Aspirin Eugenol Ester Alleviates Gastric Injury by Inhibiting Ferroptosis and Oxidative Stress

TL;DR

Aspirin eugenol ester (AEE) protects the stomach from alcohol-induced damage by reducing inflammation, oxidative stress, and cell death.

Contribution

AEE is shown to alleviate gastric injury through inhibition of ferroptosis and oxidative stress pathways.

Findings

AEE significantly reduced ethanol-induced gastric mucosal injury and restored mucus secretion.

AEE inhibited NF-κB activation and reduced inflammatory cytokine expression.

AEE enhanced antioxidant levels and suppressed ferroptosis by modulating Nrf-2/GPX4 pathways.

Abstract

Gastric ulcer (GU) is a common upper gastrointestinal disorder characterized by oxidative stress and inflammatory responses, which significantly impact the patient’s quality of life and pose a serious challenge to public health. Excessive and chronic alcohol consumption are considered primary contributing factors to gastric ulcer. Pharmacodynamic and pharmacological experiments showed that aspirin eugenol ester (AEE) had good anti-inflammatory and anti-oxidant effects. Therefore, it was speculated that AEE could alleviate ethanol-induced gastric mucosal injury through anti-inflammatory and antioxidant pathways. This study aimed to systematically evaluate the effect of AEE on ethanol-induced gastric mucosal injury using in vivo and in vitro experiments. In a gastric injury model induced by ethanol, H&E staining, AB-PAS staining, RT-PCR, immunohistochemistry, and a series of other molecular biological assays and omics techniques were employed to investigate AEE potential mechanisms. The results revealed extensive necrosis in the gastric mucosa of the ethanol group with a marked reduction in mucus secretion on the mucosal surface and a significantly decreased expression of ZO-1, claudin-1, and occludin, while AEE exhibited a significant protective effect when compared to ethanol group. AEE inhibited NF-κB pathway activation and reduced the expression of inflammatory cytokines. AEE significantly enhanced superoxide Dismutase (SOD) levels and reduced the ethanol-induced increases in reactive oxygen species (ROS), malondialdehyde (MDA), and lipid peroxidation (LPO) levels by reversing the ethanol-induced decline in Nrf-2 expression. AEE also mitigated cellular damage by inhibiting ferroptosis in the cells. AEE significantly improved the metabolic profiles of gastric tissue and serum. AEE exerts gastric protective effects by synergistically modulating multiple pathways and biological processes. AEE can enhance antioxidant capacity and inhibit ferroptosis by activating the Nrf-2/GPX4 pathway, alleviate inflammatory responses by suppressing the NF-κB pathway, and simultaneously maintain gastric mucosal barrier integrity through regulation of metabolic reprogramming and enhancement of tight junction function.

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Full text

1. Background

Gastric mucosa serves as the first line of defense within the stomach cavity, performing vital functions in resisting gastric acid, digestive enzymes, mechanical friction from food, and invasion by pathogenic microorganisms. This protective function relies on a complex physiological structure, including the gastric mucus–bicarbonate barrier, epithelial cell barrier, adequate mucosal blood flow, cellular renewal and repair capabilities, and immune defense mechanisms. When this equilibrium is disrupted, the gastric mucosa becomes vulnerable to damage, triggering a series of pathological changes ranging from mild inflammation to severe ulcers and even increasing the risk of gastric cancer [1,2]. Gastric ulcer (GU) is a common upper gastrointestinal disorder characterized by damage to the gastric mucosa due to an imbalance between protective and destructive factors in the stomach, accompanied by increased oxidative stress, inflammation, bleeding, erosion, and gastric wall ulcer formation [3]. GU is closely associated with factors such as Helicobacter pylori infection, alcohol consumption, and excessive use of nonsteroidal anti-inflammatory drugs [4,5,6,7]. Globally, GU ranks among the most prevalent upper gastrointestinal diseases, with a lifetime prevalence of 5–10% [5,8]. In China, approximately 6.1% of the population will be diagnosed with GU during their lifetime [9]. Without timely intervention, GU may progress to gastrointestinal bleeding and perforation, severely impacting the quality of life for patients and posing a significant challenge to public health.

Excessive and long-term alcohol consumption can cause acute and chronic damage to the gastric mucosa, leading to elevated hydrochloric acid concentrations in gastric juice and resulting in mucosal injury, which is considered the primary cause of GU [10,11]. Ethanol (EtOH) is a small-molecule, low-polarity organic solvent that permeates the lipid bilayer of gastric mucosal cells via passive diffusion [12,13]. It directly corrodes the gastric mucosa and mucosal tissues, damaging the gastric barrier. The mechanism of EtOH-induced gastric mucosal injury is multifaceted, and current research primarily categorizes it into direct and indirect effects. Direct damage includes mucosal disruption, dissolution of the mucus HCO_3_^−^-phospholipid layer, disruption of tight junctions, gastrointestinal bleeding, epithelial detachment, gastric mucosal edema, and ulcer formation. Indirect mechanisms involve EtOH-induced barrier disruption, leading to oxidative stress in gastric tissue, inflammatory responses, and impaired gastric acid secretion [14,15,16,17]. These pathological outcomes not only diminish the quality of life but also impose a significant burden on global healthcare systems. Currently, the treatment strategy for EtOH-induced gastric injury primarily focuses on alleviating symptoms [18]. Commonly used drugs include proton pump inhibitors, H2 receptor antagonists, antacids, acid neutralizers, and gastric mucosal protectants, which neutralize gastric acid or inhibit gastric acid secretion [4,19,20]. However, the potential side effects of long-term use, such as chronic kidney disease, allergic reactions, constipation, vomiting, malabsorption, and dysbiosis, limit their application [21,22,23,24]. Therefore, identifying highly effective and low-toxicity drugs for the prevention and treatment of gastric ulcers is of significant importance.

EtOH-induced oxidative stress, inflammatory responses, and apoptosis play pivotal roles in gastric mucosal injury [25]. During metabolism, EtOH generates substantial reactive oxygen species (ROS), which disrupt the body’s antioxidant balance and suppress endogenous antioxidant defense mechanisms in the gastric mucosa, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) [26,27]. ROS, including superoxide anion (O_2_^−^), hydrogen peroxide (H_2_O_2_), and hydroxyl radicals (·OH), disrupt membrane integrity through multiple pathways, such as lipid peroxidation (LPO), protein oxidation, and DNA damage, impairing mitochondrial function and triggering a cascade of cellular injury [28]. Multiple studies showed that inhibiting oxidative stress protects the gastric mucosa, reduces inflammation, and impedes inflammatory cell infiltration. For example, Dan-Shen-Yin particles mitigate EtOH-induced gastric mucosal damage by suppressing oxidative stress, inflammation, and apoptosis [13]. Kaempferia galanga L., kaempferol, and luteolin exhibit protective effects against EtOH-induced gastric ulcers. Their mechanism involves regulating the mucosal barrier, oxidative stress, and gastric mucosal mediators, inhibiting the TRPV1 signaling pathway and gastric acid secretion, and ultimately reducing the gastric ulcer index [29]. Sargassum siliquastrum attenuates EtOH-induced gastric mucosal injury by suppressing oxidative stress and inflammatory responses [30]. Inflammation is another key mechanism underlying EtOH-induced gastric injury. Oxidative stress and inflammation form a vicious cycle. ROS activate multiple transcription factors, such as nuclear factor kappa-B (NF-κB), which promotes the expression and release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). This activation initiates and amplifies the inflammatory response. Infiltration of inflammatory cells generates additional ROS, further exacerbating oxidative stress. Notably, key factors in EtOH-induced gastric injury, such as ROS, LPO increase, and GSH depletion, highly overlap with the core features of ferroptosis. In recent years, the discovery of ferroptosis, a novel iron-dependent form of programmed cell death, has opened new perspectives for understanding the pathological processes of various diseases [31,32]. Ferroptosis is characterized by the iron-dependent accumulation of LPO, which differs from traditional apoptosis and necrosis [33]. Excessive intracellular iron ion (Fe^2+^) accumulation catalyzes the production of massive amounts of lipid peroxides via the Fenton reaction. Concurrent impairment of the cell’s own lipid peroxide repair system leads to a lethal LPO cascade, resulting in oxidative damage and cell death [34]. Ferroptosis contributes to EtOH-induced gastric mucosal injury [35]. Therefore, effective therapeutic strategies must simultaneously counteract both oxidative stress and inflammatory responses.

Eugenol has various pharmacological activities such as anti-inflammatory, antipyretic, and antioxidant, but the instability and pungent odor exhibited by eugenol limits its application [36,37,38]. Aspirin is a nonsteroidal anti-inflammatory drug (NSAID) widely used for its anti-inflammatory, antiplatelet, antipyretic, and analgesic effects. However, long-term use may cause side effects, including gastric ulcers, renal failure, impaired platelet function, and bleeding complications [39,40]. Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterification of aspirin with eugenol using a prodrug principle [41,42] (Figure 1). AEE maintains the pharmacological activity of aspirin and eugenol, masks the carboxyl group of aspirin, and reduces the side effects of aspirin on the gastrointestinal tract when it enters the body. Previous studies showed that AEE has various pharmacological activities, such as anti-inflammatory and anti-oxidative stress. For example, AEE reduced paraquat-induced hepatotoxicity by inhibiting oxidative stress and maintaining mitochondrial function [43]. AEE could significantly enhance the cellular SOD and glutathione peroxidase activities in HUVEC [44]. AEE protected vascular endothelial cells from oxidative damage by regulating NOS and Nrf-2 signaling path ways [45]. These studies indicated that AEE could improve oxidative stress, mitochondrial dysfunction, and immune–inflammatory system activation. Therefore, it was speculated that AEE could alleviate EtOH-induced gastric mucosal injury through anti-inflammatory and antioxidant pathways.

This study aims to systematically evaluate the protective effects of AEE against EtOH-induced gastric mucosal injury through in vivo and in vitro experimental models. This research focuses on investigating whether AEE exerts its effects by regulating key pathways involved in ferroptosis and oxidative stress. By integrating omics technologies and molecular biological detection methods, this study elucidates the mechanism of action of AEE. The findings provide a theoretical basis for the clinical application of this treatment in addressing gastric mucosal injury and offer valuable insights for the development of similar chemically synthesized drugs.

2. Materials and Methods

2.1. Chemicals and Reagents

AEE (purity 99.5%) was prepared at the Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS (Lanzhou, China). Anhydrous ethanol (EtOH) and carboxymethylcellulose sodium (CMC-Na) (30036328) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Human gastric mucosal epithelial cells (GES-1) were obtained from the Shanghai Chinese Academy of Sciences Cell Bank (Shanghai, China). Ferrostatin-1 (Fer-1, which is ferroptosis inhibitor) (HY-100579), RSL-3 (Ferroptosis agonist) (HY-100218A), (R)-Sulforaphane (Nrf-2 agonist) (HY-13755A), and ML385(Nrf-2 inhibitor) (HY-100523) were purchased Med Chem Express (Shanghai, China). Fetal Bovine Serum (AUS-01E-02) was purchased Cell-Box (Changsha, China). DMEM glucose medium and trypsin were purchased from Gibco (Shanghai, China). Cell Counting Kit-8 (C6050) was purchased from NCM Biotech (Suzhou, China). Nrf-2 protein (CSB-EP614961HU1) was purchased from Huamei Bioengineer Company (Wuhan, China). Biotin (21343) and desalting columns (89882) were purchased from Thermo Fisher Scientific (Waltham, MA USA). PrimeScript™ RT Master Mix (Perfect Real Time) (RR036Q), TaKaRa MiniBEST Universal RNA Extraction Kit (9767), PrimeScript™ RT Master Mix (RR036A) and TB Green^®^ Premix Ex Taq™ II FAST qPCR (CN830A) were purchased from Takara (Nanjing, China). Other analytical-grade reagents were purchased from Sinopharm Group (Shanghai, China).

2.2. In Vitro Experiment

2.2.1. Cell Culture and Treatments

GES-1 cells were cultured in DMEM with 10% Fetal Bovine Serum under normoxic conditions (37 °C, 5% CO_2_). Cells were observed under an inverted microscope and passaged with 0.25% trypsin when confluency reached over 95%. First, cells were treated with different concentrations of EtOH (1–9%) for 2 h to select an appropriate EtOH concentration for inducing a cellular injury model. Cells were then treated with a medium containing different drugs at various concentrations for 24 h to determine the appropriate drug concentrations for subsequent experiments.

2.2.2. Cell Viability Assay and Morphological Observation

GES-1 cell survival rates exposed to different drugs were determined according to the manufacturer’s instructions for the CCK-8 assay (Beyotime, Shanghai, China). Finally, the number of viable cells was assessed by measuring the absorbance at 450 nm (Multiskan™ FC; Thermo Scientific™, Waltham, MA USA). Subsequently, morphological changes were observed and photographed using an inverted microscope.

2.2.3. EtOH Stimulation in Rat Microglia Cells

GES-1 cells (1 × 10^5^ cells) were cultured in 12-well plates. Cells were treated with different concentrations of AEE (16, 32, and 64 μM) for 24 h, which were selected based on the cytotoxicity assay results, followed by exposure to EtOH (5%) for 2 h. Cells and cell-free supernatants were collected and stored at −80 °C for subsequent experiments.

2.2.4. Cell SOD Assay

The level of SOD (E2010) in the cells was measured using the appropriate kit (Applygen Technologies, Inc., Beijing, China) according to the manufacturer’s instructions. Briefly, after cell collection, wash with pre-chilled PBS. Add the extraction solution in proportion and sonicate on ice. Then, centrifuge at 8000× g, 4 °C for 10 min to obtain the supernatant as the test sample. In a 96-well plate, sequentially add the sample, working solution, and reaction starter. Mix thoroughly and incubate at 37 °C in the dark for 30 min. Measure absorbance at 450 nm.

2.2.5. Measurement of ROS in Cell

The level of reactive oxygen species (ROS) (S0033S) in cells was measured using the appropriate reagent (Bi yun tian, Hangzhou, China) according to the manufacturer’s instructions. Firstly, dilute DCFH-DA appropriately with PBS at a ratio of 1:1000 to achieve a final concentration of 10 μM. Subsequently, remove the cell culture medium and add an appropriate volume of the diluted DCFH-DA. Incubate at 37 °C in a cell culture incubator for 20 min. Wash cells three times with PBS to thoroughly remove any DCFH-DA that did not enter the cells. Observe directly using a laser confocal microscope.

2.2.6. Measurement of Nuclear Morphological Changes

Cell nuclear morphology was observed using Hoechst 33342 (C1022, Biyuntian, Hangzhou, China) fluorescent staining. Briefly, after washing cells with PBS, incubate them in a 5 μg/mL Hoechst 33342 working solution at 37 °C in the dark for 15 min. Following incubation, wash cells twice with PBS to remove unbound dye. Observe immediately under a fluorescence microscope.

2.2.7. Measurement of Intracellular Fe2+ Content

The intracellular and tissue ferrous iron (Fe^2+^) content was determined using the Ferrous Ion Content Assay Kit (E1046, Applygen Technologies, Inc., Beijing, China) following the manufacturer’s protocol. Briefly, cell samples were washed twice with phosphate-buffered saline (PBS) and lysed with 300 μL of extraction solution via ultrasonication. The resulting lysates and homogenates were centrifuged at 10,000× g for 10 min at 4 °C to collect the supernatant. For quantification, 200 μL of supernatant was mixed with 100 μL of colorimetric working solution in a 1.5 mL tube and incubated at 37 °C for 10 min, and then 200 μL of the reaction mixture was transferred to a 96-well plate. The absorbance was measured at 593 nm, and Fe^2+^ concentrations were calculated based on the absorbance values.

2.2.8. Lipid Peroxidation Assay

First, aspirate the cell culture medium and wash the cells once with PBS. Add BDPY 581/591 C11 (Bi yun tian, Hangzhou, China) staining working solution and incubate at 37 °C in a cell culture incubator for 10 min. After incubation, aspirate the supernatant and wash twice with PBS. Detect using a multifunctional fluorescence microplate reader. The BODIPY 581/591 C11 probe displays distinct spectral properties depending on its oxidation state: in the reduced form, it exhibits excitation/emission maxima at 581/591 nm (red fluorescence), whereas upon oxidation by lipid hydroperoxides, its excitation/emission maxima shift to approximately 488/510 nm (green fluorescence). The extent of lipid peroxidation was quantified ratiometrically by calculating the ratio of red to green fluorescence intensity.

2.2.9. Transcriptomic Sequencing and Analysis

GES-1 cells were collected from the Model group (exposed to 5% EtOH for 2 h) and AEE group (pre-incubated with 32 μM AEE for 24 h and then exposed to 5% EtOH for 2 h). Total RNA was extracted from the cells using a total RNA extraction kit. A bipartite 150 bp sequencing library was constructed using the Illumina NovaSeq X Plus platform (Illumina, San Diego, CA, USA) for high-throughput sequencing. Differential expression analysis between the Model and AEE groups was performed using DESeq2 1.26.0, with significantly differentially expressed genes identified based on p < 0.05 and |log2FC| ≥ 0.585. GO and KEGG enrichment analyses were performed using Fisher’s exact test, with p < 0.05 indicating significant enrichment based on the differentially expressed genes.

2.2.10. RT-qPCR Detection of Cellular Gene Expression

Total RNA was extracted from GES-1 cells using a kit, and the RNA concentration was measured using NanoDrop (Nanodrop 2000, Thermo Fisher Scientific, USA). According to the kit instructions, 2000 ng of RNA from each sample was reverse-transcribed into cDNA based on the RNA concentration. qPCR was performed using a fast qPCR kit (Takara, Nanjing, China). The qPCR program was as follows: pre-denaturation at 95 °C for 30 s, followed by 40 cycles of PCR at 95 °C for 5 s and 60 °C for 10 s. GAPDH mRNA were used as an internal reference. The samples were quantified using the comparative Ct method (2^−ΔΔCT^) for the relative quantification of gene expression. The PCR primer sequences are listed in Table S1.

2.2.11. ELISA Detection of Relevant Indicators in GES-1 Cells

The levels of Nrf-2 (JL18367), GPX4 (JL46163), FTH (JL52623), P53 (JL17317), VEGF (JL18341), and ZO-1 (JL19531) in cells were measured using the appropriate kits (Shanghai Jianglai Biotech, Shanghai, China) according to the manufacturer’s instructions.

2.2.12. Agonist and Inhibitor Treatment of GES-1 Cells

GSE-1 cells were divided into four groups: Control, RSL-3, Fer-1 + RSL-3, and AEE + RSL-3. In the RSL-3 group, cells were first cultured in complete medium for 24 h and then incubated in complete medium containing 10 μM RSL-3 for 24 h. The Fer-1 + RSL-3 group was incubated in complete medium containing 100 μM Fer-1 for 24 h, followed by incubation in complete medium containing 10 μM RSL-3 for 24 h. The AEE + RSL-3 group was cultured in complete medium containing 32 μM AEE for 24 h, followed by incubation in complete medium containing 10 μM RSL-3 for 24 h. The Control group was cultured in complete medium replaced after 24 h.

GSE-1 cells were divided into three groups: Control, ML385, and (R)-Sulforaphane. In the ML385 group, cells were cultured for 24 h in complete medium containing 10 μM ML385. The (R)-Sulforaphane group was cultured for 24 h in complete medium containing 10 μM (R)-Sulforaphane. The Control group was cultured in complete medium.

GSE-1 cells were divided into four groups: Control, ML385, (R)-Sulforaphane + ML385, and AEE + ML385. The ML385 group was first cultured in complete medium for 24 h, then incubated in complete medium containing 10 μM ML385 for 24 h. The (R)-Sulforaphane + ML385 group was incubated in complete medium containing 10 μM (R)-Sulforaphane for 24 h, followed by incubation in complete medium containing 10 μM ML385 for 24 h. The AEE + ML385 group was cultured in complete medium containing 32 μM AEE for 24 h, followed by incubation in complete medium containing 10 μM ML385 for 24 h. The Control group was cultured in complete medium replaced after 24 h.

GSE-1 cells were divided into four groups: Control, RSL3, (R)-Sulforaphane + RSL3, and AEE + RSL3. In the RSL3 group, cells were first cultured in complete medium for 24 h, then incubated in complete medium containing 10 μM RSL3 for 24 h. The (R)-Sulforaphane + RSL3 group was incubated in complete medium containing 10 μM (R)-Sulforaphane for 24 h, followed by incubation in complete medium containing 10 μM RSL3 for 24 h. The AEE + ML385 group was incubated in complete medium containing 32 μM AEE for 24 h, and then in complete medium containing 10 μM RSL3 for 24 h. The Control group was cultured in complete medium replaced after 24 h.

2.3. In Vivo Experiment

2.3.1. Animal Experiment and Study Design

Forty-eight male Sprague Dawley (SD) rats (8 weeks old) weighing 220–240 g were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou, China). The rats were housed in an environment as previously described [46]. The rats were acclimatized for six days before starting the study. The rats were randomly divided into six groups (n = 8), such as Control group (0.5% CMC-Na), EtOH group (0.5% CMC-Na), EtOH + AEE low-dose group (54 mg∙kg^−1^∙d^−1^ b.w.), EtOH + AEE medium-dose group (108 mg∙kg^−1^∙d^−1^ b.w.), EtOH + AEE high-dose group (216 mg∙kg^−1^∙d^−1^ b.w.), and the EtOH + Omeprazole group (30 mg∙kg^−1^∙d^−1^ b.w.) serving as the drug control group. All the administered drugs were prepared as suspensions in 0.5% CMC-Na solution. To eliminate the influence of the vehicle solvent CMC-Na on the experiment, Control and EtOH groups received an equal volume of 0.5% CMC-Na solution. Briefly, SD rats were given either the vehicle alone (0.5% CMC-Na) or the vehicle combination containing drugs by gavage once a day for fourteen consecutive days. On the 15th day of the experiment, all rats were fasted for 12 h. Subsequently, rats in each group, except the Control group, were administered EtOH (5 mL∙kg^−1^) via oral gavage and euthanized 1 h later. Blood samples were collected from the abdominal aorta. Tissue samples were carefully obtained, rapidly frozen in liquid nitrogen or fixed in paraformaldehyde, and stored at −80 °C for further analysis.

Twenty-four SD male rats (8 weeks old) weighing 220–240 g were acclimatized for six days before the start of the study. The rats were randomly divided into three groups (n = 8), such as Control group (0.5% CMC-Na), EtOH group (0.5% CMC-Na), and EtOH + AEE group (54 mg∙kg^−1^∙d^−1^ b.w.). Briefly, the rats in the EtOH group were administered 60% EtOH (5 mL·kg^−1^) once a day for five consecutive days. The Control and AEE groups received equivalent volumes of ultrapure water. Subsequently, AEE were administered in AEE group rats once daily for three consecutive days. Control and EtOH groups received equivalent volumes of 0.5% CMC-Na. At the end of the experiment, rats were euthanized by intraperitoneal injection of sodium pentobarbital, followed by sample collection, rapidly frozen in liquid nitrogen or fixed in paraformaldehyde, and stored at −80 °C for further analysis.

Thirty male C57BL/6J mice (8 weeks old) weighing 20~22 g were randomly divided into three groups (n = 10), such as Control group (0.5% CMC-Na), Model group (0.5% CMC-Na), and AEE group (108 mg∙kg^−1^∙d^−1^ b.w.). Briefly, all mice were acclimated for six days on a standard Lieber–DeCarli diet. Control group mice received the standard Lieber–DeCarli diet, while Model and AEE groups were fed an alcohol-infused Lieber–DeCarli diet. AEE were administered in AEE group mice by gavage, while the Control and Model groups received an equal volume of 0.5% CMC-Na solution once daily for eleven consecutive days. On the 18th day, the Model and AEE groups received a 31.5% EtOH solution (5 g·kg^−1^) by gavage, while the Control group received an equivalent volume of maltodextrin. All mice were euthanized 9 h later, followed by sample collection, rapidly frozen in liquid nitrogen or fixed in paraformaldehyde, and stored at −80 °C for further analysis.

2.3.2. Body Temperature Detection and Organ Index

Rat body temperature was rectally recorded using an electronic thermometer at the time of euthanasia. The spleen, liver, lungs, and kidneys of rats were collected for this study. Each organ was immediately rinsed with physiological saline, suctioned dry to remove surface moisture, weighed, and recorded. Organ Index = Organ weight (g)/Body weight (kg).

2.3.3. HE Staining for Histopathological Analysis

Gastric tissue was fixed in 4% paraformaldehyde solution for 24 h, followed by graded dehydration. The specimens were embedded in paraffin, sectioned at a thickness of 5 μm, and stained with hematoxylin and eosin (H&E) for histopathological evaluation. The Supplementary Material provides detailed instructions for sample handling. Pathology analysis was performed at a magnification of 400×.

2.3.4. AB-PAS Staining for Histopathological Analysis

Gastric tissue was fixed in 4% paraformaldehyde solution for 24 h, dehydrated using an automated tissue processor, embedded in paraffin and sectioned, then stained with AB-PAS solution for histopathological evaluation. The Supplementary Material provides detailed instructions for sample handling. Pathological analysis was performed at a magnification of 100×.

2.3.5. ELISA Detection of Gastric Tissue and Serum Indicators

The levels of MDA (Rat, JL53632), SOD (Rat, JL22893), GPX4 (Rat, JL48671), P65 (Rat, JL21039), Nrf-2 (Rat, JL13922), PUMA (Rat, JL15643), ZO-1 (Rat, JL21101), EGF (Rat, JL12974), ALOX15 (Rat, JL20458), COX-1 (Rat, JL21043), COX-2 (Rat, JL21044), IL-1β (Rat, JL20884), TNF-α (Rat, JL13202), TP53 (Rat, JL25554), VEGF (Rat, JL21369), Nrf-2 (Mice, JL18277), GPX4 (Mice, JL46273), FTH (Mice, JL48588), ACSL4 (Mice, JL48582), P65 (Mice, JL50999), and LPO (Mice, JL50336) in samples were measured using the appropriate kits (Shanghai Jianglai Biotech, Shanghai, China) according to the manufacturer’s instructions. The level of MUC6 (Rat, ml059553) in samples was measured using the appropriate kit (Enzyme-linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions.

2.3.6. Measurement of Tissue and Serum Fe2+ Content

The content of Fe^2+^ in tissue and serum was determined using the appropriate kit (E1045) (Applygen Technologies, Inc., Beijing, China) according to the manufacturer’s instructions.

2.3.7. Immunohistochemistry

The 4% paraformaldehyde-fixed samples were embedded in paraffin, followed by a series of processing steps, and finally sealed with neutral resin. The Supplementary Material provides detailed instructions for sample handling. All antibodies used are listed in Table S2. The number of positive cells per 400× image was counted using the Halo data analysis system.

2.3.8. Immunofluorescence

The 4% paraformaldehyde-fixed samples were paraffin-embedded, followed by a series of processing steps, and finally sealed with an antifluorescence quenching sealing solution. The Supplementary Material provides detailed instructions for sample handling. All antibodies used are shown in Table S3. The percentage of positive area in each image was calculated using the Halo data analysis system.

2.3.9. Gastric Tissue Gene Expression Detected by RT-qPCR

Total RNA was extracted from gastric tissue using the TransZol method, and its concentration was measured. β-actin served as the inner reference, with the remaining procedures following the protocol in Section 2.2.11. The PCR primer sequences are listed in Table S1.

2.3.10. Molecular Docking

The RCSB PDB database (http://www.rcsb.org/ accessed on 20 July 2025) was used to download the crystal structures of the relevant proteins and save them in pdb format. Water molecules, metal ions, and ligands were removed from the protein using PyMoL 2.4. Proteins added nonpolar hydrogen bonds and balanced charges using AutoDock Tool 1.5.7. In this study, the binding modes of AEE, Nrf-2 protein (PDB: 7X5E), GPX4 protein (PDB: 7U4K), and FTH protein (PDB: 4OYN) were explored, and the docking results between different drugs and key targets were evaluated based on the obtained binding energy values. Docking results were visualized and analyzed using PyMOL and Discovery Studio 4.0.

2.3.11. BLI Detection Combined with Affinity

In this study, Bio-Layer Interferometry (BLI) was used by immobilizing a biomolecule onto the surface of a biosensor probe (SAXT). When another molecule binds to the ligand in solution, it causes a change in the thickness of the biological layer on the probe surface, thereby altering the interference spectrum of reflected light. By continuously monitoring this spectral shift, the instrument directly measures the kinetic processes of molecular binding and dissociation, ultimately calculating binding affinity. Nrf-2 protein (dissolved in 10 mM PBS, pH 7.4) with biotin was added at a molar ratio of 1:1. The samples were then incubated at room temperature in the dark for 1 h. Unreacted free biotin was removed using a desalting column. The protein was collected as an elution peak to obtain biotin-labeled Nrf-2 protein. The SAXT was used to immobilize biotin-Nrf-2, which bound to a small molecule, to obtain a protein–small molecule kinetic binding curve. After reference subtraction of the sensor curve using Gator Prime analysis software 2.7.3.0728, a global fit was performed using a 1:1 binding model to calculate the binding rate constant (Kon), dissociation rate constant (Koff), and equilibrium dissociation constant (KD).

2.3.12. Metabolomics Analysis

Metabolite Extraction and LC-MS Detection

The Supplementary Material provides detailed instructions for sample handling and detailed parameters of the liquid–mass spectrometer.

Metabolomics Data Analysis

The Supplementary Material provides detailed methods for metabolomics analysis and metabolite identification.

2.4. Statistical Analysis

All statistical analyses were performed using GraphPad Prism software (version 9). All data were presented as mean ± SD. The differences among the different treatment groups were analyzed using one-way ANOVA followed by Duncan’s multiple-comparisons test. Student’s t-test was used to compare two groups. Statistical significance was considered at p < 0.05.

3. Results

3.1. AEE Prevented EtOH-Induced Gastric Mucosal Injury in Rats

Animal experimental protocol is illustrated in Figure 2A. As shown in Figure 2B, compared with the Control group, the rats in the EtOH group exhibited a significant increase in body temperature (p < 0.05). Compared with the EtOH group, rats in all AEE-dose groups showed a significant decrease in body temperature (p < 0.05), whereas no significant difference was observed in the Omeprazole group (p > 0.05). Compared with the Control group, there were no significant differences in liver, lung, and kidney indices among the various drug-treated groups (p > 0.05). Compared with the Control group, the spleen index of rats in the EtOH group was significantly higher (p < 0.05). Compared with the EtOH group, the spleen index was significantly decreased in the low- and high-dose AEE groups (p < 0.05), while no significant difference was observed in the medium-dose AEE group (p > 0.05). The spleen index was significantly decreased in the Omeprazole group (p < 0.05). These results indicated that the various drug treatments had no significant effect on rat liver, lung, and kidney tissues. Rats in the EtOH group exhibited significantly elevated body temperature and spleen index, suggesting that EtOH induced an inflammatory response in rats. Both AEE and Omeprazole reduced EtOH-induced inflammation in rats (Figure 2C). As shown in Figure 2D, compared with the Control group, rats in the EtOH group exhibited severe gastric ulceration, surface hemorrhage, and edema. Compared with the EtOH group, the gastric tissue pathological changes in rats from all drug groups improved, with the low-dose AEE group showing the most favorable effect. Therefore, the low-dose AEE group (54 mg∙kg^−1^) was selected for subsequent experiments, and the effects of the drugs on the gastric tissue were further evaluated using HE staining.

The rats in the Control group showed intact gastric tissue structures, comprising the mucosa, submucosa, muscularis, and serosa. The rats in the EtOH group exhibited more severe gastric tissue lesions, characterized by desquamation of the single-layered columnar epithelium in the mucosal layer. Red blood cells were observed to extravasate into the extravascular stroma. AEE and Omeprazole groups exhibited milder gastric tissue lesions in rats, characterized by minor desquamation of the single-layered columnar epithelium in the mucosal layer. However, AEE demonstrated superior efficacy compared with Omeprazole. Pathological changes in the gastric epithelial mucosa were used to further evaluate the effects of the drug on gastric tissue via AB-PAS staining. The rats in the Control group showed intact mucosal layer structure in gastric tissue, with visible mucus-secreting glycoproteins in the mucosal layer appearing purplish-red superficially. EtOH group rats exhibited necrosis of the gastric mucosal layer, shedding of gastric gland cells, absence of mucous cells, and markedly reduced superficial mucus secretion, along with visible hemorrhage and extensive red blood cell extravasation. The rats in AEE and Omeprazole groups exhibited localized necrosis of the gastric mucosal layer, shedding of gastric gland cells, reduced mucus secretion content, and lighter local staining in rat gastric tissues. However, AEE showed superior efficacy compared to Omeprazole. In summary, compared with the Control group, the EtOH group exhibited more pronounced pathological alterations in gastric tissue with significantly reduced mucus secretion. Compared with the EtOH group, the Omeprazole group showed less severe lesions and deeper mucus staining, while the AEE group demonstrated the mildest lesions, greater mucus secretion, and deeper staining.

3.2. AEE Alleviated EtOH-Induced Damage in GES-1 Cells

The results in Figure 3A revealed that the morphology of GES-1 cells, as observed under an inverted microscope, exhibited progressive shrinkage, rounding, and detachment as EtOH concentration increased. EtOH cytotoxicity was assessed using the CCK-8 assay. The results revealed that cellular toxicity increased progressively with increasing EtOH concentration. At an EtOH concentration of 5% in the culture medium, cell viability remained at approximately 50% (Figure 3B). Therefore, an EtOH concentration of 5% in the culture medium was selected for the subsequent experiments. AEE had no significant effect on GES-1 cells within the selected concentration range (p > 0.05) (Figure S1). As shown in Figure 3C,D, compared with the Control group, cell viability in the EtOH group was significantly reduced (p < 0.05). AEE partially reversed the EtOH-induced decrease in GES-1 cell viability, with the most pronounced effect observed at an AEE concentration of 32 μM (p < 0.05). Compared with the Control group, SOD levels were significantly reduced in the EtOH group (p < 0.05), and AEE could reverse this effect to some extent (p < 0.05) (Figure 3E). Based on the above results, AEE at a concentration of 32 μM was selected for subsequent experiments. The results showed that compared with the Control group, the EtOH group exhibited significantly higher ROS levels (p < 0.05), nuclear lysis, condensation and fragmentation (p < 0.05), and LPO levels (p < 0.05). AEE reversed this phenomenon (p < 0.05) (Figure 3F–H). These results indicated that AEE effectively prevented EtOH-induced damage in GES-1 cells.

3.3. Transcriptomic Analysis of GES-1 Cells

Transcriptomic analysis was performed on AEE and Model groups. The volcano plot showed that compared with the AEE group, the Model group exhibited 723 DEGs, including 309 upregulated and 414 downregulated genes (Figure 4A). The heatmap revealed that the expression patterns of DEGs between AEE and Model groups showed opposite trends (Figure 4B). KEGG analysis revealed that the DEGs between the AEE group and Model group were primarily enriched in the PI3K-Akt signaling pathway, apoptosis, TNF signaling pathway, ferroptosis, p53 signaling pathway, autophagy, NF-kappa B signaling pathway, mTOR signaling pathway, PPAR signaling pathway, VEGF signaling pathway, gastric acid secretion, and tight junction (p < 0.05) (Figure 4C). GO analysis showed that the DEGs between AEE group and Model group were primarily enriched in the regulation of the apoptotic process, response to stimulus, positive regulation of metabolic process, positive regulation of apoptotic process, and process response to oxygen (p < 0.05) (Figure 4D). This study further showed through GSEA analysis that AEE could suppress inflammatory responses and NF-κB signaling pathways and promote gastric acid secretion (Figure 4E–G). Based on these findings, EtOH induced oxidative stress, inflammatory responses, and ferroptosis in GES-1 cells. Therefore, this experiment measured cellular Fe^2+^ content. As shown in Figure 4H, compared with the Control group, the EtOH group exhibited significantly elevated Fe^2+^ levels, while AEE effectively reversed this effect. As shown in Figure 4I, compared with the Control group, the EtOH group showed significantly higher mRNA expression levels of IL-6, TNF-α, p65, and p53 (p < 0.05), while mRNA expression levels of Nrf-2, ZO-1, and VEGF were significantly reduced (p < 0.05). Compared with the EtOH group, the AEE group showed significantly decreased mRNA expression levels of IL-6, TNF-α, p65, and p53 (p < 0.05), whereas the mRNA expression levels of Nrf-2, ZO-1, and VEGF were significantly increased (p < 0.05). Next, this study selected RSL-3 (ferroptosis inducer) and Fer-1 (ferroptosis inhibitor) for validation. As shown in Figure 4J,K, compared with the Control group, cell viability in the RSL-3 group significantly decreased (p < 0.05). GES-1 cells exhibited membrane dissolution, vesicle formation, and loss of their normal morphology. However, Fer-1 and AEE significantly improved this phenomenon with some cells in the field of view maintaining normal morphology. Compared with the Control group, the RSL-3 group exhibited significantly elevated cellular Fe^2+^ content (p < 0.05). Compared with the RSL-3 group, both Fer-1 and AEE significantly reduced cellular Fe^2+^ content (p < 0.05) (Figure 4L), indicating that both AEE and Fer-1 could resist RSL-3-induced ferroptosis. These results indicated that AEE alleviates EtOH-induced damage in GES-1 cells by inhibiting inflammatory responses, oxidative stress, ferroptosis, and protecting tight junctions.

3.4. AEE Alleviated EtOH-Induced Oxidative Stress and Inflammatory Response in Rat Gastric Tissue

As shown in Figure 5A, compared with the Control group, the mRNA expression levels of IL-6, IL-1β, TNF-α, and P65 were significantly increased in the EtOH group (p < 0.05) and decreased in Nrf-2 and GPX4 (p < 0.05). Compared with the EtOH group, the AEE group exhibited significantly lower mRNA expression levels of IL-6, IL-1β, TNF-α, and P65 (p < 0.05), whereas the mRNA expression levels of Nrf-2 and GPX4 were significantly increased (p < 0.05). Next, this study employed ELISA to detect markers of oxidative stress and inflammatory response in rat serum and gastric tissue. Serum levels of IL-1β, TNF-α, COX-1, and COX-2 were significantly elevated in the EtOH group (p < 0.05), whereas AEE treatment reduced these levels (Figure S2A–D), indicating that AEE could attenuate inflammatory responses in rats. Compared with the Control group, EtOH-treated rats showed significantly elevated serum MDA and P65 levels and markedly reduced SOD levels (p < 0.05). AEE effectively reversed these changes (Figure S2E–G), indicating its capacity to mitigate oxidative stress in the rats. Notably, serum Nrf-2 levels in the EtOH group showed a trend toward elevation without significant change (p > 0.05), whereas GPX4 levels exhibited a trend toward reduction without significant change (p > 0.05). Compared with the EtOH group, Nrf-2 levels in the serum decreased in the AEE group but without a significant change (p > 0.05), while GPX4 levels significantly increased (p < 0.05) (Figure S2H,I). Compared with the Control group, IL-1β, TNF-α, COX-1, and COX-2 levels were significantly elevated in the gastric tissues of EtOH-treated rats (p < 0.05), whereas AEE effectively reduced these factors (Figure 5B–E). Compared with the Control group, the EtOH group exhibited significantly elevated levels of MDA and P65 and significantly reduced levels of SOD (p < 0.05) in gastric tissues, whereas AEE effectively reversed these changes. Interestingly, Nrf-2 and GPX4 levels were significantly decreased in the gastric tissues of rats in the EtOH group (p < 0.05). AEE significantly increased Nrf-2 and GPX4 levels (p < 0.05) (Figure 5F–J). This study further evaluated oxidative stress and inflammatory responses in rat gastric tissue using immunohistochemistry. Similar results were obtained in the immunohistochemical assessment. The overall levels of IL-1β and P65 were significantly elevated in the gastric tissues of EtOH-treated rats, whereas the overall level of Nrf-2 was significantly decreased (p < 0.05). AEE effectively reversed these changes (p < 0.05) (Figure 5K). All data indicated that AEE alleviated EtOH-induced oxidative stress and inflammatory responses in rat gastric tissue.

3.5. AEE Alleviated EtOH-Induced Ferroptosis in Rat Gastric Tissue

As shown in Figure 6A, compared with the Control group, the mRNA expression levels of Bax, P53, and ACSL4 were significantly increased in the EtOH group (p < 0.05), and the mRNA expression levels of Bcl-2 and SLC7A11 were significantly decreased (p < 0.05). Compared with the EtOH group, the AEE group exhibited significantly decreased mRNA expression levels of Bax, P53, and ACSL4 (p < 0.05), whereas the mRNA expression levels of Bcl-2 and SLC7A11 were significantly increased (p < 0.05). Next, this study detected markers associated with apoptosis and ferroptosis in rat serum and gastric tissue using ELISA. Compared with the Control group, PUMA protein levels in serum were significantly elevated in the EtOH group (p < 0.05), while ALOX15 protein levels showed an increase but without a significant change (p > 0.05). Compared with the EtOH group, serum PUMA protein levels in the AEE group were significantly decreased (p < 0.05), while ALOX15 protein levels showed a decrease but without a significant change (p > 0.05) (Figure 6B,C). Serum Fe^2+^ levels were significantly higher in the EtOH group (p < 0.05), while AEE treatment resulted in a significant decrease in Fe^2+^ levels (p < 0.05) (Figure 6D). Compared with the Control group, the EtOH group exhibited significantly elevated levels of PUMA and ALOX15 in the gastric tissues (p < 0.05). Compared with the EtOH group, the AEE group showed significantly reduced levels of PUMA and ALOX15 in the gastric tissues (p < 0.05) (Figure 6E,F). Fe^2+^ levels in the gastric tissue of EtOH group rats were significantly elevated (p < 0.05), and AEE effectively reversed this phenomenon (Figure 6G). This study further evaluated apoptosis and ferroptosis in rat gastric tissues using immunohistochemistry. Similar findings were observed in the immunohistochemical assessments (Figure 6H). The overall levels of Bax, p53, and ACSL4 proteins were significantly elevated in the gastric tissue of EtOH-treated rats, whereas the overall level of Bcl-2 protein was significantly reduced (p < 0.05). AEE effectively reversed these changes (p < 0.05). Notably, the overall expression levels of FTH protein were significantly decreased in the gastric tissues of EtOH-treated rats, whereas AEE significantly increased the overall expression levels of FTH. Collectively, these data indicated that AEE could attenuate EtOH-induced apoptosis and ferroptosis in rat gastric tissues.

3.6. AEE Alleviated EtOH-Induced Tight Junction Damage in Rat Gastric Tissue

As shown in Figure 7A, compared with the Control group, the mRNA expression levels of ZO-1 and VEGF were significantly reduced in the EtOH group (p < 0.05). Compared with the EtOH group, the mRNA expression levels of ZO-1 and VEGF were significantly higher in the AEE group (p < 0.05). Next, this study employed ELISA to detect changes in tight junction protein-related indicators in rat serum and gastric tissue. Compared with the Control group, serum MUC6 levels in the EtOH group were significantly decreased (p < 0.05), whereas ZO-1 and EGF levels showed no significant changes (p > 0.05). Compared with the EtOH group, MUC6 levels in rat serum from the AEE group were significantly elevated (p < 0.05), whereas ZO-1 and EGF levels showed no significant changes (p > 0.05) (Figure 7B–D). Compared with the Control group, ZO-1, EGF, and MUC6 levels in the gastric tissue of the EtOH group rats were significantly decreased (p < 0.05). Compared with the EtOH group, the AEE group showed significantly increased levels of ZO-1, EGF, and MUC6 in gastric tissue (p < 0.05) (Figure 7E–G). This study further evaluated the expression of tight junction proteins in rat gastric tissue using immunohistochemistry and tissue immunofluorescence. As shown in Figure 7H–K, compared with the Control group, the positive expression levels of claudin-1, occludin, ZO-1, and VEGF in the gastric tissue of EtOH-treated rats were significantly reduced (p < 0.05). Compared with the EtOH group, the AEE group exhibited significantly increased positive expression levels of claudin-1, occludin, ZO-1, and VEGF in gastric tissues (p < 0.05). These data indicated that EtOH disrupts tight junctions in rat gastric tissues and impairs their recovery function, whereas AEE effectively protects gastric tissues and alleviates EtOH-induced damage.

3.7. Metabonomic Analysis Results

As shown in Figure 8A,B, metabolomic data from the gastric tissues and serum samples of rats in AEE and Model groups were obtained using UPLC-Q-TOF/MS technology. OPLS-DA was performed on the two datasets. The results demonstrated excellent sample separation, good model fit, and strong predictive capability, indicating significant differences in the metabolic profiles of gastric tissues and serum samples between AEE and Model groups. Potential differential metabolites (DMs) were screened using the following criteria: VIP > 1, Fold change (FC) < 0.67 or >1.50, and p < 0.05. Targeted MS/MS scanning was performed on these differential metabolites to obtain the characteristic secondary ion fragments for database matching. Metabolic pathway analysis was conducted using the MetaboAnalyst 6.0 software. Gastric tissue samples were screened for 16 DMs (Table S4), which were primarily enriched in Pyruvaldehyde Degradation, Spermidine and Spermine Biosynthesis, Glutathione Metabolism, Methionine Metabolism, Pyruvate Metabolism, Glutamate Metabolism, and arachidonic acid metabolism. Serum samples were screened for 13 DMs (Table S5), which were primarily enriched in Sphingolipid Metabolism, Pantothenate and CoA Biosynthesis, Beta-Alanine Metabolism, and bile acid biosynthesis.

3.8. AEE-Treated EtOH-Induced Gastric Mucosal Injury in Rats

The animal experimental protocol is illustrated in Figure 9A. As shown in Figure 9B, compared with the Control group, rats in the EtOH group exhibited a significant decrease in body weight during the modeling period (p < 0.05), which stabilized after the cessation of modeling. Compared with the EtOH group, rats in the AEE group showed a significant increase in body weight during AEE administration (p < 0.05). The results in Figure 9C showed that compared with the Control group, the liver and spleen indices of rats in the EtOH group significantly increased (p < 0.05). Compared with the EtOH group, the liver and spleen indices of rats in the AEE group were significantly decreased (p < 0.05). Compared with the Control group, the gastric tissue in the EtOH group exhibited ulceration, surface hemorrhage, and edema. Compared with the EtOH group, gastric tissue pathology improved in the AEE group. The effects of the drugs on gastric tissue were further evaluated via HE staining. The Control group rats showed intact mucosal, submucosal, muscularis, and serosal structures in the gastric tissue, with no significant fibrous tissue proliferation or inflammatory cell infiltration. The EtOH group exhibited more severe gastric lesions, including mucosal necrosis, localized mucosal atrophy and necrosis, cellular sloughing and disintegration accompanied by hemorrhage, visible mucosal thinning, submucosal edema with exudate, and inflammatory cell infiltration. The AEE group showed milder gastric lesions, with minor desquamation of the single-layered columnar epithelium of the mucosa. Edema and fluid exudation were observed in parts of the submucosa, accompanied by minimal infiltration of inflammatory cells. In summary, the EtOH group showed marked pathological alterations compared with the Control group. Compared with the EtOH group, the AEE group demonstrated significantly reduced lesion severity (Figure 9D). As shown in Figure 9E, compared with the Control group, the mRNA expression levels of IL-1β, TNF-α, Bax, P53, P65, and ACSL4 were significantly increased in the EtOH group (p < 0.05), while mRNA expression levels of Bcl-2, Nrf-2, GPX4, ZO-1, VEGF, and SLC7A11 were significantly decreased (p < 0.05). Compared with the EtOH group, the AEE group showed significantly decreased mRNA expression levels of IL-1β, TNF-α, Bax, P53, P65, and ACSL4 (p < 0.05), while mRNA expression levels of Bcl-2, Nrf-2, GPX4, ZO-1, VEGF, and SLC7A11 were significantly increased (p < 0.05). Next, this study detected inflammatory responses, oxidative stress, ferroptosis, and tight junction-related indicators in rat serum and gastric tissue using the ELISA method. Compared with the Control group, the EtOH group exhibited significantly elevated serum IL-1β, MDA, and P53 levels (p < 0.05), significantly decreased ZO-1 and VEGF levels (p < 0.05), and no significant changes in Nrf-2 and GPX4 levels (p > 0.05). Compared with the EtOH group, the AEE group showed significantly decreased serum IL-1β, MDA, and P53 levels (p < 0.05), significantly increased VEGF and GPX4 levels (p < 0.05), and no significant changes in Nrf-2 and ZO-1 levels (p > 0.05) (Figure S3A–G). Compared with the Control group, EtOH-treated rats exhibited significantly increased IL-1β, MDA, and p53 levels in the gastric tissue (p < 0.05), whereas Nrf-2, GPX4, ZO-1, and VEGF levels were significantly decreased (p < 0.05). Compared with the EtOH group, AEE effectively reversed these indicators (Figure 9F–L). Collectively, these data indicated that AEE could treat EtOH-induced gastric mucosal injury in rats by suppressing inflammatory responses, oxidative stress, ferroptosis, and protecting tight junctions.

3.9. AEE Protected Against EtOH-Induced Gastric Mucosal Injury in Mice

The animal experimental protocol is illustrated in Figure 10A. Control group rats showed intact gastric tissue structures including the mucosa, submucosa, muscularis, and serosa with no significant inflammatory cell infiltration. EtOH group rats exhibited necrosis and sloughing of gastric mucosal epithelial cells, partial necrosis and disappearance of gastric gland cells in the lamina propria, dissolution of necrotic cells, fragmentation and dissolution of nuclei, and atrophy and reduction in size of some gastric glands. The AEE group exhibited milder gastric lesions, characterized by minor desquamation of the single-layered columnar epithelium in the mucosa, accompanied by a slight infiltration of inflammatory cells. In summary, compared with the Control group, the EtOH group showed marked pathological alterations, while AEE significantly attenuated the severity of lesions (Figure 10B). As shown in Figure 10C, compared with the Control group, the mRNA expression levels of IL-6, Bax, P53, P65, ACSL4, and LPO were significantly increased in the EtOH group (p < 0.05), while those of Bcl-2, Nrf-2, GPX4, FTH, and ZO-1 were significantly decreased (p < 0.05). Compared with the EtOH group, the AEE group exhibited significantly decreased mRNA expression levels of IL-6, Bax, P53, P65, ACSL4, and LPO (p < 0.05), while mRNA expression levels of Bcl-2, Nrf-2, GPX4, FTH, and ZO-1 were significantly increased (p < 0.05). Next, this study employed ELISA to detect changes in oxidative stress and ferroptosis-related markers in rat gastric tissue. Compared with the Control group, the EtOH group exhibited significantly elevated levels of P65, LPO, and ACSL4 (p < 0.05) and markedly decreased levels of Nrf-2, GPX4, and FTH (p < 0.05) in the gastric tissue. Compared with the EtOH group, AEE effectively reversed these indicators (Figure 10D–I). Collectively, these data indicated that AEE could protect mice from EtOH-induced gastric mucosal injury by inhibiting inflammatory response, oxidative stress, ferroptosis, and preserving tight junctions.

3.10. AEE Reduced EtOH-Induced Gastric Mucosal Injury by Regulating the Nrf2/GPX-4/FTH Signaling Pathway

An inducer and inhibitor were selected for subsequent in vitro validation experiments to further elucidate the underlying mechanism by which AEE alleviates EtOH-induced gastric mucosal injury. As shown in Figure 11A, compared with the Control group, the mRNA expression levels of Nrf-2, GPX4, and FTH were significantly decreased in the ML385 group (p < 0.05), while those of the (R)-Sulforaphane group were significantly increased (p < 0.05). These results indicated that Nrf-2 inhibition also suppressed GPX4 and FTH. Subsequently, the changes in the relevant markers in GES-1 cells were detected using ELISA. Compared with the Control group, the ML385 group showed significantly decreased levels of Nrf-2, GPX4, FTH, ZO-1, and VEGF (p < 0.05), whereas P53 levels were significantly increased (p < 0.05). The (R)-Sulforaphane group exhibited significantly increased levels of Nrf-2, GPX4, and FTH (p < 0.05), whereas P53, ZO-1, and VEGF levels showed no significant changes (p > 0.05). As shown in Figure 11B, compared with the Control group, the mRNA expression levels of Nrf-2, GPX4, and FTH were significantly decreased in the ML385 group (p < 0.05). In contrast, the mRNA expression levels of Nrf-2, GPX4, and FTH were significantly increased in AEE and (R)-Sulforaphane groups (p < 0.05). Compared with the Control group, the ML385 group showed significantly decreased levels of Nrf-2, GPX4, FTH, ZO-1, and VEGF (p < 0.05), while P53 levels were significantly increased (p < 0.05). AEE and (R)-Sulforaphane groups effectively reversed these changes. The results indicated that AEE and (R)-Sulforaphane counteracted ML385-induced suppression of Nrf-2 and activation of GPX4 and FTH, thereby inhibiting apoptosis and protecting the tight junction barrier. As shown in Figure 11C, compared with the Control group, the mRNA expression levels of Nrf-2, GPX4, and FTH were significantly decreased in the RSL-3 group (p < 0.05). In contrast, the mRNA expression levels of Nrf-2, GPX4, and FTH were significantly increased in AEE and (R)-Sulforaphane groups (p < 0.05). Compared with the Control group, the RSL-3 group showed significantly decreased levels of Nrf-2, GPX4, FTH, ZO-1, and VEGF (p < 0.05), whereas the P53 levels were significantly increased (p < 0.05). Both AEE and (R)-Sulforaphane groups effectively reversed these changes. The results indicated that AEE and (R)-Sulforaphane counteract RSL-3-induced GPX4 suppression by activating Nrf-2, thereby inhibiting apoptosis and preserving tight junction integrity. As shown in Figure 11D, molecular docking indicated that AEE interacted with multiple sites of Nrf-2 (P36, K83, R84, L86, and V90), multiple sites of GPX4 (F170, H168, P167, Y63, and C66), and multiple sites of FTH (E116, S113, Y29, L26, and R22). BLI analysis further determined the binding force between AEE and Nrf-2 protein (KD = 4.81 μM), confirming that AEE could regulate Nrf-2 expression. These suggested that AEE could alleviate EtOH-induced gastric mucosal injury by inhibiting inflammatory response, oxidative stress, ferroptosis, and protecting tight junctions through regulating the Nrf-2/GPX-4/FTH signaling pathway.

4. Discussion

Gastric ulcer is a common gastric disorder characterized by bleeding, erosion, and the formation of ulcers in the stomach wall. It primarily involves damage to the gastric mucosa, accompanied by increased oxidative stress, inflammation, LPO, and destruction of the gastric mucosal barrier [47,48]. This study comprehensively investigated the effects and mechanisms of AEE in alleviating EtOH-induced gastric mucosal injury through in vivo and in vitro experiments. The results showed that AEE alleviated EtOH-induced gastric injury in rats by regulating the oxidative stress–inflammation–ferroptosis axis, protected mucosal barrier function, and promoted tissue repair. The comprehensive effects are significantly superior to those of the traditional proton pump inhibitor Omeprazole.

The inflammatory response is a core component of EtOH-induced gastric injury [49]. This study revealed that EtOH treatment caused severe ulceration, hemorrhage, and edema in rat gastric tissue, accompanied by elevated body temperature and increased spleen index, all of which are hallmarks of systemic inflammatory response. AEE could significantly improve the histopathological characteristics of gastric tissue and reduce the spleen index. Notably, changes in the spleen index warrant particular attention, as the spleen is a vital immune organ whose elevated index often indicates activation of the immune system and persistent inflammation [50]. AEE showed no significant effects on liver, lung, or kidney indices, indicating a high safety profile. At the molecular level, this study showed that EtOH exposure led to significantly elevated expression of inflammatory cytokines (IL-6, IL-1β, and TNF-α) and p65, a key molecule in the NF-κB signaling pathway. NF-κB is a crucial transcription factor in inflammatory responses [51]. Upon stimulation with EtOH or other agents, NF-κB translocate into the nucleus, initiating the transcription of multiple inflammatory cytokines [52]. ELISA and immunohistochemical analyses confirmed that EtOH treatment markedly elevated P65 protein levels. Conversely, AEE treatment suppressed NF-κB pathway activation and reduced the expression of inflammatory cytokines. AEE possessed superior efficacy compared to the clinically used drug Omeprazole, potentially due to its multitargeted action profile. Beyond directly inhibiting NF-κB activation, it was also found that AEE suppresses COX-2 expression. COX-2, a key enzyme in prostaglandin synthesis, is overexpressed during inflammation and plays a crucial role in sustaining and amplifying the inflammatory response [53]. The inhibitory effect of AEE on COX-2 may partially explain its potent anti-inflammatory activity.

Oxidative stress represents another significant mechanism underlying EtOH-induced gastric injury [52]. The body maintains a healthy oxidative–antioxidant equilibrium, and disruption of this balance contributes to gastric ulceration. Characteristic manifestations of this imbalance include reduced levels of antioxidant enzymes, such as SOD, CAT, and GSH-Px, alongside increased LPO products, such as MDA [54]. Consequently, compounds or extracts exhibiting antioxidant activity can enhance gastric protection and promote healing. EtOH stimulation reduces the resistance of the gastric mucosa to oxidative stress, promotes lipid peroxide production, and causes oxygen radical damage. This was manifested as significantly decreased SOD and GSH-Px activity and increased MDA content in the gastric tissue, indicating that the gastric mucosa is susceptible to EtOH-induced oxidative stress [14]. This study showed that EtOH exposure led to increased ROS production, decreased SOD activity, and elevated LPO and MDA levels. AEE significantly reduced EtOH-induced increases in ROS, MDA, and LPO levels while enhancing SOD activity, indicating that AEE possesses potent antioxidant capacity. GPX4 is a selenoprotein whose active site contains selenocysteine. It utilizes glutathione (GSH) as a cofactor to reduce toxic lipid hydroperoxides within cells to non-toxic lipid alcohols [55]. This process protects cell membranes from oxidative damage, maintaining membrane structural integrity and stability, which are crucial for cellular survival. It plays a central role in maintaining the cellular redox balance and regulating ferroptosis [56]. GPX4 levels were significantly reduced in the EtOH group (p < 0.05), whereas AEE restored antioxidant activity (p < 0.05). EtOH metabolism leads to excessive ROS production, stimulating macrophages, promoting the release of inflammatory mediators, activating inflammatory pathways, and resulting in tissue inflammation and damage. Furthermore, ROS are key initiators of apoptosis, causing extensive damage to cellular components, including proteins, lipids, and DNA [57]. Apoptosis-related markers revealed a significant increase in the Bax/Bcl-2 ratio in the gastric tissues of EtOH-treated rats (p < 0.05), indicating apoptotic processes in the gastric tissues [58,59]. AEE significantly reduced Bax expression (p < 0.05), elevated Bcl-2 levels (p < 0.05), and markedly decreased the Bax/Bcl-2 ratio (p < 0.05). As a sequence-specific transcription factor, P53 regulates over 100 downstream genes by binding to DNA response elements (REs). Its core functions include cell cycle arrest, DNA repair, and apoptosis [60]. This study showed that P53 protein levels were significantly elevated in the gastric tissues of EtOH-treated rats (p < 0.05), indicating that EtOH promotes gastric mucosal cell apoptosis by activating P53. AEE treatment significantly reduced P53 levels (p < 0.05), and this inhibition may be mediated by Nrf-2 activation. Nrf-2 plays a crucial role in oxidative stress, primarily by regulating the expression of genes that protect cells and counteract antioxidants [61]. Under normal conditions, Nrf-2 binds to Keap1 and is rapidly degraded via the ubiquitin pathway. To activate the expression of HO-1, SOD2, and GSH, Nrf-2 enters the cell nucleus and binds to ARE, thereby enhancing the cell’s antioxidant capacity. Activation of Nrf-2 and suppression of oxidative stress inhibit mitochondrial autophagy and apoptosis [62]. Furthermore, Nrf-2 activation plays a vital role in suppressing DNA damage and mitigating intracellular oxidative injuries. Costunolide promotes Nrf-2 expression, reducing DNA damage and apoptosis [63]. Immunohistochemistry and ELISA results demonstrated that AEE treatment reversed the EtOH-induced decrease in Nrf-2 expression (p < 0.05), suggesting that this may represent an upstream regulatory mechanism for its antioxidant and anti-apoptotic effects. Magnolol nanoparticles gradually disintegrated and released in simulated gastric fluid, effectively reducing inflammatory response. Furthermore, they alleviated EtOH-induced gastric inflammation by decreasing NOX4 protein expression and increasing Nrf-2 protein expression levels [14]. Lacticaseibacillus paracasei NCU-21 prevented EtOH-induced gastric ulcers by activating the Nrf-2/HO-1 signaling pathway [64]. These findings were similar to the results of this study. Notably, this study conducted functional validation experiments using the Nrf-2 inhibitor ML385 and the agonist (R)-Sulforaphane. The results indicated that AEE exhibits effects similar to (R)-Sulforaphane, providing strong evidence for the central role of the Nrf-2 pathway in AEE’s antioxidant stress response. Molecular docking and BLI analysis further confirmed direct binding between AEE and Nrf-2 protein, suggesting that AEE may function as an Nrf-2 activator.

Ferroptosis is gaining increasing attention due to its role in gastrointestinal diseases [65]. Transcriptomic analysis revealed that the ferroptosis pathway was significantly enriched in EtOH-treated cells. Experimental validation demonstrated that EtOH treatment induced Fe^2+^ accumulation and elevated LPO levels in GES-1 cells and in rat gastric tissues. Concurrently, the expression of ACSL4, a key ferroptosis gene, was upregulated, whereas that of the inhibitory factors SLC7A11 and GPX4 was downregulated. FTH, an iron storage protein, serves as a core molecule that maintains intracellular iron homeostasis through its iron storage capacity and directly negatively regulates ferroptosis [66]. When FTH functions normally, it effectively sequesters excess iron and prevents LPO and ferroptosis. However, when FTH expression or function is impaired, intracellular free iron levels significantly increase, leading to exacerbated LPO and ferroptosis activation. This study showed that AEE not only upregulated GPX4 expression but also enhanced the expression of FTH. This increased expression helps reduce free iron levels, thereby suppressing ferroptosis at its source. Subsequently, in-depth functional investigations were conducted using the ferroptosis inducer RSL-3 and the inhibitor Fer-1. The results indicated that AEE exhibits protective effects similar to Fer-1, significantly inhibiting RSL-3-induced cell death, further confirming that AEE mitigates cellular damage by suppressing ferroptosis.

The gastric mucosal barrier, composed of epithelial cells, tight junctions between cells, and a surface mucus layer, serves as the first line of defense against damage from gastric acid and other harmful substances. EtOH increases permeability by inducing the release of vasoactive substances and causing vascular damage, thereby enhancing gastric necrosis. HE staining revealed extensive necrosis in the gastric mucosa of rats in EtOH group, accompanied by glandular atrophy and vascular leakage. Notably, the AEE group exhibited significant protective effects. This study found that EtOH exposure led to a significant decrease in ZO-1, claudin-1, and occludin expression (p < 0.05), whereas AEE treatment restored the expression of these tight junction proteins. Furthermore, AEE elevated VEGF expression, which promotes gastric mucosal angiogenesis and repair and plays a crucial role in maintaining mucosal integrity [67]. Second, AEE enhanced mucus secretion. AB-PAS staining revealed that EtOH exposure caused a significant reduction in mucus secretion on the gastric mucosal surface, whereas AEE treatment preserved the mucus secretion function. The mucus layer neutralizes gastric acid and protects epithelial cells, serving as a crucial component of the gastric mucosal barrier. This study also revealed that AEE enhances MUC6 expression, a primary constituent of gastric mucus. Transcriptomic enrichment of the ‘Tight junction’ and ‘Gastric acid secretion’ pathways further support the protective role of AEE in barrier function. AEE restored expression of these key proteins while suppressing oxidative stress and inflammatory responses. This dual strategy of ‘barrier repair-inflammation suppression’ effectively disrupted the vicious cycle induced by EtOH. Furthermore, in vitro experiments showed AEE counteracted the inhibitory effects of ML385 and RSL-3 on ZO-1 and VEGF, indicating its barrier-protective action is associated with the Nrf-2/GPX4/FTH pathway. This may occur because oxidative stress and ferroptosis disrupt cytoskeletal and junctional protein structures, whereas inhibiting these processes helps maintain barrier integrity. This suggests that Nrf-2 signaling plays a crucial role in maintaining barrier function by AEE. This finding expanded the understanding of Nrf-2 function, which is traditionally regarded primarily as an antioxidant transcription factor, while this study indicates that it may also participate in regulating the maintenance of cell junctions and barrier function.

Metabolomic analysis revealed that AEE treatment significantly altered the metabolic profiles of gastric tissue and serum, indicating that AEE exerts protective effects by regulating metabolic pathways via multiple targets. In gastric tissue, AEE primarily modulated pathways, including spermidine and spermine biosynthesis, glutathione metabolism, and arachidonic acid metabolism. These pathways are closely associated with oxidative stress and inflammation. For example, glutathione serves as a crucial intracellular antioxidant, and enhanced glutathione metabolism aids ROS clearance [68]. Arachidonic acid, a precursor of prostaglandins, may influence inflammatory responses through metabolic regulation [69]. In the serum, AEE primarily influences pathways including sphingolipid metabolism, pantothenic acid and CoA biosynthesis, β-alanine metabolism, and bile acid biosynthesis. Sphingolipids participate in cell membrane composition and signal transduction, and alterations in their metabolism may affect cell death and inflammatory responses [70]. These metabolic alterations correlate with transcriptomic findings. Transcriptomics reveal that AEE regulates signaling pathways, including PI3K-Akt, TNF, NF-κB, ferroptosis, and autophagy, exhibiting extensive overlap with pathways identified by metabolomics. For instance, glutathione metabolism directly participates in ferroptosis regulation [71]. Arachidonic acid metabolism is closely linked to the NF-κB inflammatory pathway, while sphingolipid metabolism correlates with autophagy and cell death. These data suggested that AEE exerts its gastric protective effects by synergistically regulating multiple pathways and biological processes. On one hand, it enhances antioxidant capacity and inhibits ferroptosis by activating the Nrf-2/GPX4 pathway. On the other hand, it alleviates inflammatory responses by suppressing the NF-κB pathway. Simultaneously, it maintains gastric mucosal barrier integrity by regulating metabolic reprogramming and enhancing tight junction function.

Notably, this study found that AEE exhibited effects similar to those of (R)-Sulforaphane, demonstrating the central role of the Nrf-2 pathway in the antioxidative stress effects of AEE treatment. Furthermore, AEE and (R)-Sulforaphane exhibited protective effects similar to those of Fer-1, significantly inhibiting RSL-3-induced cell death, further confirming that AEE and (R)-Sulforaphane mitigate cellular damage by suppressing ferroptosis. AEE and (R)-Sulforaphane counteracted the inhibitory effects of ML385 and RSL-3 on ZO-1 and VEGF, further indicating that this barrier-protective action is associated with the Nrf-2/GPX4/FTH signaling pathway. In summary, this study demonstrated that AEE alleviates EtOH-induced gastric mucosal injury by activating the Nrf-2/GPX4/FTH signaling pathway.

5. Conclusions

AEE significantly alleviates EtOH-induced gastric mucosal injury. The mechanisms include the following: (1) AEE significantly mitigates EtOH-induced gastric mucosal damage, improving oxidative stress, inflammatory response, and barrier function impairment. (2) AEE inhibits ferroptosis by regulating Nrf-2 and upregulating downstream GPX4 and FTH expressions. (3) AEE reduces the expression of inflammatory cytokines by inhibiting the NF-κB pathway. (4) AEE maintains the integrity of the gastric mucosal barrier by enhancing the expression of tight junction proteins and mucus secretion. These findings not only deepen the understanding of EtOH-induced gastric injury mechanisms but also provide crucial experimental evidence and theoretical guidance for the clinical application of AEE.