A combined transcriptomics and proteomics approach to reveal the mechanism of AEE relieving hyperlipidemia in ApoE-/- mice.

Hyperlipidemia caused by abnormal lipid metabolism has reached epidemic proportions. This phenomenon is also common in companion animals. Previous studies showed that AEE significantly improves abnormal blood lipids in hyperlipidemia rats and mice, but its mechanism is still not clear enough. In this study, the mechanism and potential key pathways of AEE on improving hyperlipidemia in mice were investigated through the transcriptome and proteome study of ApoE-/- mice liver and the verification study on high-fat HepG2 cells. The results showed that AEE significantly decreased the serum TC and LDL-C levels of hyperlipidemia ApoE-/- mice, and significantly increased the enzyme activity of CYP7A1. After AEE intervention, the results of mice liver transcriptome and proteome showed that differential genes and proteins were enriched in lipid metabolism-related pathways. The results of RT-qPCR showed that AEE significantly regulated the expression of genes related to lipid metabolism in mice liver tissue. AEE significantly upregulated the protein expression of CYP7A1 in hyperlipidemia ApoE-/- mice liver tissue. The results in vitro showed that AEE significantly decreased the levels of TC and TG, and improved lipid deposition in high-fat HepG2 cells. AEE significantly increased the expression of CYP7A1 protein in high-fat HepG2 cells. AEE regulates the expression of genes related to lipid metabolism in high-fat HepG2 cells, mainly by FXR-SHP-CYP7A1 and FGF19-TFEB-CYP7A1 pathways. To sum up, AEE can significantly improve the hyperlipidemia status of ApoE-/- mice and the lipid deposition of high-fat HepG2 cells, and its main pathway is probably the bile acid metabolism-related pathway centered on CYP7A1.

Fingolimod Inhibits Exopolysaccharide Production and Regulates Relevant Genes to Eliminate the Biofilm of K. pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) exhibits the ability to form biofilms as a means of adapting to its adverse surroundings. K. pneumoniae in this biofilm state demonstrates remarkable resistance, evades immune system attacks, and poses challenges for complete eradication, thereby complicating clinical anti-infection efforts. Moreover, the precise mechanisms governing biofilm formation and disruption remain elusive. Recent studies have discovered that fingolimod (FLD) exhibits biofilm properties against Gram-positive bacteria. Therefore, the antibiofilm properties of FLD were evaluated against multidrug-resistant (MDR) K. pneumoniae in this study. The antibiofilm activity of FLD against K. pneumoniae was assessed utilizing the Alamar Blue assay along with confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and crystal violet (CV) staining. The results showed that FLD effectively reduced biofilm formation, exopolysaccharide (EPS), motility, and bacterial abundance within K. pneumoniae biofilms without impeding its growth and metabolic activity. Furthermore, the inhibitory impact of FLD on the production of autoinducer-2 (AI-2) signaling molecules was identified, thereby demonstrating its notable anti-quorum sensing (QS) properties. The results of qRT-PCR analysis demonstrated that FLD significantly decreased the expression of genes associated with the efflux pump gene (AcrB, kexD, ketM, kdeA, and kpnE), outer membrane (OM) porin proteins (OmpK35, OmpK36), the quorum-sensing (QS) system (luxS), lipopolysaccharide (LPS) production (wzm), and EPS production (pgaA). Simultaneously, FLD exhibited evident antibacterial synergism, leading to an increased survival rate of G. mellonella infected with MDR K. pneumoniae. These findings suggested that FLD has substantial antibiofilm properties and synergistic antibacterial potential for colistin in treating K. pneumoniae infections.

Protective Effect and Mechanism of Aspirin Eugenol Ester on Lipopolysaccharide-Induced Intestinal Barrier Injury.

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.

AEE alleviates ox-LDL-induced lipid accumulation and inflammation in macrophages.

Atherosclerosis is a chronic immune inflammatory disease. Aspirin eugenol ester (AEE) is a novel safe and non-toxic compound with many pharmacological effects such as anti-inflammatory, anti-hyperlipidemic and anti-thrombotic action. In order to investigate the effect of AEE on the inhibition of aortic lipid plaque formation and macrophage-derived foam cell formation induced by oxidized low density lipoprotein (ox-LDL), in vivo atherosclerosis model by feeding ApoE-/- mice with a high-fat diet and foam cells formation in vitro model by ox-LDL-induced RAW264.7 macrophages were established. It was found that AEE decreased the levels of TC and LDL-C in serum, and the plaque formation area and lipid accumulation in the aortic intima of ApoE-/- mice. In vitro studies showed that AEE could prevent the uptake of ox-LDL and reduce the contents of TC and FC in cells. AEE enhanced the cholesterol efflux by increasing the expression of ABCA1, ABCG1 and PPARγ, which effectively alleviated excess cholesterol accumulated in the cells. Meanwhile, AEE also reduced the secretion and expression of inflammatory factors in the cells. In addition, AEE could reverse the action of PPARγ inhibitor T0070907 and/or ox-LDL. Therefore, AEE may become an effective candidate drug for the prevention of atherosclerosis.

Multi-omics reveals aspirin eugenol ester alleviates neurological disease.

BACKGROUND: Exosomes play an essential role in maintaining normal brain function due to their ability to cross the blood-brain barrier. Aspirin eugenol ester (AEE) is a new medicinal compound synthesized by the esterification of aspirin with eugenol using the prodrug principle. Aspirin has been reported to have neuroprotective effects and may be effective against neurodegenerative diseases. PURPOSE: This study wanted to investigate how AEE affected neurological diseases in vivo and in vitro. EXPERIMENTAL APPROACH: A multi-omics approach was used to explore the effects of AEE on the nervous system. Gene and protein expression changes of BDNF and NEFM in SY5Y cells after AEE treatment were detected using RT-qPCR and Western Blot. KEY RESULTS: The multi-omics results showed that AEE could regulate neuronal synapses, neuronal axons, neuronal migration, and neuropeptide signaling by affecting transport, inflammatory response, and regulating apoptosis. Exosomes secreted by AEE-treated Caco-2 cells could promote the growth of neurofilaments in SY5Y cells and increased the expression of BDNF and NEFM proteins in SY5Y cells. miRNAs in the exosomes of AEE-treated Caco-2 cells may play an important role in the activation of SY5Y neuronal cells. CONCLUSIONS: In conclusion, AEE could play positive effects on neurological-related diseases.

The Transport and Uptake of Resveratrol Mediated via Glucose Transporter 1 and Its Antioxidant Effect in Caco-2 Cells.

Resveratrol has anti-inflammatory, anti-cancer, and anti-aging pharmacological activities. There is currently a gap in academic research regarding the uptake, transport, and reduction of H2O2-induced oxidative damage of resveratrol in the Caco-2 cell model. This study investigated the role of resveratrol in the uptake, transport, and alleviation of H2O2-induced oxidative damage in Caco-2 cells. In the Caco-2 cell transport model, it was observed that the uptake and transport of resveratrol (10, 20, 40, and 80 µM) were time dependent and concentration dependent. Different temperatures (37 °C vs. 4 °C) could significantly affect the uptake and transportation of resveratrol. The apical to basolateral transport of resveratrol was markedly reduced by STF-31, a GLUT1 inhibitor, and siRNA intervention. Furthermore, resveratrol pretreatment (80 µM) improves the viability of Caco-2 cells induced by H2O2. In a cellular metabolite analysis combined with ultra-high performance liquid chromatography-tandem mass spectrometry, 21 metabolites were identified as differentials. These differential metabolites belong to the urea cycle, arginine and proline metabolism, glycine and serine metabolism, ammonia recycling, aspartate metabolism, glutathione metabolism, and other metabolic pathways. The transport, uptake, and metabolism of resveratrol suggest that oral resveratrol could prevent intestinal diseases caused by oxidative stress.

Isolation, molecular typing and antimicrobial resistance of Clostridium difficile in dogs and cats in Lanzhou city of Northwest China.

Clostridium difficile infection (CDI) in human and animals belonged usually to antibiotic-associated diarrhea, ranging in severity from mild to life-threatening intestinal tract illnesses. This study aimed to isolation and characterization, toxin genes test, molecular typing, and drug sensitivity of Clostridium difficile (C. difficile) which were isolated from clinical diseased dogs and cats. A total of 247 clinical samples were collected from five animal hospitals in Lanzhou City of Northwest China, of which dogs and cats accounted for 74.9% (185/247) and 25.1% (62/247), respectively. We successfully identified 24 C. difficile strains by 16S rRNA and Matrix-Assisted Laser Desorption/Ionization Time of Fight Mass Spectroscopy (MALDI-TOF-MS). 10.3% (19/185) of dogs and 8.1% (5/62) of cats were positive for C. difficile. Among them, 16 strains were toxic and 8 were non-toxic, with a toxic rate of 57.9% (11/19) in dogs and 100% (5/5) in cats. A total of 10 STs and 10 RTs were identified in this study. The percentages of ST42 (RT106) and ST2 (RT014/LW01) among 16 toxic strains were 41.7 and 12.5%, respectively. However, ST3 (RT001), ST1 (RT027), ST133 (LW04), and ST-UN (LW04) had only one strain. ST42 (RT106) was the most common genotype and RT027 strain was first isolated in China from pets. Antimicrobial susceptibility test showed that isolates were extremely sensitive to vancomycin and metronidazole but were resistant to erythromycin and ciprofloxacin. The drug resistant rates to clindamycin, levofloxacin, moxifloxacin and meropenem were 62.5, 20.8, 16.7, and 8.3%, respectively. In conclusion, C. difficile was quietly prevalent in dogs and cats in Lanzhou city with RT106 and RT014 as the main ribotypes. The CDI in pets should be paying more attention and further studies are needed.

Aspirin eugenol ester alleviates lipopolysaccharide-induced acute lung injury in rats while stabilizing serum metabolites levels.

Aspirin eugenol ester (AEE) was a novel drug compound with aspirin and eugenol esterified. AEE had various pharmacological activities, such as anti-inflammatory, antipyretic, analgesic, anti-oxidative stress and so on. In this study, it was aimed to investigate the effect of AEE on the acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rats. In vitro experiments evaluated the protective effect of AEE on the LPS-induced A549 cells. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) were measured in the cell supernatant. The Wistar rats were randomly divided into five groups (n = 8): control group, model group (LPS group), LPS + AEE group (AEE, 54 mg·kg-1), LPS + AEE group (AEE, 108 mg·kg-1), LPS + AEE group (AEE, 216 mg·kg-1). The lung wet-to-dry weight (W/D) ratio and immune organ index were calculated. WBCs were counted in bronchoalveolar lavage fluid (BALF) and total protein concentration was measured. Hematoxylin-Eosin (HE) staining of lung tissue was performed. Glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT), antioxidant superoxide dismutase (SOD), total antioxidant capacity (T-AOC), lactate dehydrogenase (LDH), C-reactive protein (CRP), myeloperoxidase (MPO), malondialdehyde (MDA), macrophage mobility inhibitory factor (MIF), TNF-α, IL-6, and IL-1ß activity were measured. The metabolomic analysis of rat serum was performed by UPLC-QTOF-MS/MS. From the results, compared with LPS group, AEE improved histopathological changes, reduced MDA, CRP, MPO, MDA, and MIF production, decreased WBC count and total protein content in BALF, pro-inflammatory cytokine levels, immune organ index and lung wet-dry weight (W/D), increased antioxidant enzyme activity, in a dose-dependent manner. The results of serum metabolomic analysis showed that the LPS-induced ALI caused metabolic disorders and oxidative stress in rats, while AEE could ameliorate it to some extent. Therefore, AEE could alleviate LPS-induced ALI in rats by regulating abnormal inflammatory responses, slowing down oxidative stress, and modulating energy metabolism.

Isobavachalcone From Cullen corylifolium Presents Significant Antibacterial Activity Against Clostridium difficile Through Disruption of the Cell Membrane.

Background: Clostridium difficile infection (CDI) has been widely reported in human and animals around the world over the past few decades. The high relapse rate and increasing drug resistance of CDI make the discovery of new agents against C. difficile fairly urgent. This study aims to investigate the antibacterial activity against C. difficile from traditional Chinese herb medicine Cullen corylifolium and confirm its active components. Methods: Phenolic extract from the seeds of C. corylifolium was prepared routinely and the contents of relative flavonoids were determined by High Performance Liquid Chromatography (HPLC). In vitro antibacterial activities of the phenolic extract and its major components were tested. The influence of the major components on cell membrane was investigated with membrane integrity by SEM and propidium iodid uptake assay. Cytotoxicity of the extract and its active compounds on Caco-2 cell line was assessed by CCK-8 kit. The in vivo therapeutic efficacy of IBCL was evaluated on the mice model. Results: Phenolic extract was found to be active against C. difficile with minimum inhibitory concentrations (MIC) of 8 µg/mL. As the major component of the extract, IBCL was the most active compound against C. difficile. The MIC of IBCL and 4MBCL were 4 µg/ml and 4 µg/ml, respectively. Meanwhile, PFPE, IBCL, and 4MBCL showed rapid bactericidal effect against C. difficile in 1 h, which was significant compared to antibiotic vancomycin. Mechanism studies revealed that IBCL can disrupt the integrity of the cell membrane, which may lead to the death of bacteria. PFPE was low cytotoxic against Caco-2 cells, and the cytotoxicity of IBCL and 4MBCL were moderate. Symptoms of CDI were effectively alleviated by IBCL on the mice model and weight loss was reduced. From death rates, IBCL showed better efficacy compared to vancomycin at 50 mg/kg dosage. Conclusion: As the major component of phenolic extract of C. corylifolium seeds, IBCL showed significant antibacterial activity against C. difficile in vitro and rapidly killed the bacteria by disrupting the integrity of the cell membrane. IBCL can significantly prevent weight loss and reduce death caused by CDI on the mice model. Therefore, IBCL may be a promising lead compound or drug candidate for CDI.

Florfenicol-Polyarginine Conjugates Exhibit Promising Antibacterial Activity Against Resistant Strains.

Florfenicol was widely used as antibiotic in the livestock and poultry breeding industry, resulting in a serious problem of drug resistance. In order to solve the resistance of florfenicol, this study designed and synthesized a new series of florfenicol-polyarginine conjugates and tested for antimicrobial activities. Drug-sensitive bacteria, gram-negative bacteria Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus), were sensitive to several of the compounds tested. These conjugates also showed excellent activity against drug-resistant strains such as methicillin-resistant S. aureus (MRSA) and florfenicol resistant Escherichia coli strains (2017XJ30, 2019XJ20), one of which as E6 had a minimum inhibitory concentration of 12.5 µmol/L. These conjugates did not allow bacteria to develop resistance and also decreased bacterial growth by membrane depolarization and disruption. Additionally, florfenicol succinate (C1) showed certain activity after coupling with arginine. This suggested that conjugating arginine to florfenicol succinate effectively modulated the properties of prodrugs. These new conjugates may provide useful insights for expanding the pool of antibiotics.

Uptake and Transport of Naringenin and Its Antioxidant Effects in Human Intestinal Epithelial Caco-2 Cells.

Naringenin, a flavanone, has been reported for a wide range of pharmacological activities. However, there are few reports on the absorption, transport and antioxidant effects of naringenin. The study was to explore the uptake, transport and antioxidant effects of naringenin in vitro. Cell transmembrane resistance, lucifer yellow transmission rate, and alkaline phosphatase activity were used to evaluate the successful construction of cell model. The results showed that the absorption and transport of naringenin by Caco-2 cells were time- and concentration-dependent. Different temperatures (37 and 4°C) had a significant effect on the uptake and transport of naringenin. Verapamil, potent inhibitor of P-glycoprotein, significantly inhibit naringenin transport in Caco-2 cells. The results revealed that naringenin was a moderately absorbed biological macromolecule and can penetrate Caco-2 cells, mainly mediated by the active transport pathway involved in P-glycoprotein. At the same time, naringenin pretreatment could significantly increase the viability of H2O2-induced Caco-2 cells. Twenty four differential metabolites were identified based on cellular metabolite analysis, mainly including alanine, aspartate and glutamate metabolism, histidine metabolism, taurine and hypotaurine metabolism, pyruvate metabolism, purine metabolism, arginine biosynthesis, citrate cycle, riboflavin metabolism, and D-glutamine and D-glutamate metabolism. We concluded that the transport of naringenin by Caco-2 cells is mainly involved in active transport mediated by P-glycoprotein and naringenin may play an important role in oxidative stress-induced intestinal diseases.

Investigation of the Uptake and Transport of Aspirin Eugenol Ester in the Caco-2 Cell Model.

Background: Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterification of aspirin with eugenol using the prodrug principle. AEE has the pharmacological activities of being anti-inflammatory, antipyretic, analgesic, anti-cardiovascular diseases, and anti-oxidative stress However, its oral bioavailability is poor, and its intestinal absorption and transport characteristics are still unknown. Objective: The purpose of this study was to investigate the uptake and transport mechanisms of AEE in Caco-2 cells. Methods: The effects of time, concentration, and temperature on the transport and uptake of AEE were studied. Results: The results showed that a higher concentration of salicylic acid (SA) was detected in the supernatant of cell lysates and cell culture medium, while AEE was not detected. Therefore, the content change of AEE was expressed as the content change of its metabolite SA. In the uptake experiment, when the factors of time, concentration, and temperature were examined, the uptake of SA reached the maximum level within 30 min, and there was concentration dependence. In addition, low temperature (4°C) could significantly reduce the uptake of SA in Caco-2 cells. In the transport experiment, under the consideration of time, concentration, and temperature, the transepithelial transport of SA from AP-BL and BL-AP sides was time-dependent. The amount of SA transported in Caco-2 cells increased with the increase of concentration, but the transmembrane transport rate had no correlation with the concentration. This phenomenon may be due to the saturation phenomenon of high concentration. The efflux ratio (ER) was less than 1, which indicated that their intestinal transport mechanism was passive transport. Moreover, the temperature had a significant effect on the transport of AEE. Conclusion: In summary, intestinal absorption of AEE through Caco-2 cell monolayers was related to passive transport. The uptake and transport of AEE were concentration-dependent, and temperature significantly affected their uptake and transport. The absorption and transport characteristics of AEE may contribute to the exploration of mechanisms of absorption and transport of chemosynthetic drugs in vitro.

Untargeted lipidomics and metagenomics reveal the mechanism of aspirin eugenol ester relieving hyperlipidemia in ApoE-/- mice.

Hyperlipidemia is induced by abnormal lipid metabolism, which can cause the occurrence of cardiovascular diseases and lead to grievous injury to health. Studies showed that AEE had a significant therapeutic effect on hyperlipidemia and is likely to be associated with the up-regulation of cholesterol 7-alpha hydroxylase (CYP7A1), the key enzyme for cholesterol conversion to bile acids, but no research confirmed whether the effect of AEE on hyperlipidemia was related to the gut microbiota and liver lipids. At the same time, more and more studies have shown that gut microbiota and lipids are closely related to hyperlipidemia. Hence, in this study, we investigated the effects of AEE on liver lipids through LC-MS-based untargeted lipidomics and the effects of AEE on gut microbiota based on cecal contents metagenomics by Illumina sequencing in HFD-induced hyperlipidemia ApoE-/- mice at the overall level. The results of lipidomics showed that AEE relieved hyperlipidemia by decreasing the concentration of 10 PEs and 12 SMs in the liver and regulating the pathways of glycerophospholipid metabolic pathway, sphingolipid signaling pathway, and NF-kB signaling pathway. The results of metagenomics concluded that AEE treatment changed the composition of gut microbiota and regulated the functions of lipid transport and metabolism, as well as the metabolism of bile acids and secondary bile acids. The results of the joint analysis between lipidomics and metagenomics showed that the abundance of Verrucomicrobia, Verrucomicrobiales, Candidatus_Gastranaerophilales, and Candidatus_Melainabacteria was significantly positively correlated with the concentration of SM (d18:1/18:0) and PE (16:0/18:1) in the process of AEE alleviating hyperlipidemia in mice. In conclusion, these results suggested that the effect of AEE on hyperlipidemia was closely related to the gut microbiota by the change of bile acids and liver lipids.

Artificial Intelligence to Detect Meibomian Gland Dysfunction From in-vivo Laser Confocal Microscopy.

Background: In recent years, deep learning has been widely used in a variety of ophthalmic diseases. As a common ophthalmic disease, meibomian gland dysfunction (MGD) has a unique phenotype in in-vivo laser confocal microscope imaging (VLCMI). The purpose of our study was to investigate a deep learning algorithm to differentiate and classify obstructive MGD (OMGD), atrophic MGD (AMGD) and normal groups. Methods: In this study, a multi-layer deep convolution neural network (CNN) was trained using VLCMI from OMGD, AMGD and healthy subjects as verified by medical experts. The automatic differential diagnosis of OMGD, AMGD and healthy people was tested by comparing its image-based identification of each group with the medical expert diagnosis. The CNN was trained and validated with 4,985 and 1,663 VLCMI images, respectively. By using established enhancement techniques, 1,663 untrained VLCMI images were tested. Results: In this study, we included 2,766 healthy control VLCMIs, 2,744 from OMGD and 2,801 from AMGD. Of the three models, differential diagnostic accuracy of the DenseNet169 CNN was highest at over 97%. The sensitivity and specificity of the DenseNet169 model for OMGD were 88.8 and 95.4%, respectively; and for AMGD 89.4 and 98.4%, respectively. Conclusion: This study described a deep learning algorithm to automatically check and classify VLCMI images of MGD. By optimizing the algorithm, the classifier model displayed excellent accuracy. With further development, this model may become an effective tool for the differential diagnosis of MGD.

Protective Activity of Aspirin Eugenol Ester on Paraquat-Induced Cell Damage in SH-SY5Y Cells.

Aspirin eugenol ester (AEE) is a new pharmaceutical compound esterified by aspirin and eugenol, which has anti-inflammatory, antioxidant, and other pharmacological activities. The aim of this study was to investigate the protective effect of AEE on paraquat- (PQ-) induced cell damage of SH-SY5Y human neuroblastoma cells and its potential molecular mechanism. There was no significant change in cell viability when AEE was used alone. PQ treatment reduced cell viability in a concentration-dependent manner. However, AEE reduced the PQ-induced loss of cell viability. Flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and 4'6-diamidino-2-phenylindole (DAPI) staining were used to evaluate cell apoptosis. Compared with the PQ group, AEE pretreatment could significantly inhibit PQ-induced cell damage. AEE pretreatment could reduce the cell damage of SH-SY5Y cells induced by PQ via reducing superoxide anion, intracellular reactive oxygen species (ROS), and mitochondrial ROS (mtROS) and increasing the levels of mitochondrial membrane potential (ΔΨm). At the same time, AEE could increase the activity of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) and decrease the activity of malondialdehyde (MDA). The results showed that compared with the control group, the expression of p-PI3K, p-Akt, and Bcl-2 was significantly decreased, while the expression of caspase-3 and Bax was significantly increased in the PQ group. In the AEE group, AEE pretreatment could upregulate the expression of p-PI3K, p-Akt, and Bcl-2 and downregulate the expression of caspase-3 and Bax in SH-SY5Y cells. PI3K inhibitor LY294002 and the silencing of PI3K by shRNA could weaken the protective effect of AEE on PQ-induced SH-SY5Y cells. Therefore, AEE has a protective effect on PQ-induced SH-SY5Y cells by regulating the PI3K/Akt signal pathway to inhibit oxidative stress.

The Protective Effect of Aspirin Eugenol Ester on Oxidative Stress to PC12 Cells Stimulated with H2O2 through Regulating PI3K/Akt Signal Pathway.

Aspirin eugenol ester (AEE) is a new pharmaceutical compound esterified by aspirin and eugenol, which has anti-inflammatory, antioxidant, and other pharmacological activities. This study is aimed at identifying the protective effect of AEE against H2O2-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. The results of cell viability assay showed that AEE could increase the viability of PC12 cells stimulated by H2O2, while AEE alone had no significant effect on the viability of PC12 cells. Compared with the control group, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were significantly decreased, and the content of malondialdehyde (MDA) was significantly increased in the H2O2 group. By AEE pretreatment, the level of MDA was reduced and the levels of SOD, CAT, and GSH-Px were increased in H2O2-stimulated PC12 cells. In addition, AEE could reduce the apoptosis of PC12 cells induced by H2O2 via reducing superoxide anion, intracellular ROS, and mitochondrial ROS (mtROS) and increasing the levels of mitochondrial membrane potential (ΔΨm). Furthermore, the results of western blotting showed that compared with the control group, the expression of p-PI3K, p-Akt, and Bcl-2 was significantly decreased, while the expression of Caspase-3 and Bax was significantly increased in the H2O2 group. In the AEE group, AEE pretreatment could upregulate the expression of p-PI3K, p-Akt, and Bcl-2 and downregulate the expression of Caspase-3 and Bax in PC12 cells stimulated with H2O2. The silencing of PI3K with shRNA and its inhibitor-LY294002 could abrogate the protective effect of AEE in PC12 cells. Therefore, AEE has a protective effect on H2O2-induced PC12 cells by regulating the PI3K/Akt signal pathway to inhibit oxidative stress.

Aspirin eugenol ester ameliorates paraquat-induced oxidative damage through ROS/p38-MAPK-mediated mitochondrial apoptosis pathway.

Paraquat (PQ) is an effective and commercially important herbicide that is widely used worldwide. However, PQ is highly toxic and can cause various complications and acute organ damage. Aspirin eugenol ester (AEE) is a potential new compound with anti-inflammatory and antioxidant stress pharmacological activity. The present study was to reveal the therapeutic effects and the protective effect of AEE against PQ-induced acute lung injury (ALI) with the help of PQ-induced oxidative damage in A549 cells and PQ-induced lung injury in rats. AEE might have no significant therapeutic effect on PQ-induced lung injury in rats. However, AEE had a significant protective effect on PQ-induced lung injury in rats. AEE pretreatment significantly reduced the stimulatory effect of PQ on malondialdehyde (MDA), the inhibitory effect of PQ on catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, the ratio of GSH/GSSH, the activity of caspase-3 and the overexpression of p38 mitogen-activated protein kinase (MAPK) phosphorylation in vivo. In vitro, A549 cells were treated with 250 µM PQ for 24 h. Incubation of A549 cells with PQ led to apoptosis, and increased the level of superoxide anions, reactive oxygen species (ROS), malondialdehyde and the activity of caspase-3 and up-regulation of phosphorylated p38-MAPK, reduced mitochondrial membrane potential (ΔΨm) and the activity of SOD. However, after 24 h on AEE pretreatment of A549 cells, the above-mentioned adverse reactions caused by PQ were significantly alleviated. In addition, AEE pretreatment reduced p38-MAPK phosphorylation in PQ-treated A549 cells. SB203580, the specific p38-MAPK inhibitor, and p38-MAPK shRNA attenuated the activation of the p38-MAPK signaling pathway. N-acetylcysteine (NAC) reduced the level of phosphorylated p38-MAPK and the production of intracellular ROS and inhibited apoptosis. The results showed that AEE may inhibit PQ-induced cell damage through ROS/p38-MAPK-mediated mitochondrial apoptosis pathway.

Aspirin Eugenol Ester Attenuates Paraquat-Induced Hepatotoxicity by Inhibiting Oxidative Stress.

Aspirin eugenol ester (AEE) is a new potential drug with anti-inflammatory and antioxidant stress pharmacological activity. Paraquat (PQ) is an effective and commercially important herbicide that is widely used worldwide. However, paraquat is highly toxic and can cause various complications and acute organ damage, such as liver, kidney and lung damage. The purpose of this study was to investigate whether AEE has a protective effect on hepatotoxicity induced by PQ in vivo and in vitro. Cell viability, apoptosis rate, mitochondrial function and intracellular oxidative stress were detected to evaluate the protective effect of AEE on PQ-induced BRL-3A (normal rat hepatocytes) cytotoxicity in vitro. In vivo, AEE pretreatment could attenuate oxidative stress and histopathological changes in rat liver induced by PQ. The results showed that AEE could reduce the hepatotoxicity induced by PQ in vivo and in vitro. AEE reduced PQ-induced hepatotoxicity by inhibitingoxidative stress and maintaining mitochondrial function. This study proved that AEE is an effective antioxidant and can reduce the hepatotoxicity of PQ.

Aspirin Eugenol Ester Protects Vascular Endothelium From Oxidative Injury by the Apoptosis Signal Regulating Kinase-1 Pathway.

Aspirin eugenol ester (AEE) is a new potential pharmaceutical compound possessing anti-inflammatory, anti-cardiovascular disease, and antioxidative stress activity. The pharmacological activities of AEE are partly dependent on its regulation of cell apoptosis. However, it is still unclear how AEE inhibits cell apoptosis on the basis of its antioxidative stress effect. This study aimed to reveal the vascular antioxidative mechanism of AEE in response to H2O2-induced oxidative stress in HUVECs and paraquat-induced oxidative stress in rats. In the different intervention groups of HUVECs and rats, the expression of ASK1, ERK1/2, SAPK/JNK, and p38 and the phosphorylation levels of ERK1/2, SAPK/JNK, and p38 were measured. The effects of ASK1 and ERK1/2 on the anti-apoptotic activity of AEE in the oxidative stress model were probed using the corresponding inhibitors ASK1 and ERK1/2. The results showed that in the HUVECs, 200 µM H2O2 treatment significantly increased the phosphorylation of SAPK/JNK and the level of ASK1 but decreased the phosphorylation of ERK1/2, while in the HUVECs pretreated with AEE, the H2O2-induced changes were significantly ameliorated. The findings were observed in vitro and in vivo. Moreover, inhibition of ASK1 and ERK1/2 showed that ASK1 plays a vital role in the protective effect of AEE on H2O2-induced apoptosis. All findings suggested that AEE protects the vascular endothelium from oxidative injury by mediating the ASK1 pathway.