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New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1? Release: An Involvement in its Alleviating Effect on Neuropathic Pain.

ABSTRACT: P2X7 receptors (P2X7Rs) belong to a family of ATP-gated non-selective cation channels. Microglia represent a major cell type expressing P2X7Rs. The activation of microglial P2X7Rs causes the release of pro-inflammatory cytokines such as interleukin-1? (IL-1?). This response has been implicated in neuroinflammatory states in the central nervous system and in various diseases, including neuropathic pain. Thus, P2X7R may represent a potential therapeutic target. In the present study, we screened a chemical library of clinically approved drugs (1979 compounds) by high-throughput screening and showed that the Ca²⁺ channel blocker cilnidipine has an inhibitory effect on rodent and human P2X7R. In primary cultured rat microglial cells, cilnidipine inhibited P2X7R-mediated Ca²⁺ responses and IL-1? release. Moreover, in a rat model of neuropathic pain, the intrathecal administration of cilnidipine produced a reversal of nerve injury-induced mechanical hypersensitivity, a cardinal symptom of neuropathic pain. These results point to a new inhibitory effect of cilnidipine on microglial P2X7R-mediated inflammatory responses and neuropathic pain, proposing its therapeutic potential.

SUBMITTER: Yamashita T

PROVIDER: S-EPMC7922706 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1β Release: An Involvement in its Alleviating Effect on Neuropathic Pain.

Yamashita Tomohiro T Kamikaseda Sawako S Tanaka Aya A Tozaki-Saitoh Hidetoshi H Caaveiro Jose M M JMM Inoue Kazuhide K Tsuda Makoto M

Cells 20210218 2

P2X7 receptors (P2X7Rs) belong to a family of ATP-gated non-selective cation channels. Microglia represent a major cell type expressing P2X7Rs. The activation of microglial P2X7Rs causes the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β). This response has been implicated in neuroinflammatory states in the central nervous system and in various diseases, including neuropathic pain. Thus, P2X7R may represent a potential therapeutic target. In the present study, we screened a ...[more]

PMID: 33670748

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Project description:BackgroundATP-gated P2X7 is a non-selective cation channel, which participates in a wide range of cellular functions as well as pathophysiological processes including neuropathic pain, immune response, and neuroinflammation. Despite its abundant expression in microglia, the role of P2X7 in neuroinflammation still remains unclear.MethodsPrimary microglia were isolated from cortices of P0-2 C57BL/6 wild-type or P2X7 knockout (P2X7-/-) mouse pups. Lipopolysaccharide, lipopolysaccharide plus IFN?, or IL4 plus IL13 were used to polarize microglia to pro-inflammatory or anti-inflammatory states. P2rx7 expression level in resting or activated mouse and human microglia was measured by RNA-sequencing and quantitative real-time PCR. Microglial cell death was measured by cell counting kit-8 and immunocytochemistry, and microglial secretion in wild-type or P2X7-/- microglia was examined by Luminex multiplex assay or ELISA using P2X7 agonist BzATP or P2X7 antagonist A-804598. P2X7 signaling was analyzed by Western blot.ResultsFirst, we confirmed that P2rx7 is constitutively expressed in mouse and human primary microglia. Moreover, P2rx7 mRNA level was downregulated in mouse microglia under both pro- and anti-inflammatory conditions. Second, P2X7 agonist BzATP caused cell death of mouse microglia, while this effect was suppressed either by P2X7 knockout or by A-804598 under both basal and pro-inflammatory conditions, which suggests the mediating role of P2X7 in BzATP-induced microglial cell death. Third, BzATP-induced release of IL1 family cytokines including IL1?, IL1?, and IL18 was blocked in P2X7-/- microglia or by A-804598 in pro-inflammatory microglia, while the release of other cytokines/chemokines was independent of P2X7 activation. These findings support the specific role of P2X7 in IL1 family cytokine release. Finally, P2X7 activation was discovered to be linked to AKT and ERK pathways, which may be the underlying mechanism of P2X7 functions in microglia.ConclusionsThese results reveal that P2X7 mediates BzATP-induced microglial cell death and specific release of IL1 family cytokines, indicating the important role of P2X7 in neuroinflammation and implying the potential of targeting P2X7 for the treatment of neuroinflammatory disorders.

Project description:BackgroundCentral sensitization is an important mechanism of chronic migraine (CM) and is related to the inflammatory response of microglia. The NOD-like receptor protein 3 (NLRP3) inflammasome may regulate the inflammatory process of microglia in several neurological diseases, but its role in CM is largely unknown. Therefore, the aim of this study was to identify the precise role of microglial NLRP3 in CM.MethodsAn experimental CM mouse model was established by repeated intraperitoneal (i.p) injection with nitroglycerin (NTG). We evaluated the expression levels of NLRP3 and its downstream interleukin (IL)-1β protein in the trigeminal nucleus caudalis (TNC; which is a central area relevant to migraine pain) at different time points. To further examine the effects of the NLRP3 inflammasome pathway on central sensitization of CM, we examined MCC950, an NLRP3 inflammasome-specific inhibitor, and IL-1ra, an IL-1β antagonist, whether altered NTG-induced mechanical hyperalgesia of the periorbital area and hind paw. The effect of MCC950 and IL-1ra on c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK) and calcitonin gene-related peptide (CGRP) expression in the TNC were also analyzed. The cell localization of NLRP3 and IL-1β in the TNC was evaluated by immunofluorescence staining.ResultsRepeated NTG administration induced acute and chronic mechanical hyperalgesia and increased expression of NLRP3 and IL-1β. Blockade of NLRP3 or IL-1β reduced NTG-induced hyperalgesia, and this effect was accompanied by a significant inhibition of the NTG-induced increase in p-ERK, c-Fos and CGRP levels in the TNC. Immunofluorescence staining revealed that NLRP3 and IL-1β were mainly expressed in microglia in the TNC, and the IL-1β receptor, IL-1R, was mainly expressed in neurons in the TNC.ConclusionsThese results indicate that NLRP3 activation in the TNC participates in the microglial-neuronal signal by mediating the inflammatory response. This process contributes to the central sensitization observed in CM.

Project description:Inflammatory responses play a key role in many neural pathologies, with localized signaling from the non-immune cells making critical contributions. The NLRP3 inflammasome is an important component of innate immune signaling and can link neural insult to chronic inflammation. The NLRP3 inflammasome requires two stages to contribute: priming and activation. The priming stage involves upregulation of inflammasome components while the activation stage results in the assembly and activation of the inflammasome complex. The priming step can be rate limiting and can connect insult to chronic inflammation, but our knowledge of the signals that regulate NLRP3 inflammasome priming in sterile inflammation is limited. This study examined the link between mechanical strain and inflammasome priming in neural systems. Transient non-ischemic elevation of intraocular pressure increased mRNA for inflammasome components IL-1β, NLRP3, ASC, and CASP1 in rat and mouse retinas. The elevation was greater 1 day after the insult, with the rise in IL-1β most pronounced. The P2X7 receptor was implicated in the mechanosensitive priming of IL-1β mRNA in vivo, as the antagonist Brilliant Blue G (BBG) blocked the increased expression, the agonist BzATP mimicked the pressure-dependent rise in IL-1β, and the rise was absent in P2X7 knockout mice. In vitro measurements from optic nerve head astrocytes demonstrated an increased expression of IL-1β following stretch or swelling. This increase in IL-1β was eliminated by degradation of extracellular ATP with apyrase, or by the block of pannexin hemichannels with carbenoxolone, probenecid, or 10panx1 peptide. The rise in IL-1β expression was also blocked by P2X7 receptor antagonists BBG, A839977 or A740003. The rise in IL-1β was prevented by blocking transcription factor NFκB with Bay 11-7082, while the swelling-dependent fall in NFκB inhibitor IκB-α was reduced by A839977 and in P2X7 knockout mice. In summary, mechanical trauma to the retina primed NLRP3 inflammasome components, but only if there was ATP release through pannexin hemichannels, and autostimulation of the P2X7 receptor. As the P2X7 receptor can also trigger stage two of inflammasome assembly and activation, the P2X7 receptor may have a central role in linking mechanical strain to neuroinflammation.

Dataset Information

New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1? Release: An Involvement in its Alleviating Effect on Neuropathic Pain.

Publications

New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1β Release: An Involvement in its Alleviating Effect on Neuropathic Pain.

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