Protective Effects of Licochalcone A Against Alcoholic Liver Injury in Mice by Modulating the Gut–Liver Axis and TLR4/NF-κB Pathway

Abstract

This study intended to elucidate the preventive effects of Licochalcone A (Lico A, a flavonoid from Glycyrrhiza inflata) on acute alcoholic liver injury (AALI) in mice and its mechanisms. Lico A (50, 100 mg/kg) markedly decreased the serum ALT, AST, and ALP levels (p < 0.05) and elevated the ALB and TP levels in AALI mice (p < 0.05). Lico A (100 mg/kg) markedly reduced the hepatic levels of MDA, NO, TNF-α, IL-1β, and IL-6 in AALI mice (p < 0.05), while elevating SOD, GSH, ADH, and ALDH activities (p < 0.05). Furthermore, Lico A (100 mg/kg) downregulated TLR4, MyD88, IKKβ, p-IκBα/IκBα, and p-NF-κB p65/NF-κB p65 levels in the liver tissue of AALI mice (p < 0.05) and diminished the serum LPS and DAO contents (p < 0.05). Lico A (50, 100 mg/kg) upregulated the expression of the intestinal tissue ZO-1 and Occludin in AALI mice. Pathological observation also showed that Lico A significantly improved the liver tissue and intestinal mucosa tissue damage caused by alcohol. Additionally, Lico A altered gut microbiota composition, accompanied by increased concentrations of fecal short-chain fatty acids (SCFAs), which restored microbial diversity and elevated the relative abundance of Actinomycetota, Bacteroidota, Bacillota_A_368345, Limosilactobacillus, Lactobacillus, and Bifidobacterium_388775. These results indicated that Lico A had better hepatoprotective effects on AALI, and its mechanisms may involve modulation of the gut–liver axis and the TLR4/NF-κB signaling pathway.

1. Introduction

Prolonged alcohol abuse, as well as acute binge drinking, imposes significant hepatic stress, leading to alcoholic liver injury (ALI) [1,2]. The pathogenesis of ALI is driven by ethanol-induced redox imbalance, apoptosis, and metabolic disorders [3,4,5]. Emerging research indicates the “gut–liver axis” significantly influences ALI progression [6,7]. Excessive alcohol intake compromises gut mucosal barrier functionality and induces significant intestinal microbial imbalance, thereby enhancing intestinal permeability. This promotes the entry of endotoxins, such as LPS, into the portal venous blood [8,9,10]. Once in the liver, LPS engages the TLR4 pathway in resident immune cells. This interaction initiates a potent inflammatory response, thereby strongly exacerbating hepatocyte damage [11,12,13]. Therefore, preventing ALI by interfering with the gut–liver axis has received widespread attention.

In recent years, functional foods, such as probiotics, dietary fiber, and specific phytochemicals (silymarin, etc.), have attracted widespread attention due to their preventive potential against ALI by regulating intestinal flora, repairing the intestinal barrier, and systemically inhibiting inflammatory cytokines [14,15]. As a classic example of a food-derived medicinal plant, licorice, one of the sweeteners and flavor enhancers for food, is often used in the development of health products for hepatoprotection and gastric mucosa protection, due to its anti-inflammatory and hepatoprotective activities [16]. Licochalcone A (Lico A, Figure 1A), a chalcone compound derived from Glycyrrhiza inflata, exhibits diverse pharmacological effects, including inhibitory effects on inflammation [17], free radical-scavenging capacity [18,19] and anti-neoplastic properties [20,21]. Recently, Lico A has garnered increasing interest as a bioactive food component for its hepatoprotective effects. It is reported that Lico A has preventive effects against MASLD or APAP-induced hepatotoxicity in mice [22,23]. Considering the pivotal function of nutrition and gut–liver interactions in ALI development and the capacity of food constituents to influence this pathway, this study intended to elucidate the prophylactic efficacy of Lico A against AALI via regulation of the gut–liver axis, establishing scientific validation for its development as a hepatoprotective dietary ingredient.

2. Materials and Methods

2.1. Chemicals and Reagents

The following were purchased: Lico A (purity ≥ 98%, Hubei Cuiyuan Biotechnology, Wuhan, China); DG (Diammonium Glycyrrhizinate, Chia Tai Tianqing Pharmaceutical, Lianyungang, China); ALT, AST, ALB, ALP, and TP (Shenzhen Mindray Bio-Medical Electronics, Shenzhen, China); LPS, DAO, SOD, MDA, GSH, NO, IL-1β, IL-6, and TNF-α (Wuhan Elabscience Biotechnology, Wuhan, China); ADH and ALDH (Shanghai Youxuan Biological Technology, Shanghai, China); MyD88, IκBα, p-IκBα, and GAPDH antibodies (Abcam, Cambridge, UK); TLR4, IKKβ, and NF-κB p65 antibodies (Cell Signaling Technology, Beverly, MA, USA); and p-NF-κB p65 antibody (Affinity Biosciences, Changzhou, China).

2.2. Animals

Kunming male mice (18–22 g) were maintained under the conditions of 23–25 °C, 50–60% humidity, and a 12 h light/dark photoperiod. The Xinjiang Medical University Animal Ethics Committee approved this experimental protocol (IACUC-JT-20241210-28).

2.3. Experimental Design

The AALI mouse model was constructed as per the previously reported methods [23,24]. The Kunming mice were randomly assigned to six experimental groups (n = 8): control, model, DG (positive control, 100 mg/kg), and Lico A (25, 50, 100 mg/kg) groups. The control and model groups were administered a daily oral dose of 0.5% CMC-Na solution at 10 mL/kg, while the other mice were given the drug solution at the same volume. One hour after each administration, the mice from all test groups received a daily intragastric dose of 60% ethanol at 12 mL/kg for 7 consecutive days, whereas deionized water was given to the control animals. Subsequent to the last intervention, the mice underwent a 12 h fasting period, followed by weighing and orbital blood collection. The blood rested for 1 h at room temperature and then underwent centrifugation at 3000 rpm for 15 min at 4 °C. The serum was then collected. The liver was excised and weighed, and its index was calculated. Meanwhile, liver and intestinal tissue samples were processed as required for subsequent experiments. The collected samples were kept at −80 °C.

2.4. Detection of Biochemical Indicators in Mice Serum and Liver Tissue

The serum ALT, AST, ALB, ALP, and TP levels in mice were measured using a BS-240vet biochemical analyzer (Shenzhen Mindray Animal Medical Technology, Shenzhen, China). The serum concentrations of DAO and LPS were determined by ELISA. Hepatic tissue was homogenized in PBS (1:9, m/v) to formulate a 10% liver suspension. Hepatic homogenate was used to measure the activities of ADH, ALDH, and SOD and the levels of MDA, GSH, NO, TNF-α, IL-1β, and IL-6, following the manufacturer’s instructions.

2.5. Histopathological Observation

Overnight fixation of hepatic lobe and jejunal tissues was carried out in 4% paraformaldehyde. The specimens were then processed for dehydration, paraffin-embedded, sectioned at a thickness of 5 μm, and stained with H&E. Additionally, mice colon tissues were fixed with Carnoy’s fixative, paraffin-embedded, sectioned, dewaxed, and stained with AB-PAS. Pathological changes were observed under light microscopy.

2.6. Immunohistochemical Analysis

Colon tissue sections embedded in paraffin underwent dewaxing and rehydration. Antigen recovery was achieved through microwave treatment in an EDTA buffered solution, followed by blocking of endogenous peroxidase with hydrogen peroxide and then blocking with 3% BSA. Incubation of the sections with primary antibodies against ZO-1 and Occludin (1:200) was carried out overnight at 4 °C. Upon completion of the washing procedure, incubation with secondary antibodies was carried out on the sections for 50 min under ambient conditions. Immunostaining was visualized using a DAB kit. Afterward, the slides were stained with hematoxylin, differentiated in hydrochloric acid alcohol, blued in ammonia water, dehydrated via a graded ethanol series, cleared with xylene, and coverslipped using neutral balsam. The staining intensity was analyzed quantitatively using ImageJ software (v1.8.0.112).

2.7. Western Blot

Liver tissues from mice were homogenized in 8 volumes of prepared lysis buffer. The protein content was subsequently determined using a BCA assay. The sample was mixed with loading buffer and boiled. The protein samples were resolved by SDS-PAGE, electrotransferred onto a PVDF membrane, and then incubated with a blocking buffer to block non-specific binding. The blots were incubated with specific primary antibodies at 4 °C overnight. They were subsequently treated with the secondary antibody for 1 h the next day. Finally, ECL substrate was applied for signal detection. The target proteins were quantified using ImageJ.

2.8. 16S rRNA Sequencing of Mice Gut Microbiota

Genomic DNA was isolated from jejunal contents obtained from the control, model, and Lico A-treated groups utilizing the OMEGA Soil DNA Kit (M5635-02; Omega Bio-Tek, Norcross, GA, USA). PCR amplification was performed targeting the V3–V4 region of the bacterial 16S rRNA gene. The amplified products were then quantified with the Quant-iT PicoGreen dsDNA Assay Kit using a BioTek FLx800 Microplate reader (Winooski, VT, USA). Subsequently, amplicon sequencing was carried out on the Illumina NovaSeq 6000 system with the SP Reagent Kit (500 cycles) (San Diego, CA, USA) to generate 2 × 250 bp paired-end reads. The acquired sequences were subjected to stringent processing, including quality checking, noise correction, fragment assembly, and the removal of chimeric sequences. Based on the resulting Amplicon Sequence Variants (ASVs), analyses including alpha diversity, beta diversity, taxonomic annotation, and differential analysis were performed.

2.9. Quantification of SCFAs in Mice Intestinal Contents by GC-MS

SCFAs in fecal supernatants were quantified using a GC-MS system (Thermo Trace 1300-ISQ 7000, Waltham, MA, USA). Briefly, the samples were resuspended in deionized water containing glass beads, vortexed vigorously, and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was treated with phosphoric acid and mixed with an internal standard, followed by extraction with ethyl ether. Following an additional round of centrifugation, the organic phase was collected and subjected to GC-MS analysis.

2.10. Statistical Analysis

SPSS 26.0 was used to analyze the results, and the data were expressed as mean ± SEM. Significant differences were assessed using one-way ANOVA or the Kruskal–Wallis test (p < 0.05).

3. Results

3.1. Protective Effects of Lico A on AALI Mice

In the experimental process, the control mice maintained a normal food intake and a glossy coat, indicating good health. In contrast, as the modeling period extended, the mice in the model group showed reduced food intake, an emaciated body shape, and decreased body weight, indicating that alcohol caused severe damage to the mice. The AALI mice showed a slow growth in body weight and a marked elevation in the liver index relative to the control group (p < 0.01). Upon Lico A intervention, these conditions in AALI mice were remarkably improved (p < 0.05, Figure 1B,C and Table S1). Lico A also attenuated the serum levels of ALT, AST, and ALP in AALI mice, as well as increasing the serum ALB and TP contents (p < 0.05, Figure 1D–H and Table S2). Histological examination of the control group indicated that the specimens displayed a typical hepatocellular structure with no visible abnormalities, while the liver tissue of the model group mice exhibited hyaline degeneration of liver cells, loose cytoplasm, lipid vacuole degeneration, and inflammatory cell infiltration. Lico A (25, 50, 100 mg/kg) alleviated the liver tissue damage caused by alcohol to different degrees (Figure 1I and Figure S1). Collectively, these findings demonstrated that Lico A exerts a significant protective effect against AALI.

3.2. Effects of Lico A on ADH and ALDH Levels in AALI Mice

Compared with the control group, AALI mice exhibited markedly reduced levels of hepatic homogenate ADH and ALDH enzymes (p < 0.01). Lico A (100 mg/kg) markedly elevated hepatic ADH and ALDH activities in AALI mice (p < 0.05, Figure 2 and Table S3).

3.3. Effects of Lico A on Hepatic Homogenate MDA, NO, SOD, and GSH Levels in AALI Mice

Figure 3 and Table S4 clearly demonstrate that treatment with Lico A (100 mg/kg) substantially decreased the MDA and NO concentrations and elevated the SOD activity and GSH levels in the liver tissue of AALI mice (p < 0.05).

3.4. Effect of Lico A on Hepatic Homogenate TNF-α, IL-1β, and IL-6 Levels in AALI Mice

The expression levels of TNF-α, IL-1β, and IL-6 in the hepatic homogenate of AALI mice were considerably higher than those in the control group (p < 0.01). Lico A (25, 50, 100 mg/kg) notably reversed the increases in these proinflammatory cytokines in AALI mice (p < 0.05, Figure 4 and Table S5).

3.5. Effects of Lico A on the Serum LPS and DAO Levels in AALI Mice

The serum levels of LPS and DAO were remarkably elevated in AALI mice compared to the control group (p < 0.01), and this elevation was reversed by treatment with Lico A (100 mg/kg) (p < 0.05, Figure 5 and Table S6).

3.6. Effects of Lico A on the TLR4/NF-κB Pathway in AALI Mice

Lico A significantly suppressed the TLR4/NF-κB pathway in the liver tissue of AALI mice. Figure 6 illustrates that the levels of TLR4, MyD88, IKKβ, p-NF-κB p65/NF-κB p65, and p-IκBα/IκBα in liver tissue were elevated in AALI mice in comparison to the control group (p < 0.01), whereas Lico A (100 mg/kg) suppressed this elevation in AALI mice (p < 0.05, Figure S2 and Table S7).

3.7. Effects of Lico A on Intestinal Histopathological Features in AALI Mice

To systematically evaluate intestinal barrier damage in AALI mice, we first performed H&E staining on jejunal tissues to assess the structural pathological changes and inflammatory infiltration. Figure 7 and Figure S3 demonstrate that the intestinal mucosa of the control group remained intact, exhibiting a well-organized structure. The intestinal tissue in AALI mice exhibited a certain degree of mucosal damage, characterized by mucosal necrosis, erosion, scattered inflammatory cell infiltration, amyloid deposition, and edema. Lico A (25, 50, 100 mg/kg) alleviated these injuries caused by alcohol to different degrees.

Subsequently, to further investigate the status of the chemical and functional defense barriers, AB-PAS staining was conducted on colon tissues to analyze the mucus layer and goblet cell function. As illustrated in Figure 7 and Figure S4, Table S8, the crypt structure in the control group was clearly defined, with abundant goblet cells present in each crypt. The acidic mucus and neutral mucus secreted by these cells were distinctly visible. In contrast, the model group exhibited significant pathological alterations, including focal desquamation of the mucosal epithelium, goblet cell depletion, and reduced mucus secretion, suggesting compromised integrity of the colonic mucosal barrier. In contrast to the model group, the Lico A groups showed a notable upregulation of the count of goblet cells and varying degrees of alleviation in intestinal tissue damage, suggesting that Lico A has a certain preventive and therapeutic effect on AALI mice.

3.8. Effects of Lico A on Colon Tissue ZO-1 and Occludin Protein Expression in AALI Mice

Lico A enhanced intestinal epithelial tight junctions and preserved intestinal mucosal barrier integrity. As shown in Figure 8, Figures S5 and S6, Table S9, unlike the control mice, AALI mice exhibited significantly downregulated ZO-1 and Occludin levels (p < 0.01), while Lico A (50, 100 mg/kg) markedly upregulated the levels of these proteins (p < 0.05).

3.9. Effect of Lico A on the Intestinal Flora in AALI Mice

Disruption of the intestinal microbial balance caused by excessive alcohol consumption can subsequently result in liver damage. To delve into how Lico A affects the alteration of gut microbiota due to alcohol consumption, the gut microbial composition of the mice was profiled using 16S rRNA. Compared with the control group, AALI mice exhibited significantly lower gut microbiota alpha diversity (p < 0.01), a deficiency that was effectively ameliorated by Lico A treatment (p < 0.05, Figure 9A–E and Table S10).

The PCoA analysis of intestinal flora revealed that the samples within each group exhibited good clustering, indicating small differences and high similarity among them. The dispersion of samples among groups was higher, with the control group clearly separated from the model and Lico A groups, while the model and Lico A groups exhibited considerable similarity, with a small overlap, which points to discrepancies in the intestinal microbiota composition across the experimental groups (Figure 9F,G). The data revealed that administration of Lico A reshaped the intestinal flora composition and distribution in mice.

As shown in Figure 10A and Table S11, the gut microbiota exhibited a phylum-level composition predominantly consisting of Actinomycetota, Bacteroidota, Bacillota and Pseudomonadota. The AALI mice exhibited a distinct shift in gut microbiota composition compared to the control group, characterized by significantly lower abundances of Bacteroidota and Bacillota_A_368345 (p < 0.05) and a markedly higher abundance of Pseudomonadota (p < 0.01). Lico A administration effectively reshaped the intestinal microbiota composition in AALI mice, enriching beneficial flora while suppressing the proliferation of potentially deleterious bacteria (p < 0.01).

As shown in Figure 10B and Table S12, at the genus level, Staphylococcus, Limosilactobacillus, Lactobacillus and Bifidobacterium_388775 constituted the dominant members of the gut microbiota. The abundance of Limosilactobacillus, Lactobacillus, and Bifidobacterium_388775 was notably reduced in the AALI group relative to the control group (p < 0.05). A relative increase in the abundance of Staphylococcus was also observed. Lico A administration induced a notable shift in the intestinal microbiota composition of mice, characterized by a markedly higher abundance of beneficial genera and a concomitant reduction in potentially opportunistic bacteria (p < 0.01).

As a common differential method, LEfSe analysis facilitates the simultaneous comparison of all taxonomic levels, thereby identifying more reliable potential biomarkers. In this study, 48 biomarkers were screened through LEfSe analysis. Referring to Figure 11, we can observe that a total of 57 taxa at different levels were identified, exhibiting varying degrees of abundance. Notably, the highest count of differentiated taxa was observed in the control group. Specifically, c_Actinomycetes, o_Mycobacteriales, g_Ligilactobacillus, g_Lactobacillus, and g_Limosilactobacillus were the most abundant in the control group, while g_Enterococcus_B and f_Enterococcaceae dominated in the model group. Additionally, f_Aerococcaceae, g_Ruoffia, and g_Aerococcus dominated in the Lico A group.

Statistically significant variations relative to the control group are presented in Figure 12 and Table S13; AALI mice exhibited significantly lower levels of acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acids in their feces (p < 0.01). Lico A administration markedly reversed the reduction in these SCFAs in AALI mice (p < 0.05).

4. Discussion

AALI comprises acute inflammation and dysfunction of the liver triggered by excessive alcohol intake and involves multiple pathogenic mechanisms including abnormal alcohol metabolism, oxidative stress, inflammatory responses, and gut–liver axis disorder caused by the impairment of the intestinal barrier function and intestinal flora [25,26]. The liver, the principal site for alcohol metabolism, can suffer varying degrees of damage from excessive drinking. Currently, Lico A has not been officially approved as a clinical therapeutic agent for AALI and, therefore, does not hold an established position within the contemporary medical framework. However, our study provides a theoretical basis for its potential future application by revealing for the first time that Lico A attenuates AALI by regulating the gut–liver axis, highlighting its potential as a preventive dietary supplement or adjunctive therapy, particularly in light of the intestinal-targeted interventions available for AALI.

The increment in liver function indicators (AST, ALT, etc.), steatosis, and inflammatory cell infiltration are typical characteristics of initial biochemical and pathological alterations in alcoholic liver disease [27,28,29]. In this study, Lico A treatment suppressed the elevation of the liver injury markers ALT, AST, and ALP in AALI mice while concurrently increasing the ALB and TP levels. Meanwhile, pathological observation revealed that Lico A significantly alleviated hepatic lipid deposition and injury-related inflammation in AALI mice. The metabolic clearance of alcohol in the liver relies on the synergistic action of ADH and ALDH [30]. Insufficient activities of these enzymes lead to the accumulation of ethanol and its toxic intermediate product, acetaldehyde, which directly damages the structure and function of hepatocytes [31]. Lico A significantly enhanced the hepatic ADH and ALDH activities, accelerated the conversion of ethanol into harmless products, and facilitated the excretion of these metabolites from the body. This reduced the sustained attack of toxic substances on hepatocytes at the source. Alcohol metabolism is commonly accompanied by the generation of ROS. Excess ROS provokes oxidative stress by overwhelming antioxidant defenses, and this process is also one of the core drivers of AALI progression [32,33]. Lico A effectively enhanced liver tissue SOD and GSH activities in AALI mice to strengthen antioxidant defense, while significantly reducing the MDA and NO levels, thereby curbing alcohol-induced oxidative stress.

Alcohol intake primarily targets the intestine, causing disruption of the intestinal barrier and subsequent hyperpermeability, which promotes the translocation of LPS produced by intestinal flora disturbance to the liver and systemic circulation. The binding of LPS and TLR4 activates the MyD88 protein, which subsequently activates the IKK complex. This causes IκB phosphorylation and degradation, permitting NF-κB nuclear translocation and leading to the overexpression of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 [34,35,36]. These cytokines can initiate and amplify the inflammatory cascade reaction in the liver, thereby promoting immune cell infiltration and liver cell necrosis [37]. Therefore, improvement in the gut–liver axis including restoration of the intestinal flora balance, the repair of intestinal mucosal barrier, and the suppression of the TLR4/NF-κB pathway is vital for AALI mitigation. This study demonstrated a protective effect of Lico A on the intestinal barrier. Lico A effectively alleviated damage to intestinal mucosal tissue, reducing serum LPS and DAO levels. Moreover, Lico A significantly downregulated the TLR4, MyD88, IKKβ, p-NF-κB p65/NF-κBp65, and p-IκBα/IκBα levels and further downregulated TNF-α, IL-1β, and IL-6 expression, thereby blocking the cascade amplification of inflammation.

The 16S rRNA gene sequencing results confirmed the alcohol-induced disruption of intestinal microbial homeostasis, mirroring prior findings that alcohol intake leads to dysbiosis of the gut microbiota [38,39]. Lico A restored the overall microbial abundance and diversity, promoting a more stable microbiota structure. Consistent with the modulatory effects observed with other natural products, cinnamon treatment has been demonstrated to similarly alter gut microbiota composition in lupus mice by mitigating the Firmicutes/Bacteroidota ratio decline, reducing Lachnospiraceae abundance, and boosting advantageous species such as Lactobacillus and Limosilactobacillus [40]. In addition, SCFAs, as crucial metabolites from the intestinal fermentation of dietary fiber, supply energy to the gut and are vital for preserving barrier integrity, immune homeostasis, and suppressing inflammation [41,42]. Recent studies have underscored the critical function of SCFAs in ameliorating ALI [43,44], and our findings align with this concept. Lico A markedly increased the fecal concentrations of SCFAs in mice with AALI. These findings indicate that the hepatoprotective effect of Lico A against AALI is mediated by the modulation of gut microbiota metabolism, leading to increased SCFA production.

Although this study systematically elucidated the multiple mechanisms by which Lico A ameliorates AALI via the gut–liver axis, it focused only on an acute model of AALI. Therefore, whether Lico A exerts similar protective effects in chronic alcoholic liver injury warrants further investigation. First, this study lacks direct verification of the impact of gut microbiota on the hepatoprotective effects of Lico A via fecal microbiota transplantation experiments. Future studies could employ germ-free or antibiotic-treated mice receiving microbiota from Lico A-treated donors to confirm whether specific microbiota or their metabolites mediate this effect. Second, while Lico A was observed to suppress the TLR4/NF-κB pathway, its critical role in gut–liver axis regulation requires further validation using gene knockout animals or specific inhibitors to determine whether it constitutes the core anti-inflammatory mechanism.

5. Conclusions

Lico A can exert a protective effect on AALI through repairing the intestinal barrier, improving intestinal flora, blocking TLR4/NF-κB signaling pathways, inhibiting proinflammatory cytokine expression, and exerting antioxidant effects. These results provide supporting data for the application of Lico A as a health product in the prevention of alcoholic liver damage, as well as for the development and utilization of G. inflata.