subjected to daily oral gavage administration of XZTZ at the prescribed dosages for a period of 3 weeks. In a previous study by our group, the analysis of the main metabolites of XZTZ using High Performance Liquid Chromatography revealed the following six metabolites in the extract of XZTZ: mannitol (1.23 min) from Shan zha, citric acid (1.7 min) from Shan zha and He ye, quercetin 3-O-β-D-glucuronopyranoside (16.90 min) from He ye, naringin (19.33 min) from Zhi qiao, hesperidin (20.94 min) from Zhi qiao, and alisol A (32.24 min) from Ze xie (Qiu et al., 2023). The chemical composition of the drug in XZTZ complies with the ConPhyMP statement and has been validated for classification at “http://www.plantsoftheworldonline.org”.

Kits for testing the levels of ALT, AST, MDA, SOD, CAT, and GSH-Px were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA kit was purchased from the Beyotime Institute of Biotechnology (Jiangsu, China).

The traditional Lieber-DeCarli alcohol liquid diet and an isocaloric control diet were purchased from Trophic Animal Feed High-tech Co., Ltd (Nantong, China). Twenty male C57BL/6J mice (8 weeks old, 21.00 ± 1.00 g) were provided by the Animal Experiment Center of Zhejiang Chinese Medical University. Animal experiments were reviewed by the Laboratory Animal Management and Ethics Committee (approval number: IACUC-20220321-08). Mice were housed in an environment of 55% ± 5% relative humidity with 23°C ± 2°C and 12 h of light-dark cycle (lights on at 7:30 a.m.). To acclimate the mice, they were continuously free access to regular food and water for 3 days following their acquisition. After 3-day of acclimatization, mice were randomly assigned to four groups for 4 weeks of intervention: a) PF group, fed Lieber-DeCarli liquid diets containing isocaloric maltose dextrin; b) AF group, fed alcohol-containing modified Lieber-DeCarli liquid diets; c) AF with XZTZ-L (AF + XZTZ-L) group; d) AF with XZTZ-H (AF + XZTZ-H) group. After 12 h of fasting, mice were anesthetized with pentobarbital solution (80 mg/kg body weight (Guo et al., 2023)) and euthanized. Blood samples were collected and stored at −80°C until being assayed. Liver tissue was rapidly excised, weighed, and stored at −80°C for further analysis. The Growth parameters of mice among four groups are shown in Table 2.

The liver was fixed in a 4% buffered paraformaldehyde (Biosharp Biotechnology, Shanghai, China) for 24 h. Then it was embedded with an optimum cutting temperature embedding agent and sliced on a cryostat. The 4 µm thick liver sections were stained with H&E.

The myeloperoxidase (MPO) staining (equivalent to monocytes and neutrophils), the 4 µm thick liver sections were de-paraffinized, and re-hydrated in descending grades of alcohol, followed by heat mediated antigen retrieval procedure. According to the manufacturer’s instruction, sections were incubated in BloxALL solution (Vector Laboratories, Burlingame, CA, United States) to block endogenous peroxidase activity. Then, sections were incubated with anti-MPO antibody (Servicebio, Wuhan, GB12224) overnight at 4°C in a humidified chamber. The secondary antibody was HRP-labeled goat anti-mouse antibodies (Servicebio, Wuhan, G23301) and the DAB chromogenic agent kit (Servicebio, Wuhan, GB12224) was used for histochemical chromography. The MPO staining was evaluated with a light fluorescence microscope at a 20-fold magnification. Three slides were randomly selected for each tissue, and the MPO-positive cells were quantifed by ImageJ software.

Total RNA was extracted from mice livers using TRIzol reagent (Invitrogen, UK), based on the manufacturer’s protocol. qRT-PCR was performed with the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, United States). Genes levels were normalized to that of 18S and were calculated by the 2^−ΔΔCT method. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of all primers used in this study are listed in Table 3.

Protein concentrations of mice livers were detected using BCA protein assay kits. Samples (approximately 20 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The primary antibodies were used as follows: cleaved-Caspase3 (Cell Signaling Techonology, Cat. No. 9664S, rabbit monoclonal, 1:1000), Bcl-2 (Santa Cruz, Cat. No. sc-7382, mouse monoclonal, 1:500), CYP2E1 (Abcam, Cat. No. ab28146, rabbit polyclonal, 1:5000), Nrf2 (Cell Signaling Techonology, Cat. No. 12721S, rabbit monoclonal, 1:1000), Keap1 (Abcam, Cat. No. ab227828, rabbit polyclonal, 1:2000), p-JNK (Cell Signaling Techonology, Cat. No. 9255S, mouse monoclonal, 1:1000), JNK (Cell Signaling Techonology, Cat. No. 9252S, rabbit monoclonal, 1:1000), p-P38 (Cell Signaling Techonology, Cat. No. 4511S, rabbit monoclonal, 1:1000), P38 (Cell Signaling Techonology, Cat. No. 8690S, rabbit monoclonal, 1:1000), p-P65 (Cell Signaling Techonology, Cat. No. 3033S, rabbit monoclonal, 1:1000), P65 (Cell Signaling Techonology, Cat. No. 8242S, rabbit monoclonal, 1:1000), p-IκBα (Cell Signaling Techonology, Cat. No. 2859S, rabbit monoclonal, 1:1000), IκBα (Cell Signaling Techonology, Cat. No. 4814S, mouse monoclonal, 1:1000), LaminB (Cell Signaling Techonology, Cat. No. 17416S, rabbit monoclonal, 1:1000), and β-Actin (Santa Cruz, Cat. No. sc-4778, mouse monoclonal, 1:5000). β-Actin and LaminB were used as the internal control. The secondary antibodies were anti-rabbit (Boster Biological Technology, ba1054, 1:5000) and anti-mouse (Boster Biological Technology, ba1050, 1:5000). Moreover, a nuclear extraction kit (KeyGEN BioTECH, KGP1100) was used for Nrf2 protein. Bands were quantified by the ImageJ software.

All data were expressed as the mean ± SD. Statistical analysis was performed using unpaired Student’s t-test with GraphPad Prism 8.02 software (GraphPad Software, San Diego, CA). The p-value <0.05 was statistically significant.

In our study, we developed a mouse alcohol-induced liver injury model to evaluate potential recuperative effect of the XZTZ treatment (Figure 1A, Supplementary Figure S1). The increased activities of serum ALT and AST are gold indicators of liver injury. As shown in Figures 1B,C, the activities of serum ALT and AST in the AF group were obviously increased by 2.03-fold and 1.91-fold compared with that in the PF group. The elevations of ALT and AST induced by alcohol were effectively inhibited by the XZTZ, accompanied by a dose-response relationship. We also observed the hepatic pathophysiological changes by H&E staining, and found that XZTZ significantly alleviated alcohol-induced liver injury (Figure 1D). In addition, the elevated hepatic protein level of cleaved-Caspase3 and the decreased hepatic protein level of Bcl-2 induced by alcohol was rescued by the XZTZ (Figure 1E).

Oxidative stress is an essential pathological mechanism of alcohol-induced liver injury. Hence, we measured the levels of oxidative stress products and the activities of antioxidant enzymes in alcohol-fed mice livers. As shown in Figure 2A, the hepatic level of MDA was increased in the AF group compared with that in the PF group, while the intervention of the XZTZ significantly reversed the MDA increase induced by alcohol. Also, the activities of SOD, CAT, and GSH-Px were disrupted by alcohol, while the intervention of the XZTZ clearly rescued the activities of the above antioxidant enzymes (Figures 2B–D). Furthermore, CYP2E1 was analyzed in mice model induced by alcohol. Compared with that in the PF group, the expression of CYP2E1 in the liver tissue of mice was significantly increased in the AF group, while the XZTZ treatment decreased its expression (Figure 2E).

To further discover the molecular mechanism of the XZTZ on inhibiting oxidative stress, the nuclear protein level of Nrf2 and the total protein level of Keap1 were measured by Western blot. As shown in Figure 3A, compared with the PF group, the hepatic protein level of nuclear-Nrf2 was decreased and the hepatic protein level of Keap1 was increased in the AF group, whereas the levels of Nrf2 and Keap1 were obviously reversed by the XZTZ pretreatment. Moreover, we measured the gene expression levels of HO-1, GCLC, and GPX-1 by qRT-PCR in mice livers. The result showed that the XZTZ groups had significantly increased HO-1, GCLC, and GPX-1 mRNA expressions compared with the AF group (Figure 3B).

The inflammation is also a critical pathological process of alcohol-induced liver injury. The MPO staining in our study was performed to visualize monocytes and neutrophils in the different groups. As shown in Figure 4A, there was a robust increase of MPO-positive cells in the AF group compared to other groups, while MPO-positive cells in XZTZ groups were significantly increased compared to the PF group. Additionally, pro-inflammatory cytokines including TNFA, IL-6, IL-1B, and MCP-1 in mice livers were significantly increased in the AF group, while the XZTZ supplementation reduced their expressions (Figure 4B).

To further explore the molecular mechanism of the XZTZ on alleviating inflammatory response, the expression of proteins involved in MAPKs and NF-κB pathways was measured by Western blot. As shown in Figure 5A, the XZTZ pretreatment significantly reduced alcohol-stimulated both JNK and P38 phosphorylation in a dose-dependent manner. Furthermore, the ratios of p-P65/P65 and p-IκBα/IκBα also decreased clearly after pretreatment with the XZTZ (Figure 5B).