Identification of osteoporosis ferroptosis-related markers and potential therapeutic compounds based on bioinformatics methods and molecular docking technology.

RESEARCH BACKGROUND AND PURPOSE: Osteoporosis (OP) is one of the most common bone diseases worldwide, characterized by low bone mineral density and susceptibility to pathological fractures, especially in postmenopausal women and elderly men. Ferroptosis is one of the newly discovered forms of cell death regulated by genes in recent years. Many studies have shown that ferroptosis is closely related to many diseases. However, there are few studies on ferroptosis in osteoporosis, and the mechanism of ferroptosis in osteoporosis is still unclear. This study aims to identify biomarkers related to osteoporosis ferroptosis from the GEO (Gene Expression Omnibus) database through bioinformatics technology, and to mine potential therapeutic small molecule compounds through molecular docking technology, trying to provide a basis for the diagnosis and treatment of osteoporosis in the future. MATERIALS AND METHODS: We downloaded the ferroptosis-related gene set from the FerrDb database ( http://www.zhounan.org/ferrdb/index.html ), downloaded the data sets GSE56815 and GSE7429 from the GEO database, and used the R software "limma" package to screen differentially expressed genes (DEGs) from GSE56815, and intersected with the ferroptosis gene set to obtain ferroptosis-related DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed by the R software "clusterProfiler" package. The random forest model was further screened to obtain essential ferroptosis genes. R software "corrplot" package was used for correlation analysis of essential ferroptosis genes, and the Wilcox test was used for significance analysis. The lncRNA-miRNA-mRNA-TF regulatory network was constructed using Cytoscape software. The least absolute shrinkage and selection operator (LASSO) was used to construct a disease diagnosis model, and a Receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic performance, and then GSE7429 was used to verify the reliability of the diagnosis model. Molecular docking technology was used to screen potential small molecule compounds from the Drugbank database. Finally, a rat osteoporosis model was constructed, and peripheral blood mononuclear cells were extracted for qRT-PCR detection to verify the mRNA expression levels of crucial ferroptosis genes. RESULT: Six DEGs related to ferroptosis were initially screened out. GO function and KEGG pathway enrichment analysis showed that ferroptosis-related DEGs were mainly enriched in signaling pathways such as maintenance of iron ion homeostasis, copper ion binding function, and ferroptosis. The random forest model identified five key ferroptosis genes, including CP, FLT3, HAMP, HMOX1, and SLC2A3. Gene correlation analysis found a relatively low correlation between these five key ferroptosis genes. The lncRNA-miRNA-mRNA-TF regulatory network shows that BAZ1B and STAT3 may also be potential molecules. The ROC curve of the disease diagnosis model shows that the model has a good diagnostic performance. Molecular docking technology screened out three small molecule compounds, including NADH, Midostaurin, and Nintedanib small molecule compounds. qRT-PCR detection confirmed the differential expression of CP, FLT3, HAMP, HMOX1 and SLC2A3 between OP and normal control group. CONCLUSION: This study identified five key ferroptosis genes (CP, FLT3, HAMP, HMOX1, and SLC2A3), they were most likely related to OP ferroptosis. In addition, we found that the small molecule compounds of NADH, Midostaurin, and Nintedanib had good docking scores with these five key ferroptosis genes. These findings may provide new clues for the early diagnosis and treatment of osteoporosis in the future.

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.

Targeting vulnerable microcircuits in the ventral hippocampus of male transgenic mice to rescue Alzheimer-like social memory loss.

BACKGROUND: Episodic memory loss is a prominent clinical manifestation of Alzheimer's disease (AD), which is closely related to tau pathology and hippocampal impairment. Due to the heterogeneity of brain neurons, the specific roles of different brain neurons in terms of their sensitivity to tau accumulation and their contribution to AD-like social memory loss remain unclear. Therefore, further investigation is necessary. METHODS: We investigated the effects of AD-like tau pathology by Tandem mass tag proteomic and phosphoproteomic analysis, social behavioural tests, hippocampal electrophysiology, immunofluorescence staining and in vivo optical fibre recording of GCaMP6f and iGABASnFR. Additionally, we utilized optogenetics and administered ursolic acid (UA) via oral gavage to examine the effects of these agents on social memory in mice. RESULTS: The results of proteomic and phosphoproteomic analyses revealed the characteristics of ventral hippocampal CA1 (vCA1) under both physiological conditions and AD-like tau pathology. As tau progressively accumulated, vCA1, especially its excitatory and parvalbumin (PV) neurons, were fully filled with mislocated and phosphorylated tau (p-Tau). This finding was not observed for dorsal hippocampal CA1 (dCA1). The overexpression of human tau (hTau) in excitatory and PV neurons mimicked AD-like tau accumulation, significantly inhibited neuronal excitability and suppressed distinct discrimination-associated firings of these neurons within vCA1. Photoactivating excitatory and PV neurons in vCA1 at specific rhythms and time windows efficiently ameliorated tau-impaired social memory. Notably, 1 month of UA administration efficiently decreased tau accumulation via autophagy in a transcription factor EB (TFEB)-dependent manner and restored the vCA1 microcircuit to ameliorate tau-impaired social memory. CONCLUSION: This study elucidated distinct protein and phosphoprotein networks between dCA1 and vCA1 and highlighted the susceptibility of the vCA1 microcircuit to AD-like tau accumulation. Notably, our novel findings regarding the efficacy of UA in reducing tau load and targeting the vCA1 microcircuit may provide a promising strategy for treating AD in the future.

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.

P301S-hTau acetylates KEAP1 to trigger synaptic toxicity via inhibiting NRF2/ARE pathway: A novel mechanism underlying hTau-induced synaptic toxicities.

BACKGROUND: Human Tau (hTau) accumulation and synapse loss are two pathological hallmarks of tauopathies. However, whether and how hTau exerts toxic effects on synapses remain elusive. METHODS: Mutated hTau (P301S) was overexpressed in the N2a cell line, primary hippocampal neurons and hippocampal CA3. Western blotting and quantitative polymerase chain reaction were applied to examine the protein and mRNA levels of synaptic proteins. The protein interaction was tested by co-immunoprecipitation and proximity ligation assays. Memory and emotion status were evaluated by a series of behavioural tests. The transcriptional activity of nuclear factor-erythroid 2-related factor 2 (NRF2) was detected by dual luciferase reporter assay. Electrophoresis mobility shift assay and chromosome immunoprecipitation were conducted to examine the combination of NRF2 to specific anti-oxidative response element (ARE) sequences. Neuronal morphology was analysed after Golgi staining. RESULTS: Overexpressing P301S decreased the protein levels of post-synaptic density protein 93 (PSD93), PSD95 and synapsin 1 (SYN1). Simultaneously, NRF2 was decreased, whereas Kelch-like ECH-associated protein 1 (KEAP1) was elevated. Further, we found that NRF2 could bind to the specific AREs of DLG2, DLG4 and SYN1 genes, which encode PSD93, PSD95 and SYN1, respectively, to promote their expression. Overexpressing NRF2 ameliorated P301S-reduced synaptic proteins and synapse. By means of acetylation at K312, P301S increased the protein level of KEAP1 via inhibiting KEAP1 degradation from ubiquitin-proteasome pathway, thereby decreasing NRF2 and reducing synapse. Blocking the P301S-KEAP1 interaction at K312 rescued the P301S-suppressed expression of synaptic proteins and memory deficits with anxiety efficiently. CONCLUSIONS: P301S-hTau could acetylate KEAP1 to trigger synaptic toxicity via inhibiting the NRF2/ARE pathway. These findings provide a novel and potential target for the therapeutic intervention of tauopathies.

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.

Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling.

BACKGROUND: Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer's disease (AD). This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy. METHODS: The primary hippocampal neurons, N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy, which was analysed by Student's two-tailed t-test. The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1 (mTORC1) activity and the vacuolar H+-ATPase (v-ATPase) activity, respectively, which were analysed by One-way ANOVA with post hoc tests. The Western blotting, co-immunoprecipitation and immunofluorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations, as analysed by Student's two-tailed t-test or One-way ANOVA with post hoc tests. The autophagosome formation was detected by immunofluorescence staining and transmission electron microscopy. The amino acids (AA) levels were detected by high performance liquid chromatography (HPLC). RESULTS: We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits. Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1 (PRD-TIA1) and this association significantly increased the intercellular level of amino acids (Leucine, P = 0.0038; Glutamic acid, P = 0.0348; Alanine, P = 0.0037; Glycine, P = 0.0104), with concordant upregulation of mTORC1 activity [phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1), P < 0.0001; phosphorylated 70 kDa ribosomal protein S6 kinase 1 (p-p70S6K1), P = 0.0001, phosphorylated unc-51-like autophagy-activating kinase 1 (p-ULK1), P = 0.0015] and inhibition of autophagosome formation [microtubule-associated protein light chain 3 II (LC3 II), P = 0.0073; LC3 puncta, P < 0.0001]. As expected, this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation. Importantly, we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1, downregulating the endogenous TIA1 expression by shRNA, or downregulating tau protein level by a small proteolysis targeting chimera (PROTAC) could remarkably attenuate tau-induced autophagy impairment. CONCLUSIONS: Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway, and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treatment and that of related tauopathies.

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.

A Magnetic Resonance Angiography-Based Study Comparing Machine Learning and Clinical Evaluation: Screening Intracranial Regions Associated with the Hemorrhagic Stroke of Adult Moyamoya Disease.

OBJECTIVES: Moyamoya disease patients with hemorrhagic stroke usually have a poor prognosis. This study aimed to determine whether hemorrhagic moyamoya disease could be distinguished from MRA images using transfer deep learning and to screen potential regions that contain rich distinguishing information from MRA images in moyamoya disease. MATERIALS AND METHODS: A total of 116 adult patients with bilateral moyamoya diseases suffering from hemorrhagic or ischemia complications were retrospectively screened. Based on original MRA images at the level of the basal cistern, basal ganglia, and centrum semiovale, we adopted the pretrained ResNet18 to build three models for differentiating hemorrhagic moyamoya disease. Grad-CAM was applied to visualize the regions of interest. RESULTS: For the test set, the accuracies of model differentiation in the basal cistern, basal ganglia, and centrum semiovale were 93.3%, 91.5%, and 86.4%, respectively. Visualization of the regions of interest demonstrated that the models focused on the deep and periventricular white matter and abnormal collateral vessels in hemorrhagic moyamoya disease. CONCLUSION: A transfer learning model based on MRA images of the basal cistern and basal ganglia showed a good ability to differentiate between patients with hemorrhagic moyamoya disease and those with ischemic moyamoya disease. The deep and periventricular white matter and collateral vessels at the level of the basal cistern and basal ganglia may contain rich distinguishing information.

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.

Blood lead level in Chinese adults with and without coronary artery disease.

BACKGROUND: The Trial to Assess Chelation Therapy study found that edetate disodium (disodium ethylenediaminetetraacetic acid) chelation therapy significantly reduced the incidence of cardiac events in stable post-myocardial infarction patients, and a body of epidemiological data has shown that accumulation of biologically active metals, such as lead and cadmium, is an important risk factor for cardiovascular disease. However, limited studies have focused on the relationship between angiographically diagnosed coronary artery disease (CAD) and lead exposure. This study compared blood lead level (BLL) in Chinese patients with and without CAD. METHODS: In this prospective, observational study, 450 consecutive patients admitted to Beijing Anzhen Hospital with suspected CAD from November 1, 2018, to January 30, 2019, were enrolled. All patients underwent coronary angiography, and an experienced heart team calculated the SYNTAX scores (SXscore) for all available coronary angiograms. BLLs were determined with atomic absorption spectrophotometry and compared between patients with angiographically diagnosed CAD and those without CAD. RESULTS: In total, 343 (76%) patients had CAD, of whom 42% had low (0-22), 22% had intermediate (23-32), and 36% had high (≥ 33) SXscore. BLLs were 36.8 ± 16.95 µg/L in patients with CAD and 31.2 ± 15.75 µg/L in those without CAD (P = 0.003). When BLLs were categorized into three groups (low, middle, high), CAD prevalence increased with increasing BLLs (P < 0.05). In the multivariate regression model, BLLs were associated with CAD (odds ratio (OR): 1.023, 95% confidence interval (CI): 1.008-1.039; P = 0.0017). OR in the high versus low BLL group was 2.36 (95% CI: 1.29-4.42,P = 0.003). Furthermore, BLLs were independently associated with intermediate and high SXscore (adjusted OR: 1.050, 95% CI: 1.036-1.066; P < 0.0001). CONCLUSION: BLLs were significantly associated with angiographically diagnosed CAD. Furthermore, BLLs showed excellent predictive value for SXscore, especially for complex coronary artery lesions.

[Preparation of Antibiotic-Loaded Copper-Doped Hydroxyapatite Microspheres and Evaluation of Their Antibacterial and Osteogenic Effect].

OBJECTIVE: To explore the preparation method of copper (Cu)-doped hydroxyapatite (HA) microspheres loaded with vancomycin (Van), and evaluate their antibacterial and osteogenic effects in vitro. METHODS: The Cu doped HA microspheres (Cu-HA) with molar doping ratios of 1%, 5%, 10%, and 20% were prepared by hydrothermal synthesis. The microscopic morphology changes were observe with scanning electron microscope. X-ray diffractometer (XRD) was used to study the phase composition and analyze the crystallinity of the sample. Cu-HA with a molar doping ratio of 10% was selected for analysis of the elemental composition of the sample with energy dispersive X-ray spectroscopy (EDS), and was then coated with polydopamine (PDA) as the medium to prepare Cu-HA-PDA. XRD and Fourier infrared spectrometer were used to examine the coating effect of the sample. Van was load on Cu-HA-PDA to prepare Cu-HA-PDA-Van. HA, Cu-HA, HA-PDA, and Cu-HA-PDA-Van were added to α medium at 10 mg/mL to prepare different groups of extract solutions.The main components of the extract solutions were examined, and the Van concentration was checked. We examined the toxic effect of material extract solutions on osteogenic precursor cells and the proliferation and differentiation of bone marrow mesenchymal stem cells, and checked the expression of osteocalcin ( OCN), runt-related transcription factor 2 ( RUNX-2), and alkaline phosphatase ( ALP), the osteogenic related genes. Sterilized HA, Cu-HA, HA-PDA, Cu-HA-PDA, Cu-HA-DPA-Van microsphere materials were prepared, and the colony counting method was used to evaluate the antibacterial effect of the materials for Staphylococcus aureus. RESULTS: Various types of Cu-doped HA (Cu-HA) were successfully synthesized. As the proportion of Cu increased, the morphology gradually changed from being strip or belt-shaped to a uniform spherical shape. Cu-HA of 10% molar doping ratio showed a clearly microspherical shape and a petal-like porous micro-nano morphology on the surface. EDS and XRD analyses showed that the main structure of the material was still made up of hydroxyapatite crystals and Cu was successfully doped with HA. The infrared spectrometer showed that the PDA was successfully coated on the surface of the material. Examination of the main components of the extract solution once again verified that the Cu element had successfully entered and replaced part of the Ca element in the HA. The 10 mg/mL Cu-HA-PDA-Van extract solution contained 0.27 mg/mL of Van. In vitro cell experiments and bone-formation-related gene testing showed that Cu-HA-Van had good biological activity and promoted bone differentiation. The minimum inhibitory concentration (MIC) of Cu-HA-PDA-Van microspheres was 16 µg/mL. Compared with Cu-HA, HA-PDA and pure HA, Cu-HA-Van microspheres had significant and long-lasting antibacterial effects. CONCLUSION: Cu element was used to control the microscopic morphology of HA, and the Cu-HA-PDA-Van microspheres prepared by successfully coating of PDA and loading of Van had good antibacterial properties and biological activity.

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.