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Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Published on: July 29, 2014
Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated
Jie Qu1, Xue-You Tao1,2,3, Peng Teng1
1Neuroprotective Drug Discovery Key Laboratory of Nanjing Medical University, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu, 211166, China.
Background:
Long-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.
Methods:
CD-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.
Results:
Morphine 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.
Conclusions:
Our 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.
Insights
Morphine tolerance is linked to neuroinflammation via heat shock protein 70 (HSP70) and toll-like receptor 4 (TLR4). Glibenclamide blocks HSP70 release, reducing neuroinflammation and improving morphine tolerance.
Area of Science:
- Neuroscience
- Pharmacology
- Immunology
Background:
- Long-term morphine use leads to analgesic tolerance, limiting its effectiveness.
- Morphine-induced neuroinflammation, involving toll-like receptor 4 (TLR4) and NOD-like receptor protein 3 (NLRP3) inflammasome, is crucial for tolerance.
- Heat shock protein 70 (HSP70), a damage-associated molecular pattern, is released and activates TLR4, contributing to inflammation.
Purpose of the Study:
- To investigate alternative pathways causing morphine-induced TLR4 activation in microglia.
- To explore the role of HSP70 in morphine tolerance.
- To evaluate the potential of glibenclamide in improving morphine tolerance by targeting HSP70 release and NLRP3 inflammasome activation.
Main Methods:
- Tail-flick tests in CD-1 mice to assess morphine tolerance.
- In vitro studies using BV-2 microglial and SH-SY5Y neural cell lines.
- Immunofluorescence staining for microglial activation and HSP70 levels.
- Western blot and real-time PCR for neuroinflammation markers and signaling pathways (MAPK, NF-κB).
Main Results:
- Morphine induced HSP70 release from neurons, activating microglia and TLR4.
- HSP70 triggered TLR4-mediated inflammation, activating p38 MAPK, NF-κB, and the NLRP3 inflammasome.
- Anti-HSP70 antibody partially reduced morphine tolerance.
- HSP70 release is regulated by the MOR/AKT/KATP/ERK pathway.
- Glibenclamide inhibited morphine-induced HSP70 release and suppressed neuroinflammation, attenuating morphine tolerance.
Conclusions:
- Morphine-induced extracellular HSP70 is an alternative pathway for TLR4-NLRP3 activation in analgesic tolerance.
- HSP70 release is regulated by the MOR/AKT/KATP/ERK signaling pathway.
- KATP channels represent a promising therapeutic target, and glibenclamide is a potential compound for treating morphine tolerance.
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