Divanillyl sulfone suppresses NLRP3 inflammasome activation via inducing mitophagy to ameliorate chronic neuropathic pain in mice

Shao et al. Journal of Neuroinflammation (2021) 18:142 https://doi.org/10.1186/s12974-021-02178-z RESEARCH Open Access Divanillyl sulfone suppresses NLRP3 inflammasome activation via inducing mitophagy to ameliorate chronic neuropathic pain in mice Shuai Shao†, Cheng-Bo Xu†, Cheng-Juan Chen, Gao-Na Shi, Qing-Lan Guo, Yu Zhou, Ya-Zi Wei, Lei Wu, Jian-Gong Shi* and Tian-Tai Zhang* Abstract Background: Chronic neuropathic pain is a frequent sequel to peripheral nerve injury and maladaptive nervous system function. Divanillyl sulfone (DS), a novel structural derivative of 4,4′-dihydroxydibenzyl sulfoxide from a traditional Chinese medicine Gastrodia elata with anti-nociceptive effects, significantly alleviated neuropathic pain following intrathecal injection. Here, we aimed to investigate the underlying mechanisms of DS against neuropathic pain. Methods: A chronic constrictive injury (CCI) mouse model of neuropathic pain induced by sciatic nerve ligation was performed to evaluate the effect of DS by measuring the limb withdrawal using Von Frey filament test. Immunofluorescence staining was used to assess the cell localizations and expressions of Iba-1, ASC, NLRP3, and ROS, the formation of autolysosome. The levels of NLRP3-related proteins (caspase-1, NLRP3, and IL-1β), mitophagy-related proteins (LC3, Beclin-1, and p62), and apoptosis-related proteins (Bcl-XL and Bax) were detected by Western blotting. The apoptosis of BV-2 cell and caspase activity were evaluated by flow cytometry. Results: DS significantly alleviated the neuropathic pain by increasing the mechanical withdrawal threshold and inhibiting the activation of NLRP3 in CCI-induced model mice. Our findings indicated that DS promoted the mitophagy by increasing the LC3II and Beclin 1 and decreasing the levels of p62 protein in BV-2 cell. This is accompanied by the inhibition of NLRP3 activation, which was shown as inhibited the expression of NLRP3 in lysates as well as the secretion of mature caspase-1 p10 and IL-1β p17 in supernatants in cultured BV-2 microglia. In addition, DS could promote mitophagy-induced improvement of dysfunctional mitochondria by clearing intracellular ROS and restoring mitochondrial membrane potential. Conclusion: Together, our findings demonstrated that DS ameliorate chronic neuropathic pain in mice by suppressing NLRP3 inflammasome activation induced by mitophagy in microglia. DS may be a promising therapeutic agent for chronic neuropathic pain. Keywords: Chronic neuropathic pain, Microglia, Mitophagy, NLRP3 inflammasome, Divanillyl sulfone * Correspondence: ; † Shuai Shao and Cheng-Bo Xu contributed equally to this work. State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shao et al. Journal of Neuroinflammation (2021) 18:142 Background Neuralgia is a type of chronic pain that manifests as spontaneous pain, hyperalgesia, and allodynia [1, 2]. It is caused by injury or disease in the somatosensory nervous system that includes the central nerves, spinal cord, posterior root of the spinal cord, and peripheral nerves. Pre-clinical studies show that the basis of neuropathic pain is the plasticity of nerve cells [3, 4]. In addition, some non-neuronal cells of the central nervous system (CNS), especially microglia, have also been implicated in triggering neuropathic pain [5–8]. Microglia is a kind of primary innate immune cells in the CNS, and is activated by various pathological stimuli. The activated microglia interacts with the astroglia or neurons to induce neuroinflammation and facilitate transmission of pain signals [9–11]. Chronic neuropathic pain is characterized by infiltration of immune cells into the dorsal root ganglia (DRG), and the activation of microglia in spinal cord and brain, eventually leading to a neuroinflammatory response [12]. Pro-inflammatory factor of interleukin-1β (IL-1β) plays an important role in the microglial inflammatory signaling mediated neuropathic pain [13]. Multiple mechanisms participate in the central neuronal excitation mediated by microglial inflammation in peripheral nerve injury (PNI)-induced neuropathic pain. In microglia, the activation of toll-like receptor 2 (TLR2) and/or TLR4 can promote nuclear factor-κB (NF-κB) signal to induce IL-1β transcription [14], triggering receptor expressed on myeloid cells 2 (TREM2) and via transcription factors interferon regulatory factor 1/5/8 (IRF1/5/8) [15]. In addition, P2X purinoceptor 7 (P2X7), P2Y purinoceptor 12 (P2Y12), and CX3C-chemokine receptor 1 (CX3CR1) on microglial cells also lead to IL-1β secretion via p38 MAPKs as well [16]. The important thing is that nucleotide-binding oligomerization domain (NOD), leucine-rich repeat, and pyrin domains-containing protein (NLRP)-type inflammasomes promote pro-IL-1β processing and mature IL1β secretion. Of NLR family, NLR family pyrin domaincontaining 3 (NLRP3) is the most extensively studied and well-characterized inflammasome sensor molecule. Meanwhile, NLRP3 inflammasome can also be activated by TLRs receptor and P2X7. IL-1β acts as mediators between microglia and neurons and assumes roles as neuromodulator when it acts on spinal dorsal horn (SDH) neurons to increase the strength of synaptic connectivity and excitatory synaptic transmission in the process of neuropathic pain [17, 18]. Thereby, the NLRP3 inflammasome is the most recognized contributor and plays an irreplaceable role to the transmission of pain signals [19, 20]. NLRP3 inflammasome is a multi-protein complex consisting of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) Page 2 of 18 and danger-associated molecular patterns (DAMPs) during the course of natural immune response, which then activates caspase-1 and promotes the m (...truncated)