This study aimed to evaluate the effects of aspirin eugenol ester (AEE) on growth performance, oxidative liver damage, inflammation, and liver metabolomics in broilers under high-stocking-density (HSD) stress. A total of 360 broilers were divided into four groups: normal density (ND, 14/m²), high density (HD, 22/m²), ND-AEE (ND + 0.01% AEE), and HD-AEE (HD + 0.01% AEE). HSD decreased total antioxidant capacity, increased malondialdehyde (MDA) levels, and elevated the expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) mRNA, which contributed to the reduced performance of broilers. Specifically, HSD caused abnormalities in linoleic acid metabolism, leading to elevated levels of Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) synthesis, which aggravated inflammation, increased liver lipid levels, and impaired ATP production. AEE counteracted the decline in broiler production performance induced by HSD by enhancing total antioxidant capacity, reducing MDA levels, protecting the liver from oxidative damage, and maintaining mitochondrial oxidative phosphorylation. AEE positively regulated the linoleic acid metabolism by promoting the synthesis of γ-linolenic acid and phosphatidylcholine, which reduced the synthesis of COX-2 and mPGES-1. AEE alleviated the metabolic imbalance caused by HSD stress and enhanced the efficiency of mitochondrial fatty acid oxidation, which reduced excess lipid accumulation in the liver and promoted ATP production. In summary, this study provides strong support for the dietary addition of AEE to alleviate liver oxidative damage, inflammation, and energy metabolism disorders caused by HSD stress.

Intestinal inflammation is a complex and recurrent inflammatory disease. Pharmacological and pharmacodynamic experiments showed that aspirin eugenol ester (AEE) has good anti-inflammatory, antipyretic, and analgesic effects. However, the role of AEE in regulating intestinal inflammation has not been explored. This study aimed to investigate whether AEE could have a protective effect on LPS-induced intestinal inflammation and thus help to alleviate the damage to the intestinal barrier. This was assessed with an inflammation model in Caco-2 cells and in rats induced with LPS. The expression of inflammatory mediators, intestinal epithelial barrier-related proteins, and redox-related signals was analyzed using an enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence staining, and RT-qPCR. Intestinal damage was assessed by histopathological examination. Changes in rat gut microbiota and their functions were detected by the gut microbial metagenome. AEE significantly reduced LPS-induced pro-inflammatory cytokine levels (p < 0.05) and oxidative stress levels in Caco-2 cells and rats. Compared with the LPS group, AEE could increase the relative expression of Occludin, Claudin-1, and zonula occludens-1 (ZO-1) and decrease the relative expression of kappa-B (NF-κB) and matrix metalloproteinase-9. AEE could significantly improve weight loss, diarrhea, reduced intestinal muscle thickness, and intestinal villi damage in rats. Metagenome results showed that AEE could regulate the homeostasis of the gut flora and alter the relative abundance of Firmicutes and Bacteroidetes. Flora enrichment analysis indicated that the regulation of gut flora with AEE may be related to the regulation of glucose metabolism and energy metabolism. AEE could have positive effects on intestinal inflammation-related diseases.

Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterifying aspirin with eugenol using the pro-drug principle. Pharmacological and pharmacodynamic experiments showed that AEE had excellent thromboprophylaxis and inhibition of platelet aggregation. This study aimed to investigate the effect of AEE on the liver of thrombosed rats to reveal its mechanism of thromboprophylaxis. Therefore, a multi-omics approach was used to analyze the liver. Transcriptome results showed 132 differentially expressed genes (DEGs) in the AEE group compared to the model group. Proteome results showed that 159 differentially expressed proteins (DEPs) were identified in the AEE group compared to the model group. Six proteins including fibrinogen alpha chain (Fga), fibrinogen gamma chain (Fgg), fibrinogen beta chain (Fgb), orosomucoid 1 (Orm1), hemopexin (Hpx), and kininogen-2 (Kng2) were selected for parallel reaction monitoring (PRM) analysis. The results showed that the expression of all six proteins was upregulated in the model group compared with the control group. In turn, AEE reversed the upregulation trend of these proteins to some degree. Metabolome results showed that 17 metabolites were upregulated and 38 were downregulated in the model group compared to the control group. AEE could reverse the expression of these metabolites to some degree and make them back to normal levels. The metabolites were mainly involved in metabolic pathways, including linoleic acid metabolism, arachidonic acid metabolism, and the tricarboxylic acid (TCA) cycle. Comprehensive analyses showed that AEE could prevent thrombosis by inhibiting platelet activation, decreasing inflammation, and regulating amino acid and energy metabolism. In conclusion, AEE can have a positive effect on thrombosis-related diseases.

Broilers grown in a high-density (HD) stocking environment may experience intense competition that may adversely affect their growth relative to animals reared at a normal density (ND). The growth performance of HD broilers is increased by aspirin eugenol ester (AEE), although the mechanism by which this compound modulates hypothalamus-regulated feeding behavior is unclear. The aims of this study were to determine the effects of including AEE in the basal diet on the hypothalamic transcriptome and to examine in parallel the impact of these modifications on broiler production performance in HD conditions. Three hundred sixty one-day-old male Arbor Acres broilers were randomly divided into four groups: an ND group (14 broilers/m²), HD group (22 broilers/m²), ND-AEE group, and HD-AEE group. Each treatment group had 10 replicates, with 7 broilers per replicate in the ND and ND-AEE groups and 11 broilers per replicate in the HD and HD-AEE groups. Broiler growth performance was monitored, and hypothalamus samples were collected for transcriptome analysis on day 28. The HD group exhibited a reduced body weight (p < 0.01) at this timepoint compared to the ND group. However, the addition of AEE significantly improved average daily feed intake, average daily gain, and feed conversion ratio in the HD group from days 22 to 28 compared to the HD group without AEE (p < 0.05). The transcriptome results showed that 20 signaling pathways were commonly enriched among the groups (ND vs. HD, HD vs. HD-AEE). Several potential candidate genes were identified as involved in chicken central nervous system development and regulation of feed intake. Thus, the current study provides new insights into hypothalamic transcription patterns that are associated with the ameliorative effects of AEE in HD broilers.

Platelet activation is closely related to thrombosis. Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterifying aspirin with eugenol using the pro-drug principle. Pharmacological and pharmacodynamic experiments showed that AEE has excellent anti-inflammatory, antioxidant, and inhibitory platelet activation effects, preventing thrombosis. However, the regulatory network and action target of AEE in inhibiting platelet activation remain unknown. This study aimed to investigate the effects of AEE on platelets of thrombosed rats to reveal its regulatory mechanism via a multi-omics approach. The platelet proteomic results showed that 348 DEPs were identified in the AEE group compared with the model group, of which 87 were up- and 261 down-regulated. The pathways in this result were different from previous results, including mTOR signaling and ADP signaling at P2Y purinoceptor 12. The metabolomics of heart and abdominal aortic tissue results showed that the differential metabolites were mainly involved in steroid biosynthesis, the citric acid cycle, phenylalanine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, and glutathione metabolism. Molecular docking results showed that AEE had a better binding force to both the COX-1 and P2Y12 protein. AEE could effectively inhibit platelet activation by inhibiting COX-1 protein and P2Y12 protein activity, thereby inhibiting platelet aggregation. Therefore, AEE can have a positive effect on inhibiting platelet activation.

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.