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

Jie Qu,Xue-You Tao,Yan Zhang,Peng Teng,Ci-Liang Guo,Wen-Tao Liu,Chun-Yi Jiang,Liang Hu,Yan-Ning Qian

doi:10.1186/s12974-017-0997-0

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.

Highlights

Long-term use of morphine induces analgesic tolerance, which limits its clinical efficacy
We found that morphine promoted the efflux of heat shock protein 70 (HSP70) into extracellular environment in a concentration-dependent manner (Fig. 1a); morphine decreased HSP70 protein level in SH-SY5Y cells (Fig. 1b)
One study reported that H2S exerted a protective effect against cerebral hypoxia-induced neuronal cell death via KATP/PKC/ERK1/2/HSP90 pathway [55], and they proved that glibenclamide abolished the effect of NaHS on ERK1/ 2 phosphorylation in SH-SY5Y cells, indicating that ERK1/ 2 activation was downstream to KATP channel; we investigated whether or not glibenclamide could inhibit elevated ERK1/2 phosphorylation induced by morphine

Summary

Introduction

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. 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. The mechanism of morphine tolerance is complicated, which involves many aspects, such as receptors, ion channels, and neuroinflammation [1,2,3]. Chronic morphine administration activated microglia in the spinal cord [4,5,6,7,8,9,10], and the activated microglia released proinflammatory factors such as IL-1β, tumor necrosis factor-α (TNF-α), and IL-6 [11] and upregulated the expression of cell-surface receptors, such as TLR4, which participated in the neuroinflammatory process [12,13,14,15,16,17]

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