Loganin prevents CXCL12/CXCR4-regulated neuropathic pain via the NLRP3 inflammasome axis in nerve-injured rats

Abstract

Background

Neuropathic pain has been shown to be modulated by the activation of the chemokine C-X-C motif ligand 12 (CXCL12)/chemokine CXC receptor 4 (CXCR4) dependent nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. Loganin, an iridoid glycoside, was proven to prevent neuropathic pain, but its underlying mechanisms related to NLRP3 activation are still unknown.

Purpose

This study investigated the underlying mechanisms of loganin's effect on chronic constriction injury (CCI)-induced NLRP3 inflammasome activation in the spinal cord.

Methods

Sprague-Dawley rats were randomly divided into four groups: sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was administered intraperitoneally starting the day after surgery. Paw withdrawal threshold (PWT) and latency (PWL) were assessed before CCI and on days 1, 3, 7 and 14 after CCI. Spinal cords were collected for western blots and immunofluorescence studies.

Results

Loganin prevented CCI-attenuated PWT and PWL, suggesting improved mechanical allodynia and thermal hyperalgesia. The expression of CXCL12, CXCR4, thioredoxin-interacting protein (TXNIP), NLRP3 inflammasome (NLRP3, ASC, and caspase-1), IL-1β, and IL-18 were enhanced on day 7 after CCI, and all were reduced after loganin treatment. Dual immunofluorescence also showed that increased CXCL12, CXCR4, and NLRP3 were colocalized with NeuN (neuronal marker), GFAP (astrocyte marker), and Iba1 (microglial marker) on day 7 in the ipsilateral spinal dorsal horn (SDH). These immunoreactivities were attenuated in loganin-treated rats. Moreover, loganin decreased the assembly of NLRP3/ASC inflammasome after CCI in the ipsilateral SDH. Loganin appears to attenuate CCI-induced neuropathic pain by suppressing CXCL12/CXCR4-mediated NLRP3 inflammasome.

Conclusion

Our findings suggest that loganin might be a suitable candidate for managing CCI-provoked neuropathic pain.

Introduction

Neuropathic pain is identified as pain arising from a lesion or disease affecting the somatosensory system either at the level of the peripheral nervous system (PNS) or the central nervous system (CNS), and it is often refractory to all currently available treatments, including opioids and nonsteroidal anti-inflammatory drugs (Tashima et al., 2016). Clarifying the precise mechanisms of neuropathic pain in the PNS or CNS is necessary for discovering new pharmacotherapeutic agents to address it. Diverse animal models of neuropathic pain indicate that microglia in the CNS and macrophages in the PNS are crucial to nerve injury-induced pain hypersensitivity and are a likely target of future therapeutics (Masuda et al., 2014; Tashima et al., 2016). Peripheral and central sensitization, predominantly in the dorsal root ganglion and spinal dorsal horn respectively, are essential to the development and prolongation of neuropathic pain (Ji and Suter, 2007; Kuner, 2010). The central sensitization mechanism has been recognized as the primary element in neuropathic pain. While peripheral sensitization after nerve injuries increases pain sensitivity, central sensitization reducing the pain threshold and amplifying the pain response ultimately causes neuropathic pain (Kuner, 2010). Neuropathic pain is mediated by neuroinflammatory mechanisms affecting the nervous system tissue, controlled by inflammatory responses to the initial insult. However, neuropathic pain's pathogenesis is still not fully understood and its treatment still faces enormous challenges.

Chemokine CXC receptor 4 (CXCR4) has been proven to have glia-modulatory and neuro-modulatory characteristics in the CNS (Li and Ransohoff, 2008). A growing body of evidence has demonstrated that CXCR4 is involved in various nociceptive responses such as neuropathic pain or cancer pain in glial cells of the spinal cord or dorsal root ganglion (Knerlich-Lukoschus et al., 2011; Pan et al., 2018). In a recent study, chemokine C-X-C motif ligand 12 (CXCL12) and its receptor CXCR4 were upregulated after partial sciatic nerve ligation in mice (Luo et al., 2016a) and spinal nerve ligation in rats (Liu et al., 2019). The CXCL12/CXCR4 axis is also crucial for the central sensitization mechanisms of pathological pain (Liu et al., 2019). The CXCL12/CXCR4 axis was recently demonstrated to play an essential role in the pathogenesis of neuropathic pain, hyperalgesia, cancer pain, and opioid tolerance in the CNS or PNS under pathological conditions (Luo et al., 2016b).

Thioredoxin-interacting protein (TXNIP) is ubiquitously expressed in various cells and acts endogenously as a suppressor of reactive oxygen species (ROS) scavenging protein thioredoxin. TXNIP is an essential molecular nutrient sensor of oxidative stress and inflammation in the modulation of energy metabolism (Zhou et al., 2010), and is associated with Alzheimer's disease, depression, stroke, and spinal or brain injury (Mahmood et al., 2013; Pan et al., 2018). TXNIP is linked to the regulation of neuropathic pain and modulated by CXCR4, since CXCR4 protein can stabilize TXNIP by binding to its structure to prevent its degradation, resulting in an increase of TXNIP protein in the spinal cord of neuropathic pain mice (Pan et al., 2018).

Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome activation is affected by multiple cellular proteins or factors. TXNIP bound to NLRP3 is necessary for NLRP3 formation and activation and TXNIP inhibition markedly decreased NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1 levels, IL-1β and IL-18 (Abais et al., 2014; Zhou et al., 2010). The TXNIP/NLRP3 axis has been confirmed to be a key player in CNS dysfunction in several diseases, such as brain ischemic stroke, multiple sclerosis, type 2 diabetes, and hippocampus injury (Pan et al., 2018; Rutz et al., 2015; Zhou et al., 2010) where nerve injury-induced TXNIP enhancement and NLRP3 activation form a NLRP3 and TXNIP complex (Pan et al., 2018). The NLRP3 inflammasome cascade is thus an essential signal pathway associated with CXCR4 receptor in the development of neuropathic pain.

Loganin is an iridoid glycoside extracted from Cornus officinalis Sieb. et Zucc. (Gao et al., 2021). It possesses anti-oxidative activities and can decrease blood glucose, apoptosis, and hyperglycemia-induced inflammation in RSC96 Schwann cells (Cheng et al., 2020) and diabetic db/db mice (Park et al., 2011). Loganin improves LPS-activated intestinal inflammation by suppressing the TLR4/NFκB and JAK/STAT3 signaling cascades (Wang et al., 2020). Loganin has protective effects on neuronal cells and in neurodegenerative diseases. In SH-SY5Y neuronal cells, loganin improves hydrogen peroxide-induced cell toxicity due to inhibition of mitogen-activated protein kinases phosphorylation (Kwon et al., 2011). It also attenuated Aβ_1-42-induced inflammation by regulating the TLR4/TRAF6/NFκB axis in BV-2 microglial cells (Cui et al., 2018). Loganin was demonstrated to restore survival protein on motor neurons through the Akt/mTOR pathway in spinal muscular atrophy (SMA) mice (Tseng et al., 2016). Besides, loganin and its analogs had anti-nociceptive effects attributed to its interaction with the spinal glucagon-like peptide-1 receptors (Gong et al., 2014). We recently found that loganin improves chronic constriction injury (CCI)-induced neuropathic pain by reducing TNFα/IL-1β−mediated NFκB activation (Chu et al., 2020).

To determine if loganin can be used to prevent peripheral nerve injury-induced neuropathic pain, we first investigated the neuroprotective role of loganin using the CCI model to study the molecular signaling cascades linked to CXCL12/CXCR4-mediated NLRP3 inflammasome activation.