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Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms.The FEBS Journal Jun 2024Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target....
Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target. Extracellular vesicles (EVs) may act as potent anti-inflammatory agents and also modulators of autophagy in target cells. However, the molecular mechanisms by which EVs modulate autophagy flux in human microglia remain largely unexplored. In the present study, we investigated the effects of EVs derived from human oral mucosa stem cells on the autophagy in human microglia. We demonstrate that EVs promoted autophagy and autophagic flux in human microglia and that this process was dependent on the integrity of lipid rafts. Lipopolysaccharide (LPS) also activated autophagy, but combined treatment with EVs and LPS suppressed autophagy response, indicating interference between these signaling pathways. Blockage of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody suppressed EV-induced autophagy. Furthermore, inhibition of the EV-associated heat shock protein (HSP70) chaperone which is one of the endogenous ligands of the TLR4 also suppressed EV-induced lipid raft formation and autophagy. Pre-treatment of microglia with a selective inhibitor of αvβ3/αvβ5 integrins cilengitide inhibited EV-induced autophagy. Finally, blockage of purinergic P2X4 receptor (P2X4R) with selective inhibitor 5-BDBD also suppressed EV-induced autophagy. In conclusion, we demonstrate that EVs activate autophagy in human microglia through interaction with HSP70/TLR4, αVβ3/αVβ5, and P2X4R signaling pathways and that these effects depend on the integrity of lipid rafts. Our findings could be used to develop new therapeutic strategies targeting disease-associated microglia.
PubMed: 38840471
DOI: 10.1111/febs.17192 -
Biological & Pharmaceutical Bulletin 2024Motile cilia in the ependymal cells that line the brain ventricles play pivotal roles in cerebrospinal fluid (CSF) flow in well-defined directions. However, the...
Motile cilia in the ependymal cells that line the brain ventricles play pivotal roles in cerebrospinal fluid (CSF) flow in well-defined directions. However, the substances and pathways which regulate their beating have not been well studied. Here, we used primary cultured cells derived from neonatal mouse brain that possess motile cilia and found that adenosine (ADO) stimulates ciliary beating by increasing the ciliary beat frequency (CBF) in a concentration-dependent manner, with the ED value being 5 µM. Ciliary beating stimulated by ADO was inhibited by A receptor (AR) antagonist MRS1754 without any inhibition by antagonists of other ADO receptor subtypes. The expression of AR on the cilia was also confirmed by immunofluorescence. The values of CBF were also increased by forskolin, which is an activator of adenylate cyclase, whereas they were not further increased by the addition of ADO. Furthermore, ciliary beating was not stimulated by ADO in the presence of a protein kinase A (PKA) inhibitors. These results altogether suggest that ADO stimulates ciliary beating through AR on the cilia, and activation of PKA.
Topics: Animals; Cilia; Receptor, Adenosine A2B; Cyclic AMP-Dependent Protein Kinases; Adenosine; Brain; Mice; Animals, Newborn; Cells, Cultured; Signal Transduction; Adenosine A2 Receptor Antagonists; Colforsin; Ependyma
PubMed: 38839362
DOI: 10.1248/bpb.b23-00913 -
PloS One 2024New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for...
New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.
Topics: Humans; Animals; Receptor, Adenosine A2A; HEK293 Cells; Mice; Adenosine A2 Receptor Antagonists; Single-Chain Antibodies; Macaca fascicularis; Peptide Library
PubMed: 38837964
DOI: 10.1371/journal.pone.0301223 -
Purinergic Signalling Jun 2024The A adenosine receptor (AR) is one of the four adenosine-activated G protein-coupled receptors. In addition to adenosine, protein kinase C (PKC) was recently found to... (Review)
Review
The A adenosine receptor (AR) is one of the four adenosine-activated G protein-coupled receptors. In addition to adenosine, protein kinase C (PKC) was recently found to activate the AR. The AR is coupled to both G and G, as well as G proteins in some cell types. Many primary cells and cell lines, such as bladder and breast cancer, bronchial smooth muscle, skeletal muscle, and fat cells, express the AR endogenously at high levels, suggesting its potentially important role in asthma, cancer, diabetes, and other conditions. The AR has been characterized as both pro- and anti-inflammatory, inducing cell type-dependent secretion of IL-6, IL-8, and IL-10. Theophylline and enprofylline have long been used for asthma treatment, although it is still not entirely clear if their AR antagonism contributes to their therapeutic effects or side effects. The AR is required in ischemic cardiac preconditioning by adenosine. Both AR and protein kinase C (PKC) contribute to cardioprotection, and both modes of AR signaling can be blocked by AR antagonists. Inhibitors of PKC and AR are in clinical cancer trials. Sulforaphane and other isothiocyanates from cruciferous vegetables such as broccoli and cauliflower have been reported to inhibit AR signaling via reaction with an intracellular AR cysteine residue (C210). A full, AR-selective agonist, critical to elucidate many controversial roles of the AR, is still not available, although agonist-bound AR structures have recently been reported.
PubMed: 38833181
DOI: 10.1007/s11302-024-10025-y -
Scientific Reports Jun 2024Evidence has shown that both sleep loss and daily caffeine intake can induce changes in grey matter (GM). Caffeine is frequently used to combat sleepiness and impaired... (Randomized Controlled Trial)
Randomized Controlled Trial
Repeated caffeine intake suppresses cerebral grey matter responses to chronic sleep restriction in an A adenosine receptor-dependent manner: a double-blind randomized controlled study with PET-MRI.
Evidence has shown that both sleep loss and daily caffeine intake can induce changes in grey matter (GM). Caffeine is frequently used to combat sleepiness and impaired performance caused by insufficient sleep. It is unclear (1) whether daily use of caffeine could prevent or exacerbate the GM alterations induced by 5-day sleep restriction (i.e. chronic sleep restriction, CSR), and (2) whether the potential impact on GM plasticity depends on individual differences in the availability of adenosine receptors, which are involved in mediating effects of caffeine on sleep and waking function. Thirty-six healthy adults participated in this double-blind, randomized, controlled study (age = 28.9 ± 5.2 y/; F:M = 15:21; habitual level of caffeine intake < 450 mg; 29 homozygous C/C allele carriers of rs5751876 of ADORA2A, an A adenosine receptor gene variant). Each participant underwent a 9-day laboratory visit consisting of one adaptation day, 2 baseline days (BL), 5-day sleep restriction (5 h time-in-bed), and a recovery day (REC) after an 8-h sleep opportunity. Nineteen participants received 300 mg caffeine in coffee through the 5 days of CSR (CAFF group), while 17 matched participants received decaffeinated coffee (DECAF group). We examined GM changes on the 2nd BL Day, 5th CSR Day, and REC Day using magnetic resonance imaging and voxel-based morphometry. Moreover, we used positron emission tomography with [F]-CPFPX to quantify the baseline availability of A adenosine receptors (AR) and its relation to the GM plasticity. The results from the voxel-wise multimodal whole-brain analysis on the Jacobian-modulated T1-weighted images controlled for variances of cerebral blood flow indicated a significant interaction effect between caffeine and CSR in four brain regions: (a) right temporal-occipital region, (b) right dorsomedial prefrontal cortex (DmPFC), (c) left dorsolateral prefrontal cortex (DLPFC), and (d) right thalamus. The post-hoc analyses on the signal intensity of these GM clusters indicated that, compared to BL, GM on the CSR day was increased in the DECAF group in all clusters but decreased in the thalamus, DmPFC, and DLPFC in the CAFF group. Furthermore, lower baseline subcortical AR availability predicted a larger GM reduction in the CAFF group after CSR of all brain regions except for the thalamus. In conclusion, our data suggest an adaptive GM upregulation after 5-day CSR, while concomitant use of caffeine instead leads to a GM reduction. The lack of consistent association with individual AR availability may suggest that CSR and caffeine affect thalamic GM plasticity predominantly by a different mechanism. Future studies on the role of adenosine A receptors in CSR-induced GM plasticity are warranted.
Topics: Humans; Caffeine; Male; Adult; Gray Matter; Receptor, Adenosine A1; Positron-Emission Tomography; Female; Magnetic Resonance Imaging; Double-Blind Method; Sleep Deprivation; Young Adult; Receptor, Adenosine A2A
PubMed: 38830861
DOI: 10.1038/s41598-024-61421-8 -
Expert Opinion on Therapeutic Patents Apr 2024The purinergic P2X7 receptor (P2X7R) is expressed on the surface of many different types of cells, including immune cells. Targeting P2X7R with antagonists has been... (Review)
Review
INTRODUCTION
The purinergic P2X7 receptor (P2X7R) is expressed on the surface of many different types of cells, including immune cells. Targeting P2X7R with antagonists has been studied for its potential therapeutic effects in a variety of inflammatory illnesses.
AREA COVERED
Many chemical substances, including carboxamides, benzamides and nitrogen containing heterocyclic derivatives have demonstrated promising inhibitory potential for P2X7 receptor. The chemistry and clinical applications of P2X7R antagonists patented from 2018- present are discussed in this review.
EXPERT OPINION
Purinergic receptor inhibitor discovery and application has demonstrated the potential for therapeutic intervention, as demonstrated by pharmacological research. Few chemical modalities have been authorized for use in clinical settings, despite the fact that breakthroughs in crystallography and chemical biology have increased the knowledge of purinergic signaling and its consequences in disease. The many research projects and pharmaceutical movements that sustain dynamic P2X receptor programs over decades are evidence of the therapeutic values and academic persistence in purinergic study. P2X7R is an intriguing therapeutic target and possible biomarker for inflammation. Although several companies like Merck and AstraZeneca have published patents on P2X3 antagonists, the search for P2X7R antagonists has not stopped. Numerous pharmaceutical companies have disclosed different scaffolds, and some molecules are presently being studied in clinical studies.
Topics: Humans; Patents as Topic; Receptors, Purinergic P2X7; Purinergic P2X Receptor Antagonists; Animals; Inflammation; Drug Development; Anti-Inflammatory Agents
PubMed: 38828613
DOI: 10.1080/13543776.2024.2363885 -
BioRxiv : the Preprint Server For... May 2024Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During...
Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine drives fast excitation and delayed inhibition of behavior and circuit activity. We find that the inhibitory arm of this feedforward motif is implemented by astroglial purinergic signaling. Neuromodulator imaging, behavioral pharmacology, and perturbations of neurons and astroglia reveal that norepinephrine triggers astroglial release of adenosine triphosphate, extracellular conversion into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. This work, along with a companion piece by Lefton and colleagues demonstrating an analogous pathway mediating the effect of norepinephrine on synaptic connectivity in mice, identifies a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in norepinephrine-mediated behavioral and brain state transitions.
PubMed: 38826423
DOI: 10.1101/2024.05.23.595576 -
Kidney International May 2024Platelets are anucleated cells that circulate in the bloodstream. Historically, platelets were thought to perform a singular function-stop bleeding via clotting....
Platelets are anucleated cells that circulate in the bloodstream. Historically, platelets were thought to perform a singular function-stop bleeding via clotting. Although platelets do play a key role in hemostasis and thrombosis, recent studies indicate that platelets also modulate inflammation, and this platelet-induced inflammation contributes to the pathophysiology of various diseases such as atherosclerosis and diabetes mellitus. Thus, in recent years, our understanding of platelet function has broadened. In this review, we revisit the classic role of platelets in hemostasis and thrombosis and describe the newly recognized function of platelets in modulating inflammation. We cover the potential use of purinergic receptor antagonists to prevent platelet-modulated inflammation, particularly in patients with chronic kidney disease, and finally, we define key questions that must be addressed to understand how platelet-modulated inflammation contributes to the pathophysiology of chronic kidney disease.
PubMed: 38821448
DOI: 10.1016/j.kint.2024.03.033 -
Phytomedicine : International Journal... Jul 2024Gouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the...
BACKGROUND
Gouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the limitations of current therapies. Astilbin, a flavonoid from Smilax glabra Roxb, has demonstrated potential in traditional Chinese medicine for its anti-inflammatory properties. However, the anti-GA effect and its underlying mechanism have not been fully elucidated.
PURPOSE
This study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo.
STUDY DESIGN
Firstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway.
METHODS
MSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting.
RESULTS
Under MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R.
CONCLUSION
This study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.
Topics: Extracellular Traps; Humans; Interleukin-8; Receptors, Purinergic P2; Neutrophils; Arthritis, Gouty; HL-60 Cells; Flavonols; Animals; Uric Acid; Receptors, Interleukin-8B; Male; Histones; Anti-Inflammatory Agents; Mice
PubMed: 38820662
DOI: 10.1016/j.phymed.2024.155754 -
Neural Regeneration Research Feb 2025Alzheimer's disease is the most frequent form of dementia characterized by the deposition of amyloid-beta plaques and neurofibrillary tangles consisting of...
Alzheimer's disease is the most frequent form of dementia characterized by the deposition of amyloid-beta plaques and neurofibrillary tangles consisting of hyperphosphorylated tau. Targeting amyloid-beta plaques has been a primary direction for developing Alzheimer's disease treatments in the last decades. However, existing drugs targeting amyloid-beta plaques have not fully yielded the expected results in the clinic, necessitating the exploration of alternative therapeutic strategies. Increasing evidence unravels that astrocyte morphology and function alter in the brain of Alzheimer's disease patients, with dysregulated astrocytic purinergic receptors, particularly the P2Y1 receptor, all of which constitute the pathophysiology of Alzheimer's disease. These receptors are not only crucial for maintaining normal astrocyte function but are also highly implicated in neuroinflammation in Alzheimer's disease. This review delves into recent insights into the association between P2Y1 receptor and Alzheimer's disease to underscore the potential neuroprotective role of P2Y1 receptor in Alzheimer's disease by mitigating neuroinflammation, thus offering promising avenues for developing drugs for Alzheimer's disease and potentially contributing to the development of more effective treatments.
PubMed: 38819047
DOI: 10.4103/NRR.NRR-D-23-02103