Based on the prodrug principle, aspirin eugenol ester (AEE) was synthesized, which can reduce the side effects of aspirin and eugenol. As a good candidate for new antithrombotic and anti-inflammatory medicine, it is essential to evaluate its preventive effect on thrombosis. Preventive effect of AEE was investigated in κ-carrageenan-induced rat tail thrombosis model. AEE suspension liquids were prepared in 0.5% sodium carboxymethyl cellulose (CMC-Na). AEE was administrated at the dosage of 18, 36 and 72 mg/kg. Aspirin (20 mg/kg), eugenol (18 mg/kg) and 0.5% CMC-Na (30 mg/kg) were used as control drug. In order to compare the effects between AEE and its precursor, integration of aspirin and eugenol group (molar ratio 1:1) was also designed in the experiment. After drugs were administrated intragastrically for seven days, each rat was injected intraperitoneally with 20 mg/kg BW κ-carrageen dissolved in physiological saline to induce thrombosis. The length of tail-thrombosis was measured at 24 and 48 hours. The blank group just was given physiological saline for seven days without κ-carrageenan administrated. The results indicated that AEE significantly not only reduced the average length of thrombus, PT values and FIB concentration, but also reduced the red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT) and platelet (PLT). The effects of AEE on platelet aggregation and anticoagulant in vitro showed that AEE could inhibit adenosine diphosphate (ADP)-induced platelet aggregation as dose-dependence but no notable effect on blood clotting. From these results, it was concluded that AEE possessed positive effect on thrombosis prevention in vivo through the reduction of FIB, PLT, inhibition of platelet aggregation and the change of TT and PT values.

Fatty liver hemorrhagic syndrome (FLHS) is a metabolic disease that causes decreased egg production and even death in laying hens, which brings huge economic losses to the poultry industry. However, the pathogenesis of FLHS is unclear. The purpose of the present study was to identify the changes in lipid profile and the lipid species related to FLHS. In the present study, the FLHS disease model in Chinese commercial Jing Fen laying hens was induced by a high-energy low-protein diet. A lipidomics approach based on ultra-performance liquid chromatography-mass spectrometry coupled with multivariate statistical analysis was performed for the qualitative and quantitative analyses of the liver lipids. The results showed that a total of 29 lipid subclasses, including 1,302 lipid species, were detected and identified. Among them, the proportions of phosphatidylserine (Control/FLHS, 33.1% vs. 29.1%), phosphatidylethanolamine (22.7% vs. 15.5%), phosphatidylcholine (15.7% vs. 11.7%) and phosphatidylinositol (7% vs. 6%) were reduced, while triacylglycerol (7.1% vs. 18.3%) and diglyceride (3.9% vs. 11.7%) were increased. Between the Control and FLHS groups, distinct changes in lipid profile were observed in the score plots of principal component analysis and orthogonal partial least squares discriminant analysis. Twelve differential lipid species mainly involved in glycerophospholipid metabolism and linoleic acid metabolism were identified and considered to be related to the pathogenesis of FLHS. Fatty acid chain length and unsaturation were reduced, while the mRNA levels of elongation of very long chain fatty acids-2 (ELOVL2) were increased in the liver of laying hens with FLHS. Collectively, this study characterized the liver lipid profile and explored the changes in lipid species related to FLHS, which provided insights into the pathogenesis of FLHS from the view of lipid metabolism.

Bacteroides fragilis (B. fragilis), a crucial commensal bacterium within the gut, has shown connections with hepatic lipid metabolism and inflammation regulation. Nonetheless, the role of B. fragilis in the progression of fatty liver hemorrhagic syndrome (FLHS) remains unknown. This study aims to explore the ameliorative effects of B. fragilis on FLHS in laying hens, as well as its underlying mechanisms. This is the first study to employ a chicken FLHS model, combining microbiomics and oxylipin metabolomics to investigate the mechanism of action of intestinal symbiotic bacteria. Exp. 1: 40 laying hens at 25 weeks old were randomly divided into five treatment groups (eight replicates per group and one hen per replicate), including the control group (basal diet), the high-energy and low-protein (HELP) group, and the HELP group with three different levels (10⁸, 10⁹, and 10¹⁰ CFU) of B. fragilis. Exp. 2: 18 chickens at 25 weeks old were randomly divided into three treatment groups (six replicates per group and one hen per replicate) including the control group (basal diet), the model group (HELP diet), and the arachidonic acid (AA) group (HELP diet with 0.3% AA). The experiment period of Exp. 1 and Exp. 2 were 8 weeks. B. fragilis significantly improved body weight of seventh week (P = 0.006), liver lipid degeneration, blood lipid levels (triglycerides, cholesterol, and low-density lipoprotein cholesterol; P < 0.05), and liver function (alanine aminotransferase and aminotransferase; P < 0.05) in laying hens. B. fragilis downregulated the expression of lipid synthesis-related genes fatty acid synthase, acetyl-CoA carboxylase, and liver X receptor α, and inflammation-related genes tumor necrosis factor α, interleukin (IL)-1β, IL-6, and IL-8 in the liver of FLHS-affected hens (P < 0.05), while upregulating the expression of lipid oxidation-related genes carnitine palmitoyl transferase-1, peroxisome proliferator activated receptor (PPAR) α, and PPARγ (P < 0.05). The in-depth analysis indicated alterations in oxylipin pathways triggered by B. fragilis, as evidenced by changes in the expression of pivotal genes arachidonate 15-lipoxygenase, arachidonate 5-lipoxygenase (P < 0.05), subsequently causing modifications in relevant metabolites. This included a decrease in pro-inflammatory substances such as 15-oxoETE (P = 0.004), accompanied by an increase in AA (P = 0.008). B. fragilis regulated the homeostasis of intestinal flora by increasing the abundance of Bacteroides and decreasing the abundance of Succinatimonas and Faecalicoccus (P < 0.05). The integrated analysis revealed a robust positive correlation between Bacteroides abundance and AA levels (P = 0.007). This relationship was corroborated through in vitro experiments. Subsequently, the beneficial effect of AA in mitigating FLHS was confirmed in laying hens with FLHS, further supported by reverse transcription-polymerase chain reaction analysis demonstrating gene expression patterns akin to B. fragilis intervention. This study demonstrated that B. fragilis exerts an anti-FLHS effect through modulation of oxylipin metabolism and gut microbiota stability, with a pivotal role played by AA.

Based on the prodrug principle, aspirin and eugenol, as starting precursors, were esterified to synthesize aspirin eugenol ester (AEE). The aim of the present study was to evaluate the antithrombotic effect of AEE in an animal disease model. In order to compare the therapeutic effects of AEE and its precursors, aspirin, eugenol and a combination of aspirin and eugenol were designed at the same molar quantities as the AEE medium dose in the control group. After oral administration of AEE (dosed at 18, 36 and 72 mg/kg) for seven days, rats were treated with k-carrageenan to induce tail thrombosis. Following the same method, aspirin (20 mg/kg), eugenol (18 mg/kg) and 0.5 % CMC-Na (30 mg/kg) were administered as control drug. Different drug effects on platelet aggregation, hemorheology, TXB2/6-keto-PGF1α ratio and blood biochemistry were studied. AEE significantly inhibited ADP and AA-induced platelet aggregation in vivo. AEE also significantly reduced blood and plasma viscosity. Moreover, AEE down-regulated TXB2 and up-regulated 6-keto-PGF1α, normalizing the TXB2/6-keto-PGF1α ratio and blood biochemical profile. In comparison with aspirin and eugenol, AEE produced more positive therapeutic effects than its precursors under the same molar quantity. It may be concluded that AEE was a good candidate for new antithrombotic and antiplatelet medicine. Additionally, this study may help to understand how AEE works on antithrombosis in different ways.