TMAO Promotes NLRP3 Inflammasome Activation of Microglia Aggravating Neurological Injury in Ischemic Stroke Through FTO/IGF2BP2.

Objective: Stroke is a kind of cerebrovascular disease with high mortality. TMAO has been shown to aggravate stroke outcomes, but its mechanism remains unclear. Materials and Methods: Mice were fed with 0.12% TMAO for 16 weeks. Then, mice were made into MCAO/R models. Neurological score, infarct volume, neuronal damage and markers associated with inflammation were assessed. Since microglia played a crucial role in ischemic stroke, microglia of MCAO/R mice were isolated for high-throughput sequencing to identify the most differentially expressed gene following TMAO treatment. Afterward, the downstream pathways of TMAO were investigated using primary microglia. Results: TMAO promoted the release of inflammatory cytokines in the brain of MCAO/R mice and promoted the activation of OGD/R microglial inflammasome, thereby exacerbating ischemic stroke outcomes. FTO/IGF2BP2 inhibited NLRP3 inflammasome activation in OGD/R microglia by downregulating the m6A level of NLRP3. TMAO can inhibit the expression of FTO and IGF2BP2, thus promoting the activation of NLRP3 inflammasome in OGD/R microglia. In conclusion, these results demonstrated that TMAO promotes NLRP3 inflammasome activation of microglia aggravating neurological injury in ischemic stroke through FTO/IGF2BP2. Conclusion: Our results demonstrated that TMAO promotes NLRP3 inflammasome activation of microglia aggravating neurological injury in ischemic stroke through FTO/IGF2BP2. These findings explained the molecular mechanism of TMAO aggravating ischemic stroke in detail and provided molecular mechanism for clinical treatment.

Diminazene Ameliorates Neuroinflammation by Suppression of Astrocytic miRNA-224-5p/NLRP3 Axis in Alzheimer's Disease Model.

Purpose: ACE2/Ang(1-7)/Mas Receptor, the momentous component of the renin-angiotensin system, has been shown to be involved in Alzheimer's disease (AD). We had previously found that enhancing brain ACE2 activity ameliorated cognitive impairment and attenuated brain neuroinflammation in SAMP8 mice, an animal model of AD. However, the exact mechanism of action of Diminazene (DIZE) has not been revealed. Methods: APP/PS1 mice were injected intraperitoneally with DIZE. Cognitive functions, neuronal and synaptic integrity, and inflammation-related markers were assessed by Morris water maze, Nissl staining, Western blotting and ELISA, respectively. Since astrocytes played a crucial role in AD-related neuroinflammation whilst miRNAs were reported to participate in modulating inflammatory responses, astrocytes of APP/PS1 mice were then isolated for high-throughput miRNAs sequencing to identify the most differentially expressed miRNA following DIZE treatment. Afterward, the downstream pathway of this miRNA in the anti-inflammatory action of DIZE was investigated using primary astrocytes. Results: The results showed that DIZE alleviated cognitive impairment and neuronal and synaptic damage in APP/PS1 mice. Simultaneously, DIZE suppressed the secretion of pro-inflammatory cytokines and the expression of NLRP3 inflammasome. Importantly, miR-224-5p was significantly up-regulated in the astrocytes of APP/PS1 mice treated by DIZE, and NLRP3 is one of the targets of miR-224-5p. Upregulation of miR-224-5p inhibited the expression of NLRP3 in Aß1-42-stimulated cells, whereas miR-224-5p downregulation reversed this effect. Furthermore, the inhibition of miR-224-5p could reverse the inhibitory effect of DIZE on astrocytic NLRP3 inflammasome. Conclusion: These findings firstly suggested that DIZE could inhibit astrocyte-regulated neuroinflammation via miRNA-224-5p/NLRP3 pathway. Furthermore, our study reveals the underlying mechanism by which DIZE suppresses neuroinflammatory responses in AD mice and uncovers the potential of DIZE in AD treatment.

Enhanced metabolic activation of and platelet response to clopidogrel in T cell-deficient mice through induction of Cyp2c and Cyp3a and inhibition of Ces1.

BACKGROUND: T cells and platelets reciprocally coordinate mutual functions through crosstalk or interaction. However, it is not known whether metabolic activation of and platelet response to clopidogrel could be changed if T cells were deficient or impaired in some cases and, if any, how it would work. OBJECTIVES: The objective of this study was to dissect the potential changes in platelet responses to and metabolic activation of clopidogrel in the case of T cell deficiency and to elucidate their mechanisms involved. METHODS: BALB/c athymic nude mice or euthymic mice (controls) pretreated with cyclosporine A (CsA), thymosin α1 (Tα1), or their combination were used to investigate the changes in ADP-induced platelet activation and aggregation, systemic exposure of clopidogrel and its metabolites, and mRNA/protein expression and activity levels of clopidogrel-metabolizing enzymes in the liver, respectively. RESULTS: Nude mice exhibited significantly enhanced antiplatelet effects of clopidogrel due to increased formation of clopidogrel active metabolite in the liver, where the enzyme activity levels of Cyp2c and Cyp3a were significantly elevated compared with control mice. Furthermore, the effects of CsA pretreatment on the metabolism of clopidogrel in euthymic mice were identical to those seen in athymic mice. As expected, concomitant use of Tα1 reversed all the observed effects of CsA on clopidogrel metabolism and relevant metabolic enzymes. CONCLUSIONS: T cell deficiency or suppression enhances the antiplatelet effects of clopidogrel due to the boosted metabolic activation of clopidogrel in the liver through a dramatic induction of Cyp2c and Cyp3a in mice, suggesting that the metabolism of substrate drugs of Cyp2c and Cyp3a may be enhanced by T cell impairment.

Choline and trimethylamine N-oxide impair metabolic activation of and platelet response to clopidogrel through activation of the NOX/ROS/Nrf2/CES1 pathway.

BACKGROUND: Trimethylamine N-oxide (TMAO), a gut microbe-generated metabolite, elicits thrombotic events by enhancing platelet reactivity; however, no studies have reported the effects of TMAO on the metabolism of and response to clopidogrel. OBJECTIVES: To determine whether choline and TMAO could significantly impair metabolic activation of and platelet response to clopidogrel in choline- or TMAO-fed mice and the mechanisms involved. METHODS: Male mice were fed with vehicle control (Ctrl), TMAO, choline alone or in combination with 3,3-dimethyl-1-butanol, N-acetyl-L-cysteine, or ML385 for 14 days and then treated with Ctrl or a single oral dose of clopidogrel. Plasma TMAO, protein levels of clopidogrel-metabolizing enzymes in the liver, plasma concentrations of clopidogrel and its metabolites, and adenosine diphosphate-induced platelet aggregation and activation were measured. In addition, HepG2 cells were treated with Ctrl or TMAO alone or in combination with N-acetyl-L-cysteine, ML385, or apocynin, and CES1, reactive oxygen species (ROS), and Nrf2 protein levels were measured, respectively. RESULTS: TMAO significantly increased Ces1 protein expression and activity and clopidogrel hydrolysis in the liver as well as intracellular ROS and CES1 levels and Nrf2 nucleus translocation in HepG2 cells but decreased the formation of clopidogrel active metabolite and impaired platelet response to clopidogrel. Furthermore, concomitant use of 3,3-dimethyl-1-butanol, N-acetyl-L-cysteine, or ML385 effectively reversed choline- or TMAO-induced impairment of inhibition of platelet aggregation by clopidogrel in mice, respectively. CONCLUSIONS: Choline and TMAO impair the metabolic activation of and platelet response to clopidogrel through the activation of the NOX-dependent ROS/Nrf2/CES1 pathway, suggesting novel strategies for overcoming clopidogrel resistance from bench to bedside.

Is platelet responsiveness to clopidogrel attenuated in overweight or obese patients and why? A reverse translational study in mice.

BACKGROUND AND PURPOSE: Overweight or obese patients exhibit poorer platelet responses to clopidogrel. However, the mechanisms behind this phenotype remain to be elucidated. Here, we sought to discover whether and why obesity could affect the metabolic activation of and/or platelet response to clopidogrel in obese patients and high-fat diet-induced obese mice. EXPERIMENTAL APPROACH: A post hoc stratified analysis of an observational clinical study was performed to investigate changes in residual platelet reactivity with increasing body weight in patients taking clopidogrel. Furthermore, high-fat diet-induced obese mice were used to reveal alterations in systemic exposure of clopidogrel thiol active metabolite H4, ADP-induced platelet activation and aggregation, the expression of genes involved in the metabolic activation of clopidogrel, count of circulating reticulated and mature platelets, and proliferation profiles of megakaryocytes in bone marrow. The relevant genes and potential signalling pathways were predicted and enriched according to the GEO datasets available from obese patients. KEY RESULTS: Obese patients exhibited significantly attenuated antiplatelet effects of clopidogrel. In diet-induced obese mice, systemic exposure of clopidogrel active metabolite H4 was reduced but that of its hydrolytic metabolite was increased due to down-regulation of certain P450s but up-regulation of carboxylesterase-1 in the liver. Moreover, enhanced proliferation of megakaryocytes and elevated platelet count also contributed. CONCLUSION AND IMPLICATIONS: Obesity attenuated metabolic activation of clopidogrel and increased counts of circulating reticulated and mature platelets, leading to impaired platelet responsiveness to the drug in mice, suggesting that clopidogrel dosage may need to be adjusted adequately in overweight or obese patients.

Short-term standard alcohol consumption enhances platelet response to clopidogrel through inhibition of Nrf2/Ces1 pathway and induction of Cyp2c in mice.

AIMS: Drinking alcohol is prevalent worldwide; however, it is unknown whether alcohol could affect the antiplatelet effects of clopidogrel in patients when taking both concomitantly. This study was designed to investigate the influence of short-term standard alcohol consumption on the metabolic activation of and platelet response to clopidogrel in mice as well as the mechanisms involved. MAIN METHODS: Male C57BL/6J mice were administered with normal saline (vehicle control) or alcohol at 2 g/kg/day for 7 days, and then gavaged with vehicle control or a single dose of clopidogrel at 10 mg/kg. Inhibition of ADP-induced platelet aggregation and activation by clopidogrel, plasma concentrations of clopidogrel and its active metabolite H4, and changes in mRNA and protein expression of genes related to clopidogrel metabolism and its regulation were measured in mice pretreated with or without alcohol. KEY FINDINGS: Compared with vehicle control, alcohol pretreatment significantly reduced hydrolysis of clopidogrel as a result of significant down-regulation of Nrf2-mediated Ces1 expression (responsible for the formation of clopidogrel carboxylate), increased metabolic activation of clopidogrel due to significant up-regulation of Cyp2c (for the formation of active thiol metabolite H4), and consequently enhanced inhibition of ADP-induced platelet aggregation and activation by clopidogrel. SIGNIFICANCE: Short-term standard alcohol consumption would significantly enhance suppression of ADP-induced platelet aggregation and activation by clopidogrel through significant inhibition of Nrf2/Ces1 signaling pathway and induction of Cyp2c, suggesting that alcohol may interact with drugs that are predominantly metabolized by CES1 or CYP2C in patient care, including clopidogrel.

P-glycoprotein deficiency enhances metabolic activation of and platelet response to clopidogrel through marked up-regulation of Cyp3a11 in mice: Direct evidence for the interplay between P-glycoprotein and Cyp3a.

Variability in P-glycoprotein (P-gp) efflux transporting activity was supposed to be involved in altered intestinal absorption and bioavailability of clopidogrel in patients; however, reliable evidence is still lacking. In this study, we sought to determine whether P-gp could play an important role in the metabolic activation of and platelet response to clopidogrel in mice. Abcb1a/1b knock-out (KO) and wild-type (WT) mice were used to evaluate differences in the intracellular accumulation of clopidogrel in the intestine, liver, and brain tissues and in systemic exposure of clopidogrel and its main metabolites as well as the mechanisms involved. Results indicated that, compared with WT mice, KO mice exhibited an 84% increase in systemic exposure of clopidogrel active thiol metabolite H4 and a 14.5% rise of suppression of ADP-induced platelet integrin αIIbß3 activation, paralleled by a 41% decrease in systemic exposure of clopidogrel due to enhanced systemic clearance. Furthermore, KO mice displayed a 45% increase in Cyp3a11 but a 23% decrease in Ces1 at their protein levels compared with WT mice. Concurrently, intracellular clopidogrel concentrations in the tissues examined did not differ significantly between KO and WT mice. We conclude that although P-gp does not transport clopidogrel and its major metabolites in mice, P-gp-deficient mice exhibit elevated formation of the active metabolite H4 and enhanced antiplatelet effect of clopidogrel through up-regulation of Cyp3a11 and down-regulation of Ces1, suggesting that P-gp activity may correlate inversely with the formation of H4 and antiplatelet efficacy of clopidogrel in clinical settings due to P-gp and CYP3A4 interplay.

Vicagrel enhances aspirin-induced inhibition of both platelet aggregation and thrombus formation in rodents due to its decreased metabolic inactivation.

Both aspirin and vicagrel are effective antiplatelet drugs, with the potential for concomitant use as another dual-antiplatelet therapy for the prevention of recurrent thrombotic or ischemic events. Because they both are the substrates of carboxylesterase 2 (CES2), aspirin attenuated the metabolic activation of and platelet response to vicagrel in mice treated with the two drugs concomitantly. In this study, we sought to clarify whether vicagrel could affect platelet responses to aspirin and their underlying mechanisms. Plasma levels of aspirin and salicylic acid were determined by liquid chromatography-tandem mass spectrometry, inhibition of arachidonic acid (AA)-induced whole-blood platelet aggregation by aspirin was assessed with an aggregometer, and their antithrombotic effects were evaluated by arteriovenous shunt thrombosis model. The results showed that concomitant use of vicagrel (5, 10, or 20 mg/kg) led to an average of 55% and 77% increases in systemic exposure of aspirin (Cmax and AUC0-t) and 2.8-fold increase in suppression of AA-induced platelet aggregation in mice when compared with use of aspirin alone. In the rat thrombus formation model, vicagrel (1 mg/kg) enhanced inhibition of thrombosis formation by aspirin (5 mg/kg), but not vice versa. We conclude that vicagrel increases platelet responses to aspirin and also enhances inhibition of thrombus formation of aspirin due to decreased CES2-catalyzed aspirin inactivation in rodents, and that an integrated net effect on thrombus formation in vivo is superior to inhibition of AA- or ADP-induced platelet aggregation ex vivo by either of the two drugs if taken concomitantly.

W1, a Novel Oral Antiplatelet Agent With Less Resistance Than Clopidogrel.

Clopidogrel (CLO) is a clinical antiplatelet agent, about which there are major concerns because its antiplatelet efficiency decreases with insufficient metabolic activation, leading to "clopidogrel resistance." We aimed to determine the antiplatelet effects of W1, a novel molecule composed of 2-O-clopidogrel and aspirin (1:1 ratio), on platelet aggregation ex vivo and thrombus formation in vivo, and its susceptibility to CLO resistance in combination with other therapies in rats. Platelets were prepared, and an arteriovenous shunt thrombosis model was established using Wistar rats to measure platelet aggregation and thrombus formation, respectively. W1 markedly inhibited adenosine 5'-diphosphate (ADP)-induced platelet aggregation and thrombus formation dose dependently (0.3, 1, and 3 mg/kg). W1 (3 mg/kg) acted rapidly at 0.5 hours and lasted for 72 hours. W1 prolonged bleeding and clotting times in mice, confirming its antithrombotic properties. Compared with CLO 10 mg/kg, the positive control, W1 3 mg/kg exerted equivalent effects on the above specifications. In addition, cyclic adenosine monophosphate levels, measured in rat platelets, increased rapidly after prostaglandin E1 (alprostadil) stimulation of the vehicle control (0.5% methyl cellulose suspension) and W1 (3 mg/kg)-treated groups. ADP (50 µm) reduced the control levels more remarkably than W1 did (P < 0.05 in 3 minutes or P < 0.001 at 5 minutes), suggesting that W1 suppressed ADP-induced cyclic adenosine monophosphate reduction. This was associated with a significant platelet reactivity inhibition measured using the vasodilator-stimulated phosphoprotein assay. CLO or W1 coadministration with or without omeprazole and amlodipine to rats to investigate the pharmacodynamic interactions revealed that W1 exhibited more stable and potent antithrombotic effects than CLO did. In conclusion, both W1 and CLO showed antiplatelet and antithrombotic effects, while the former exhibited less CLO resistance in combination with omeprazole or amlodipine, 2 drugs that inhibit CLO metabolism. Therefore, this study implies that W1 may be a promising oral antiplatelet agent for reducing CLO resistance after percutaneous coronary intervention.