Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated neuroinflammation

Abstract

BackgroundLong-term use of morphine induces analgesic tolerance, which limits its clinical efficacy. Evidence indicated morphine-evoked neuroinflammation mediated by toll-like receptor 4 (TLR4) - NOD-like receptor protein 3 (NLRP3) inflammasome was important for morphine tolerance. In our study, we investigated whether other existing alternative pathways caused morphine-induced activation of TLR4 in microglia. We focused on heat shock protein 70 (HSP70), a damage-associated molecular pattern (DAMP), which was released from various cells upon stimulations under the control of KATP channel and bound with TLR4-inducing inflammation. Glibenclamide, a classic KATP channel blocker, can improve neuroinflammation by inhibiting the activation of NLRP3 inflammasome. Our present study investigated the effect and possible mechanism of glibenclamide in improving morphine tolerance via its specific inhibition on the release of HSP70 and activation of NLRP3 inflammasome induced by morphine.MethodsCD-1 mice were used for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 and neural cell line SH-SY5Y were used to investigate the pharmacological effects and the mechanism of glibenclamide on morphine-induced neuroinflammation. The activation of microglia was accessed by immunofluorescence staining. Neuroinflammation-related cytokines were measured by western blot and real-time PCR. The level of HSP70 and related signaling pathway were evaluated by western blot and immunofluorescence staining.ResultsMorphine induced the release of HSP70 from neurons. The released HSP70 activated microglia and triggered TLR4-mediated inflammatory response, leading to the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 and the activation of NLRP3 inflammasome. Moreover, anti-HSP70 neutralizing antibody partly attenuated chronic morphine tolerance. The secretion of HSP70 was under the control of MOR/AKT/KATP/ERK signal pathway. Glibenclamide as a classic KATP channel blocker markedly inhibited the release of HSP70 induced by morphine and suppressed HSP70-TLR4-NLRP3 inflammasome-mediated neuroinflammation, which consequently attenuated morphine tolerance.ConclusionsOur study indicated that morphine-induced extracellular HSP70 was an alternative way for the activation of TLR4-NLRP3 in analgesic tolerance. The release of HSP70 was regulated by MOR/AKT/KATP/ERK pathway. Our study suggested a promising target, KATP channel and a new leading compound, glibenclamide, for treating morphine tolerance.Electronic supplementary materialThe online version of this article (10.1186/s12974-017-0997-0) contains supplementary material, which is available to authorized users.

A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy

“…Depending on the experiment, cell lines were treated with Wnt5a (100-200 ng/mL, R&D Systems, Bio-Techne, 645-WN-010), IFN-γ (100 ng/mL, BioAbChem, 42-IFNg), anti-PD-L1 Ab (1-2 μg/mL), HSP70 (1-10 μM, Enzo, ADI-ESP-502-D), HSP70 inhibitor (Thermo Fisher Scientific, VER155008), CLI-095 TLR4 inhibitor (3-10 μM, Invivogen, tlrl-cli95), TLR2-IN-C29 TLR2 inhibitor (1-10 μM, Glixx, GLXC-06203), MPLA TLR4 agonist (10 μM, Enzo, ALX-581-205-C100), LPS (10 ng, Millipore-Sigma, L4391-1MG), recombinant IL-1β (100-200 ng, BioLegend, IFN-γ stimulation also serves to facilitate NLRP3 priming by an alternative mechanism in tumor cells is currently being investigated. Interestingly, HSP70/TLR4 signaling, as described above, may also provide a positive feed-forward priming pathway capable of perpetuating NLRP3 activation in tumors (46). These studies reveal that NLRP3 inhibition phenocopied downstream TLR4 and CXCR2 inhibition, suppressing the recruitment of PMN-MDSCs as an adaptive resistance mechanism initiated by local CD8 + T cell activity.…”

Morphine Addiction and Oxidative Stress: The Potential Effects of Thioredoxin-1

“…An increasing number of studies have revealed that several brain regions, such as the ventral tegmental area (VTA), nucleus accumbens (NAc), and hippocampus (Hipp), are involved in morphine addiction (Kim et al, 2016). Although mechanisms underlying morphine-mediated processes remain the subject of much debate, morphine stimulation activates G protein-coupled opioid receptors and then induces significant molecular changes inside the cell, such as an inhibition of adenylate cyclase activity, and activation of potassium channels (Qu et al, 2017;Yang et al, 2019). In addition, other signalling pathways, including mitogenactivated kinases (MAPK), b-arrestin, phospholipase C, protein kinase, PI3K, and extracellular signal-regulated kinase (ERK) pathways, are also involved in morphine activity (Bianchi et al, 2010;Zhang and Pan, 2010;Dai et al, 2018;Shen et al, 2018;de Freitas et al, 2019;Dekan et al, 2019;Listos et al, 2019).…”

<p>Spinal TLR4/P2X7 Receptor-Dependent NLRP3 Inflammasome Activation Contributes to the Development of Tolerance to Morphine-Induced Antinociception</p>

“…30,31 Neuroinflammation has been heavily implicated in the formation of morphine-induced antinociceptive tolerance. 32 Inhibiting NLRP3 signaling to decrease neuroinflammation has been reported to alleviate morphineinduced antinociception tolerance in previous studies 3,5,33,34 Furthermore, with regard to neuropathic status, miR-223 has been reported to downregulate and suppress the activities of the NLRP3 inflammasome to relieve morphine-induced antinociception tolerance in rats. 35 Studies have shown that morphine can also modulate immune responses by promoting IL-1β release, while the inhibition of IL-1β prevents morphine-induced antinociception tolerance.…”