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INTRODUCTION
Clinically, pain is primarily classified into inflammatory pain (IP), which arises following tissue injury, and neuropathic pain (NP), which is caused by damage or disease of the nervous system, including cancer, metabolic disorders, and neurological diseases. Although both conditions are characterized by heightened activity in the peripheral and central nervous systems, involving multiple inflammatory signaling pathways that mediate the interactions between glial cells and neurons in the onset and maintenance of pain [1], but NP is more persistent and more difficult to treat than IP. The genetic and molecular expression patterns between these pain models display marked disparities in mice [2]. Recent studies indicate that NP affects between 6.9% and 10% of the adult population [3], with hallmark symptoms such as hyperalgesia, spontaneous pain, and mechanical allodynia [4]. Prolonged suffering from NP frequently results in severe psychiatric comorbidities, including chronic depression, anxiety, and sleep disturbances, significantly diminishing patients’ quality of life [5]. At present, there remain significant challenges in the management of NP and the development of new drugs [6].
Previous studies have confirmed that microglia-mediated neuroinflammation is a crucial pathological process associated with NP [7-10]. Blocking microglia-mediated neuroinflammatory responses has emerged as a viable intervention strategy for treating NP. This review focuses on the neuroinflammatory pathways involved in microglial activation and NP regulation. It discusses recent molecular mechanisms of intervention targeting microglia, as well as the efficacy of related compounds, drugs, agonists, and inhibitors in various NP models. The aim is to provide theoretical and experimental insights for the development of novel therapies targeting microglia.
MAIN BODY
1. Microglia involvement in neuropathic pain pathogenesis
Microglia are the resident innate immune cells in the brain, constituting approximately 20% of the glial cells in the adult brain [11]. Microglial cells in mammals are long-lived cells originating from the yolk sac, residing within the parenchyma of the central nervous system (CNS). These cells persist into adulthood and undergo self-renewal. They are considered highly dynamic and plastic, exhibiting distinct morphological, ultrastructural, transcriptomic, metabolic, and functional states in both health and disease conditions. The colony-stimulating factor 1 receptor (CSF1R) is a critical signaling pathway essential for their development and maintenance [12]. Currently, the activation process of glial cells in vivo can be dynamically and quantitatively monitored through targeting opioid receptors, transporters, and [18F] FDG PET imaging [13]. In the context of NP, microglia are the first to respond within days, followed by the activation of astrocytes within days to weeks [14]. Microglial in activation is accompanied by changes morphology (e.g., cell body enlargement and a reduction in the length and number of processes), proliferation, gene expression, and function, a phenomenon referred to as microglial proliferation [14]. Notably, these reactive and morphological changes do not directly correlate with pain, rather the pain mediators they release are the primary factors responsible for exacerbating pain [15]. Activated microglia exhibit two polarization states: M1 (pro-inflammatory) and M2 (anti-inflammatory). M1 microglia secrete numerous pro-inflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, cyclooxygenase-2 (COX-2), brain-derived neurotrophic factor (BDNF), and inducible nitric oxide synthase (iNOS), while M2 microglia release anti-inflammatory mediators such as IL-4, IL-10, and Arg-1 [16]. Studies have shown that in the chronic constriction injury (CCI) model, the activation of M1 and M2 microglial cell-associated genes occurs 1 day post-neural injury, with M1 markers remaining elevated even on day 14 post-injury [17]. Similar activation of microglia, accompanied by molecular expression changes, has also been observed around the injury site or in spinal cord and brain regions following central nervous system injuries, such as spinal cord injury (SCI) [18]. Currently, inhibiting microglial activation and promoting the shift from the M1 to the M2 phenotype is considered a novel strategy for treating NP.
Many experts have found that neuron-microglia interaction plays an essential role in NP occurrence and development [19,20]. Microglia express a variety of neurotransmitter receptors, including glutamate receptors, GABA receptors, cholinergic receptors, adrenergic receptors, dopamine receptors, and purinergic receptors. Other receptors include histamine receptors, opioid receptors, cannabinoid receptors, substance P receptors, neurotrophin receptors, chemokine receptors, interleukin receptors, interferon receptors, tumor necrosis factor receptors, and pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) receptors [21]. The receptors expressed on activated microglia interact with neurons and mediate cellular signaling through various intracellular pathways (e.g., NF-κB, PI3K/Akt/mTOR, p38MAPK, JAK2/STAT3, Nrf2/HO-1), disrupting the homeostasis between protective and harmful factors, thereby promoting the persistence of pain.
2. NF-κB pathway
The NF-κB signaling pathway plays a critical role in immune homeostasis and chronic inflammation, particularly in autoimmune diseases, tumorigenesis, chronic inflammatory diseases, and aging [22]. As a key nuclear transcription factor, nuclear factor-kappa B (NF-κB) is considered the primary signal that triggers the synthesis of inflammatory cytokines in microglia. Its activation is crucial for the onset and progression of NP [23].
1) Molecular mechanism of NF-κB activation
The NF-κB family consists of five members: p65 (RelA), RelB, c-Rel, p105/p50, and p100/p52. All members share a common amino-terminal REL homology domain (RHD) [24,25]. Under unstimulated conditions, NF-κB is localized in the cytoplasm and bound to the inhibitory protein IκB [22]. Upon stimulation, specific kinases phosphorylate IκB, leading to its ubiquitination and subsequent rapid degradation by the proteasome. This degradation exposes the nuclear localization signal on NF-κB subunits, allowing their translocation to the nucleus, where they bind to specific sequences in the promoter regions of target genes [24,26]. Ubiquitination is involved in at least three steps of the NF-κB pathway: IκB degradation, NF-κB precursor processing, and IκB kinase (IKK) activation [27].
NF-κB activation occurs mainly through two pathways: the canonical and non-canonical pathways. Here, the focus is on the canonical activation pathway, which is primarily associated with neuroinflammation. Upon activation of canonical NF-κB, the RelA/p50 heterodimer is responsible for the transcription of target genes involved in inflammation, immune response, cell proliferation, and differentiation [22]. The central event in canonical NF-κB activation is the phosphorylation of IκB molecules by IKK (which consists of two homologous catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ) [28]. Studies have demonstrated that in an IKKβ conditional gene knockout mouse model of nerve injury-induced neuropathic pain, microglial activation is reduced [29]. Intrathecal injection of PLGA nanoparticles encapsulating IKKβ siRNA has been shown to decrease microglial activation and the expression of TNF-α, IL-1β, and COX-2, thereby alleviating mechanical hypersensitivity in spinal nerve ligation (SNL) rats [30]. These results indicate that the activation of NF-κB triggers microglial activation. Upon activation, canonical NF-κB induces the production of pro-inflammatory cytokines, such as TNF-α and IL-1β, within the innate immune system, leading to an inflammatory response. On the other hand, these pro-inflammatory cytokines also activate canonical NF-κB pathway [22].
The canonical pathway can be activated through various cell surface receptors, including Toll-like receptors (TLRs) and cytokine receptors (TNF-R and IL-1R) [16,22]. Toll-like receptor 4 (TLR4), a member of the TLR family, has been extensively studied for its role in nerve injury-induced NP [31,32]. The activation of NF-κB in microglia mediated by TLR4 is a critical mechanism in neuroinflammation and central sensitization. Functional loss of the TLR4 gene reduces microglial activation, thereby lowering the concentration of inflammatory mediators [33]. IL-1R and TLR4 share similar signaling pathways. In brief, upon activation of the receptor for IL-1(IL-1 RI), it binds to IL-1 and recruits myeloid differentiation factor 88 (MyD88), which contains the TIR domain, to the receptor [34]. This, in turn, recruits IL-1 receptor-associated kinase (IRAK) and TNF receptor-associated factor 6 (TRAF6), forming the IL-1 RI complex [35]. TRAF6 mediates K63-linked auto-ubiquitination, leading to the activation of the transforming growth factor-β-activated kinase 1 (TAK1) complex [36]. Activated TAK1 phosphorylates and activates IKK, triggering the subsequent canonical NF-κB pathway and the induction of pro-inflammatory cytokine expression [37]. Research has shown that TRAF6 can activate the c-JUN/NF-κB signaling pathway to promote M1 activation of microglial cells, thereby exacerbating NP symptoms in CCI mice [38]. Another inflammatory factor, TNF-α, binds to its receptor TNFR1, which contains a death domain (DD) in the cytoplasm. Through homotypic interactions between the DD domains, TNFR1 recruits TRADD and drives the assembly of E3 ubiquitin ligases cIAP1/2, TNF receptor-associated factor 2 (TRAF2), and receptor-interacting protein kinase 1 (RIP1) [39]. RIP1 is then K63-ubiquitinated and recruited to NEMO, leading to the formation of the TAK1-IKK complex, activation of IKK, and the subsequent canonical NF-κB signaling pathway [40,41]. Thus, TLR4, IL-1, and TNF-α act as upstream regulators of the NF-κB pathway, making them potential targets for inhibiting NF-κB activation and offering promising therapeutic prospects for the treatment of NP. Currently, anti-TNF-α blockers (such as etanercept, infliximab, etc.) [42,43], IL-1β antagonists (IL-1ra) [44], TLR4 inhibitors [45,46], and NF-κB inhibitors have been investigated for the treatment of NP and related neuroinflammatory diseases. It is worth noting that these cytokines are involved in multiple physiological functions, and the existing blockers lack specificity, resulting in numerous side effects. A meta-analysis revealed that TNF-α inhibitors have limited clinical value in the treatment of disc herniation and/or sciatic nerve pain [47]. Long-term use of NF-κB inhibitors can lead to immune deficiencies [22], and therefore, the application of NF-κB pathway inhibitors in NP treatment requires further exploration.
2) Inhibiting the NF-κB pathway relieves NP by reducing microglial activation and inflammatory mediator release
(1) Inhibit the expression of Toll-like receptors
In vivo and in vitro experiments have demonstrated that mesenchymal stem cell-derived exosomes (BMSCs) can partially inhibit the activation of the TLR2/MyD88/NF-κB signaling pathway in spinal microglia, either by interfering with the expression of Rsad2 or by secreting proteins like TSG-6. This reduces microglial activation and decreases the release of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, and iNOS, thereby alleviating NP symptoms [8,48]. In addition, the TLR2/NF-κB signaling pathway can activate spinal microglial cells, promoting the secretion of pro-inflammatory cytokines and exacerbating hypersensitivity in HIV-associated neuropathic pain (HANP) mice. Blockade of TLR2 can inhibit the mechanical hypersensitivity induced by HIV-1 gp120 [49].
Studies have shown that kaempferol and its flavonoid derivatives exert multi-functional neuroprotective effects in CNS diseases through their antioxidant and anti-inflammatory properties. However, the cellular and molecular mechanisms of kaempferol’s actions in the CNS remain poorly understood. Recent studies have indicated that kaempferol treatment in CCI rats can promote the polarization of microglia from the M1 to the M2 phenotype via the TLR4/NF-κB signaling pathway, thereby reducing the release of inflammatory factors and improving hypersensitivity behaviors [50]. Wogonoside, another flavonoid compound, has been found to inhibit the TLR4/MyD88/NF-κB signaling pathway in microglia, reducing microglial activation and promoting the M1-to-M2 phenotype transition, thus alleviating weight loss and neuronal damage in the lesioned areas of SCI mice [51]. Undoubtedly, flavonoid compounds like kaempferol present a novel research direction and potential for NP treatment. Qufeng Zhitong capsules (QFZTC) are composed of seven Chinese herbal medicines. Liao et al. [52] demonstrated through network pharmacology combined with biological research that they can dose-dependently inhibit the increase in the expression of the c-Fos pain-related factor by suppressing the TLR4/MyD88/NF-κB signaling pathway in microglial cells. This inhibition reduces microglial activation, decreases the expression of inflammatory factors, and alleviates abnormal pain in CCI mice [52]. Oleanolic acid is a natural triterpenoid compound, which has been shown to dose-dependently shift SNL-induced microglial activation from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. This effect is mediated through the inhibition of the microglial TLR4-NF-κB signaling pathway, leading to reduced subsequent inflammatory responses [53]. A compound, stigmasterol, may alleviate NP symptoms by targeting the TLR4/NF-κB pathway in spinal microglia and modulating the polarization of M1/M2 microglia, thereby reducing neuroinflammation and central sensitization [54].
(2) Inhibit the upstream gene expression of NF-κB
It is well known that the gene expression regulated by NF-κB increases after nerve injury, which may contribute to the intensification of pain [55]. Multiple studies have demonstrated that NF-κB activity is elevated in the dorsal root ganglion and spinal cord in various neuropathic pain animal models [8,9,31,53]. Growing evidence suggests that NF-κB inhibitors can directly or indirectly alleviate the symptoms of NP and promote the polarization of activated microglia towards the M2 phenotype [16,50,53,55]. Studies have shown that intrathecal injection of pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, can alleviate pain behaviors induced by CCI in rats by inhibiting spinal microglial activation and TNF-α-induced upregulation of CX3CR1 [56].
FKBP5 is a member of the immunophilin family, specifically the FK506-binding protein (FKBP), and primarily promotes inflammation through the activation of the NF-κB pathway. Studies have shown that cannabidiol (CBD) can act on FKBP5 to inhibit the assembly of the IKK complex, thereby blocking the NF-κB pathway. This results in reduced microglial activation and the overexpression of FKBP5 in the dorsal horn of the lumbar spinal cord, along with decreased expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in CCI-induced rats [9]. Proto-oncogene tyrosine-protein kinase (Src) is deemed as a non-receptor tyrosine kinase that is widely expressed in various cells. Studies have found that Scr inhibition prevents functional impairment in PNL-induced NP mice by inhibiting the NF-κB pathway and reducing microglia neuroinflammation [57]. Stimulator of interferon genes (STING) is a key sensor of double-stranded DNA (dsDNA), predominantly expressed in spinal microglia. One study demonstrated that activation of the STING/TBK1/NF-κB pathway in spinal microglia could promote pain in spared nerve injury (SNI) mice via IL-6 signaling. Notably, early, but not late, intrathecal injection of C-176 (a selective STING antagonist) alleviated microglial activation and cytokine release, mitigating the pain hypersensitivity induced by SNI [58]. Thus, pharmacological blockade of STING represents a promising therapeutic target for the early treatment of neuropathic pain. Morin is a natural bioflavonoid extracted from the Musaceae plant. Studies have shown that Morin alleviates NP symptoms induced by vincristine by inhibiting the NF-κB pathway, suppressing M1 polarization of cortical microglial cells, and promoting M2 polarization, thereby reducing neuroinflammation [59]. It is worth noting that type-1 angiotensin II receptors (AT1R) can induce intense oxidative stress, which has attracted the attention of researchers. A recent study has shown that SNI leads to an increase in AT1R, and treatment with losartan potassium (an AT1R inhibitor) can alleviate oxidative stress in microglial cells by modulating the HMGB1/NF-κB signaling pathway, promoting polarization towards the M2 phenotype, and reducing abnormal pain in SNI rats [60].
Electroacupuncture (EA), as a physical therapy, has been promoted in clinical practice, yet its specific mechanisms remain unclear. A study utilizing 2 Hz EA stimulation on SNI rats for 21 days found a significant reduction in the co-expression of microglial activation markers CD11b, TLR4, and MyD88 in the spinal cord. This was associated with downregulation of spinal high mobility group box-1 (HMGB1) protein expression and inhibition of NF-κB p65 activation [61]. In another NP model, the expression of HMGB1 in the spinal cord of chemotherapy-induced peripheral neuropathy (CIPN) mice is increased. This upregulation can activate downstream TLR4 and receptor for advanced glycation end products (RAGE), further activating the microglial p38MAPK/NF-κB signaling pathway, thereby exacerbating neuroinflammation. Relevant blockers can alleviate the pain symptoms [62]. Research reports suggest that photobiomodulation (PBM) can alleviate NP symptoms induced by SCI, although the specific mechanisms are still being explored. One study indicates that PBM inhibits the activation of microglial cells in the injured tissue, leading to a decrease in NF-κB phosphorylation levels, which further suppresses the expression of the chemokine CXCL10, and reduces the occurrence of NP [63]. Physical therapy is minimally invasive and has a high safety profile, making it a treatment method worth promoting.
Chronic neuropathic pain can lead to anxiety, depression, and cognitive dysfunction. A study has shown that hippocampal cannabinoid type 2 receptor (CB2R) can alleviate abnormal pain and cognitive dysfunction in SNI rats by inducing the expression of dual-specificity phosphatase 6 (DUSP6) in microglial cells, thereby inhibiting the activation of the ERK/NF-κB pathway [64]. There is cross-talk between those pathways.
(3) Activate the negative feedback regulators of NF-κB
In the SNI model and in vitro LPS-induced BV2 cells, fibroblast growth factor 10 (FGF10) dose-dependently interferes with microglial cell proliferation via the PPAR-γ/NF-κB signaling pathway, inhibiting NF-κB phosphorylation and exerting analgesic effects, without affecting astrocyte activity [65]. Pioglitazone (a PPAR-γ agonist) exhibits similar anti-hyperalgesic and anti-microglial activation effects as FGF10, suggesting that PPAR-γ acts as a negative regulator of NF-κB to modulate microglial cell activity. Growth and differentiation factor 11 (GDF11), a member of the transforming growth factor-β (TGF-β) family, recent studies have shown that GDF11 promotes the transition of spinal microglial cells from the M1 to the M2 phenotype in SNI mice by regulating the SMAD2/NF-κB pathway, reducing the release of pro-inflammatory factors and alleviating abnormal pain. The TGF-βR1 inhibitor SB431542 inhibits the analgesic effect of GDF11 [66].
The above studies indicate that in microglia, molecules such as TLR2/TLR4, MyD88, HMGB1, FKBP5, STING, AT1R, chemokines and cytokines, act as positive feedback regulators, activating the NF-κB pathway to modulate microglial activity. In contrast, PPAR-γ, TGF-β, SMAD 2 function as negative regulators, inhibiting NF-κB phosphorylation. The overall effect is the inhibition of microglial activation or promotion of M2 polarization, thereby maintaining a balance between neuroprotective (M2) and neurotoxic (M1) microglial phenotypes. This ultimately alleviates neuroinflammation and reduces pain (Table 1).
3. PI3K/Akt/mTOR pathway
The PI3K/Akt/mTOR pathway plays a crucial role in several cellular processes essential for maintaining homeostasis. It is an important intracellular signaling pathway involved in cell growth, survival, proliferation, apoptosis, angiogenesis, and autophagy [67-69].
1) Molecular mechanism of PI3K/Akt/mTOR activation
Numerous growth factors and cytokines can activate the PI3K/Akt/mTOR signaling pathway, which is initiated by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). These receptors subsequently activate PI3K, leading to the generation of phosphoinositides. Protein kinase B (Akt) and the mammalian target of rapamycin complex 1 (mTORC1) are activated downstream [70]. PI3K consists of three classes (I, II, and III), with class I PI3Ks being the most extensively studied, as they are primarily involved in the regulation of various biological activities [71]. Class I PI3K is a heterodimer composed of a regulatory subunit (p85) and a catalytic subunit (p110). The binding between the regulatory and catalytic subunits stabilizes PI3K, providing the binding site for RTKs and GPCRs to activate PI3K [72]. Activated PI3K catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn activates downstream effectors of Akt [71]. During this process, phosphatase and tensin homolog (PTEN), a negative regulator of PI3K, limits the signaling strength by dephosphorylating PIP3 back to PIP2 [73]. Akt is a crucial messenger in the PI3K signaling pathway. In the canonical PI3K/Akt cascade, phosphoinositide-dependent kinase 1 (PDK1) and Akt are recruited to the inner surface of the cell membrane through their pleckstrin homology (PH) domains, where PDK1 activates Akt1 by phosphorylation at Thr308 [74]. Another important activation pathway of Akt is mediated by mTORC2, which interacts with the hydrophobic regulatory domain of Akt, leading to phosphorylation at Ser473 [75]. mTOR, a downstream kinase of PI3K/Akt, is activated by P-Akt and is expressed in the pain-related central nervous system. It has been identified as an effective target for opioid-induced tolerance and hyperalgesia [76]. mTOR then activates its effector molecules, including ribosomal protein S6 kinase β-1 (P70S6K), eukaryotic translation initiation factor 4E-binding protein-1 (4EBP1), and unc-51-like kinase 1 (ULK1) [77]. P70S6K and 4EBP-1 act as regulators of cell cycle progression and angiogenesis by enhancing the translation of mRNA encoding HIF-1α, cyclin D1, and c-Myc [69]. Following phosphorylation by mTORC1, ULK1 induces autophagy through phosphorylation of Beclin-1 [68]. Rapamycin, an mTORC1 inhibitor, enhances ULK1 activity [77] and is widely used as an autophagy inducer. ULK1 functions within a complex with ATG13 and FIP200, which serves as a key node in converting autophagic signaling into autophagosome biogenesis [67].
2) PI3K/Akt/mTOR pathway bidirectionally regulates NP by modulating microglial activation, neuronal apoptosis, and autophagy
(1) Inhibit the activation of microglia
In recent years, numerous studies have confirmed that the PI3K/Akt cascade modulates microglial activation, inhibits M1 polarization of microglia, or promotes the transition from M1 to M2, thereby attenuating microglia-mediated neuroinflammation and alleviating pain [78-80]. A substantial body of evidence suggests that targeting miRNAs is an effective strategy for the treatment of NP [81]. A KEGG pathway analysis revealed that the upregulated target genes of miRNAs, such as mmu-miR-133a-3p and mmu-miR-1a-3p, are involved in the PI3K/Akt signaling pathway and transcriptional dysregulation, these upregulated genes exert a negative regulatory effect on microglial activation . More recent research has focused on developing miRNA-loaded nanoparticle delivery systems targeting microglial cells, aiming to achieve more precise inhibition of microglial activation and prolong the duration of analgesia [82,83].
Purine signaling is an important target for the treatment of pathological pain, and microglial cells express a variety of purinergic receptors [84]. P2X4 receptors (P2X4R) are predominantly expressed in microglia, and research has shown that P2X4R activation drives microglial motility via the PI3K/Akt pathway. Pre-treatment of microglia with Wortmannin and LY294002, significantly blocked their chemotaxis [85]. As early as 2009, Horvath and DeLeo demonstrated that in vitro morphine stimulation activated the PI3K/Akt pathway, inducing microglial migration through the interaction between μ-opioid receptors (MOP) and P2X4R [86]. In vivo studies have shown that the chemokine monocyte chemoattractant protein-1 (MCP-1) can activate the PI3K/Akt pathway in microglia, regulate P2X4R expression in microglia, and exacerbate bone cancer pain. Intrathecal injection of anti-MCP-1 neutralizing antibody (RS-504393) significantly reduced microglial activation and mechanical allodynia in a bone cancer pain animal model [87,88].
(2) Activation of the PI3K/Akt pathway alleviates NP
BMSCs have been used in the treatment of NP due to their ability to regulate neuroinflammation [8]. One study showed that BMSCs could alleviate deafferentation-induced pain symptoms by inhibiting the M1 phenotype and promoting the M2 phenotype of microglia through the secretion of glial cell-derived neurotrophic factor (GDNF). This effect may be mediated by the suppression of the NF-κB pathway, while simultaneously promoting the activation of the PI3K/Akt pathway [89]. The potential synergistic or antagonistic interactions between these two pathways require further investigation. A study has confirmed that peripheral administration of botulinum neurotoxins (BoNT) suppresses microglia activation and the phosphorylation of the N-methyl-d-aspartate (NMDA) receptor in dorsal horn neurons [90], minocycline combined with BoNT therapy promoted the expression of Sirtuin 1 (SIRT1), which in turn inactivated the NF-κB, p53, and PI3K/Akt signaling pathways, inhibiting microglial activation and oxidative stress. This resulted in a reduction in the release of inflammatory factors (such as TNF-α, IL-1β, IL-6, and IL-8) and alleviated the symptoms of NP induced by SCI in rats [91].
In early studies, Tarassishin et al. [92] confirmed that the PI3K/Akt pathway is involved in promoting the beneficial M2 microglial polarization state. Activation of the PI3K/AKT pathway enhanced the M2 state following interferon regulatory factor 3 (IRF3) stimulation, and this effect was at least partially related to the induction of anti-inflammatory factors such as IL-1ra and IL-10 [92]. The study also suggested that this anti-inflammatory effect of PI3K/Akt appears to be specific to microglia, as it exerts the opposite effect on astrocytes [92]. The PI3K/Akt pathway plays fundamentally different roles in the inflammatory activation of astrocytes and microglia, suggesting that these two cell types may express distinct PI3K and Akt molecules with different phenotypes and functions, or that they may activate different points in the pathway (upstream or downstream). The hypothesis warrants further exploration.
Maresin 1 (MaR1), an anti-inflammatory and pro-resolving mediator, has been shown to regulate nerve growth through the activation of the PI3K/Akt/mTOR signaling pathway. It inhibits the activation of spinal astrocytes and microglia, as well as the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, thereby alleviating NP induced by neuropathic damage in CCI mice [93]. In a recent study, Han et al. [79] found that the PI3K/Akt signaling pathway in the hippocampus of mice was inhibited following SNI, accompanied by microglial M1 polarization and synaptic plasticity impairment. T0901317 (liver X receptor agonist) significantly reversed the reduction of synaptic proteins (PSD95, SYN1, BDNF reduced) and the inhibition of the PI3K/Akt pathway in the hippocampus, alleviating synaptic damage and neuroinflammation. Moreover, LY294002 exacerbated cognitive dysfunction in SNI mice and abolished the neuroprotective effect of T0901317. Another study similarly demonstrated that LY294002 significantly reversed the inhibition of LPS-induced neuroinflammation by TREM2 activation and eliminated the protective effect of TREM2 activation against LPS-induced neuronal damage [94]. Furthermore, intrathecal injection of LY294002 antagonized the analgesic effects of minocycline (a microglial inhibitor) and AMD3100 (a CXCR7 agonist) after skin/muscle incision and retraction surgery (SMIR) [95]. These findings suggest that the activation of the PI3K/Akt pathway has different effects on microglial states in various pain models, highlighting the diverse roles of this key signaling molecule in coordinating protective and potentially harmful responses.
(3) Promote autophagy and inhibit apoptosis
Autophagy has been considered a potential therapeutic target for NP [96], with ULK1 being a downstream effector molecule of the PI3K/Akt/mTOR pathway involved in the autophagic process [68]. One study demonstrated that MSC-derived extracellular vesicles (MSC-EVs) could inhibit the activation of the PI3K/Akt/mTOR signaling pathway induced by CCI/LPS, promote microglial autophagy (Beclin-1, LC3-II increased and p62 decreased), and alleviate neuroinflammation [97]. Another study showed that 4–5 weeks of treadmill exercise reduced STZ-induced NP symptoms and neuroinflammation, with the underlying mechanism likely involving the downregulation of IL-6, thereby inhibiting the mTOR signaling pathway [98]. A subsequent study found that exercise enhanced autophagy by inhibiting the BDNF/Akt/mTOR pathway and promoted the polarization of microglia from M1 to M2, thereby improving mechanical hypersensitivity in SNI mice [99]. Minocycline, a tetracycline antibiotic commonly used in clinical practice, can cross the blood-brain barrier and exert anti-inflammatory effects. It is frequently used as a microglial inhibitor in research. One study demonstrated that minocycline promotes autophagy in the injured spinal cord of SCI rats by inhibiting the activation of the PI3K/Akt/mTOR pathway. This promotes spinal cord repair and alleviates NP symptoms [100]. Intravenous injection of LY294002 further enhanced autophagy, while Insulin-like Growth Factor 1 (IGF-1) reversed the therapeutic effect of minocycline. Recent studies, through the analysis of transcriptomic data related to NP, have identified tripartite motif-containing 28 (TRIM28) as a key regulator of ferroptosis. Glycogen Synthase Kinase 3 Beta (GSK3β), a downstream target of TRIM28, can be inhibited by TRIM28, reducing ULK1 activity. This, in turn, weakens microglial autophagy, exacerbates neuroinflammation, and worsens NP symptoms [101]. Although the database used in this study pertains to mice on day 7 post-SNI, the TRIM28/GSK3β/ULK1 cascade, as an important pathway regulating microglial autophagy and ferroptosis, provides new options for alleviating NP.
In a recent study, Zhu et al. [102] confirmed that treatment with matrine alleviated damage to the dorsal root ganglion (DRG) and spinal cord neurons induced by vincristine, and inhibited neuronal apoptosis (Bcl2/Bax increased), partly through the blockade of the PI3K/Akt/mTOR pathway.
The findings of the studies outlined above suggest that various growth factors and cytokines, such as MCP-1, IL-6, TNF-α, P2X4R, BDNF, GDNF, and IL-10, can activate the PI3K/Akt/mTOR signaling pathway, thereby modulating the activation of microglia. Evidence from pharmacological and chemical blockade studies indicates that this pathway exerts a bidirectional regulatory effect on NP (Table 2). Notably, the PI3K/Akt/mTOR pathway shows differential effects across various glial cell types, and its impact on the nervous system varies between distinct NP models, exhibiting both protective and detrimental effects. Moreover, there is an interplay between the PI3K/Akt/mTOR and NF-κB pathways. The complexity and diversity of molecular actions underscore the need for further exploration of the detailed mechanisms of these key signaling molecules, which may help improve the specificity of therapeutic targeting.
4. P38 MAPK pathway
Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved serine/threonine protein kinases that play a crucial role in regulating gene expression and promoting disease progression. The MAPK family is primarily composed of three members: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK [103]. Numerous studies have highlighted that JNK is primarily expressed in astrocytes and neurons [104], while ERK plays different roles in astrocytes and microglia, respectively [105], whereas p38 MAPK is mainly expressed in microglia [106-108]. ERK5, a relatively new member of the MAPK family, is activated in spinal microglia following neural injury [109], although it has been rarely discussed in the literature. Earlier studies showed that intrathecal injection of BIX02188 (an ERK5 inhibitor) inhibited the ERK5 signaling pathway, reduced the release of inflammatory cytokines, NF-κB expression, and the extent of apoptosis in the rat spinal cord, thereby alleviating the severity of SCI [110]. Studies also demonstrate that p38 MAPK phosphorylation plays a crucial role in the release of pro-inflammatory mediators, including IL-1β, IL-6, and TNF-α, from microglia, contributing to the development of hyperalgesia and tolerance. The application of p38 inhibitors, such as FR167653, CNI-1493, and SB203580, has been shown to reverse early-stage NP [107,111].
1) Molecular mechanism of p38 MAPK activation
The p38 MAPK family consists of four isoforms: p38α, p38β, p38γ, and p38δ. Of these, p38α and p38β are particularly involved in autoimmune, inflammatory, and pain responses [112]. Like other MAPKs, the p38 MAPK pathway transduces extracellular signals via a series of phosphorylation events, where one kinase phosphorylates another (MAP3K → MAP2K → MAPK). Several pro-inflammatory cytokines, such as IL-1 and TNF-α, also contribute to the phosphorylation of p38 MAPK. Upon activation, p38 MAPK translocates to the nucleus, where it regulates gene expression; some substrates are also phosphorylated in the cytoplasm. MAP3K specifically phosphorylates and activates MAP2K (MKK3, MKK4, and MKK6), which, in turn, influences the activation of the four p38 MAPK isoforms. MKK4 selectively phosphorylates p38α and p38δ, while MKK6 activates all isoforms of p38 MAPK, and MKK3 primarily activates p38α, p38γ, and p38δ [113]. The upstream activation mechanisms of p38 MAPK involve a variety of receptors, including purinergic receptors (P2Y1Rs, P2X7, P2Y12, etc.), NMDA receptors, chemokines (such as CXCL13 and fractalkine) and their respective receptors (CXCR5 and CX3CR1), vanilloid receptors (TRPV1, TRPA1), Toll-like receptors (TLRs), and microRNAs (e.g., miR-125a-3p), as discussed in previous studies [1].
2) Inhibition of p38 MAPK signaling alleviates NP by reducing microglial activation and inflammatory cytokines release
Several studies have demonstrated the activation of p38 MAPK in spinal cord microglia across various models of neuropathic pain, including peripheral nerve injury, spinal cord injury, and chemotherapy-induced neuropathy. In the absence of pharmacological inhibitors, the first in vivo study examining the physiological role of p38 MAPK in sciatic nerve regeneration suggested that p38 MAPK may play a significant role in initiating inflammation and facilitating recovery from neural injury [114]. Additionally, prior treatment with 2% lidocaine solution before SNL-induced nerve injury reduced the upregulation of Nav1.3 and inhibited p38 MAPK activation in spinal microglia, alleviating mechanical allodynia and thermal hyperalgesia by day 3 post-surgery. Notably, p38 MAPK activation suppression persisted until day 7 post-surgery [115]. Intravenous lidocaine infusion, combined with other medications, is being investigated in clinical settings for the treatment of chronic NP. A recent retrospective study indicated that intravenous infusion of 1000 mg lidocaine (40 mg/h) over 25 hours provided minimal pain relief for cancer pain, post-herpetic neuralgia, chemotherapy-induced neuropathy, and radicular pain patients, whereas it offered more benefit for patients with myofascial pain syndrome, peripheral neuropathy, small fiber neuropathy, and vascular diseases [116]. However, further retrospective and prospective studies are needed to determine the optimal dosage, duration, and intervals for lidocaine infusion therapy, as well as to develop targeted treatments for different pain mechanisms in diverse patient populations.
Studies have shown that EA alleviates neuropathic pain symptoms by promoting the expression of programmed cell death ligand 1 (PD-L1) in microglial cells, inhibiting the p38MAPK/ERK pathway, and promoting M2 microglial polarization [117,118]. In more recent research, Kui et al. [119] further found that EA ameliorates neuroinflammation and pyroptosis to relieve chronic NP by suppressing NLRP3 inflammasome activation in microglia. Clearly, the mechanisms of EA in alleviating NP are still being explored, but its analgesic effects are undeniable.
(1) Crosstalk between neuroinflammatory pathways in NP
Early experiments have demonstrated that p38 MAPK modulates the transcriptional activity of NF-κB by regulating the nuclear translocation of NF-κB p65, thereby activating downstream signaling pathways involved in the onset and maintenance of pain [27]. In a series of in vivo and in vitro studies, Caffeic acid phenethyl ester (CAPE) was found to alleviate NP symptoms in CCI mice by inhibiting p38 MAPK and NF-κB pathways in spinal microglial cells. This reduction in microglial activation was associated with decreased release of inflammatory cytokines [23]. Duloxetine (DLX) is a selective serotonin and norepinephrine reuptake inhibitor (SNRI) that is clinically used to treat pain, although it has certain side effects. Kim et al. [120] administered DLX nanoparticles (DLX NPs) intrathecally using nanotechnology and found that DLX NPs alleviated mechanical allodynia in SNL rats by inhibiting the p38 MAPK/NF-κB pathway in microglial cells. This inhibition suppressed microglial activation and the release of inflammatory factors, significantly enhancing the analgesic effect of DLX while limiting its side effects [120]. L-thiapental (L-THP), a compound clinically used for chronic pain treatment, has an unclear molecular mechanism and target. Recent studies by Wu et al. [121] revealed that L-THP significantly reduced the expression of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis in spinal microglia of CCI rats, with no notable effect on astrocytes or neurons. The primary target was identified as Clec7a, a pattern recognition receptor expressed on microglia, which inhibited the secretion of pro-inflammatory cytokines (IL-1β and IL-18), thereby mitigating neuroinflammation and improving NP. Another commonly used clinical drug, dexmedetomidine (Dex), partially alleviates mechanical allodynia and thermal hyperalgesia in the CCI rat model by inhibiting the expression of TRPC6 in the DRG, suppressing p38 MAPK phosphorylation, and reducing microglial proliferation. This ultimately leads to the downregulation of inflammatory cytokines such as TNF-α and IL-1β [122].
Dual-specificity phosphatase 1 (DUSP1) has been shown to exert neuroprotective effects by inhibiting p38 MAPK signaling. One study demonstrated that injection of LV-DUSP1 into the medial prefrontal cortex (mPFC) of CCI rats counteracted the phosphorylation of ERK1/2 and p38 MAPK in the mPFC, suppressed mPFC cell apoptosis, and promoted the polarization of microglia from the M1 to M2 phenotype. In vitro experiments revealed that the use of SB 203580 and PD 98059 (an ERK 1/2 pathway inhibitor) attenuated the enhancing effect of DUSP1 silencing on microglial M1 polarization [111]. A recent investigation elucidates that suppression of miR-382-5p elicits upregulation of DUSP1 expression, consequently attenuating pain hypersensitivity behaviors in rodent models of CCI [123]. Corydecumine G (Cor G), the specific phthalide isoquinoline from the traditional Chinese medicine Corydalis Decumbentis Rhizoma, has been shown to inhibit spinal microglial cell activation and neuroinflammation in CCI rats by suppressing the p38MAPK/ERK pathway [124]. Sodium aescinate (SA), a triterpenoid compound from traditional Chinese medicine, has been shown to suppress microglial activation and reduce neuroinflammation by inhibiting the JNK/p38 MAPK pathway, thereby alleviating mechanical allodynia in CCI mice [125].
As a new gabapentin-class drug, mirogabalin was officially launched in Japan in 2019 for the treatment of NP. It has been shown to be more effective than the current first-line drug, pregabalin, although its mechanism of action has not been fully elucidated. One animal study demonstrated that mirogabalin, through continuous administration, inhibits the p38MAPK pathway (non-ERK/JNK pathway) in microglial cells, reducing microglial activation and the release of CCL2 and CCL5, thereby decreasing pain-like behaviors [126]. In another study, Yeo et al. [127] found that high-dose rapamycin treatment alleviates ION-pNL (infraorbital nerve injury-induced abnormal pain) by regulating MEK4, rather than MEK3/6, to inhibit p38 MAPK signaling. However, further research is needed to determine whether this regulation is direct or indirect.
Dexamethasone, as a glucocorticoid receptor agonist, is a widely used anti-inflammatory drug in clinical practice. Recent studies have shown that dexamethasone exerts mechanical and thermal antinociceptive effects by upregulating the expression of enkephalin A in spinal cord microglia. The underlying mechanism is likely mediated by the activation of the cAMP/PKA signaling pathway in spinal microglia, followed by the activation of the p38 MAPK/CREB pathway. Notably, SB203580 completely blocks the antinociceptive effects of dexamethasone and the increase in enkephalin A expression in microglia [128]. This contradicts previous studies, as the activation of p38 MAPK induced by dexamethasone may be due to the release of enkephalin A. Pre-treatment with the κ-opioid receptor antagonist GNTI did not attenuate the increase in p38 MAPK phosphorylation induced by dexamethasone. A previous study suggested that protopanaxadiol produces pain antihypersensitivity in neuropathic pain, likely through spinal microglial dynorphin A expression after glucocorticoid receptor activation. Intrathecal application of the microglial inhibitor minocycline attenuated its pain antihypersensitivity effects [129]. This indicates that the glucocorticoid receptor/dynorphin A pathway in microglial cell membranes may become a potential target for the treatment of chronic pain.
(2) Modulate purinergic receptors and chemokines receptors in microglia
One study showed that in SNI mice, the expression of P2Y6R, P2Y12R, P2Y13R, and P2Y14R in spinal microglia was upregulated, and the p38 MAPK inhibitor SB203580 significantly suppressed their expression. This suggests that p38 MAPK signaling may modulate the transcription of these purinergic receptors in spinal microglia following nerve injury [130]. In recent years, P2Y12 has gradually emerged as a target for treating NP. Earlier studies have confirmed that P2Y12 may be upstream of the MAPK signaling pathway in the cancer induced bone pain (CIBP) [131]. Yu et al. [132] demonstrated that P2Y12 is selectively expressed on microglia in the CNS. Both MRS2395 and clopidogrel (P2Y12 antagonists) inhibit microglial activation and the phosphorylation of p38 MAPK, alleviating abnormal pain induced by SNL. Immunofluorescence staining revealed co-localization of P2Y12, p-p38 MAPK, and microglia, with SB203580 showing similar effects. Magnolol, a traditional herbal compound, has been shown to inhibit microglial activation via P2Y12, subsequently blocking the upregulation of cytokines such as IL-6, TNF-α, and IL-1β, a process likely mediated by the p38 MAPK pathway. Phosphorylation levels of p38 MAPK increased approximately twofold following CCI, an effect that Magnolol effectively blocked [133]. P2X4R, a receptor specifically expressed on microglia, has been implicated in the regulation of NP. A recent study demonstrated that Daphnetin treatment suppressed P2X4 receptor activation, thereby inhibiting the p38 MAPK pathway in microglia of the CCI rat model. This led to a reduction in the activation of spinal microglia and a decrease in the expression of inflammatory cytokines, ultimately alleviating mechanical allodynia in the model [134]. P2X7R, which is primarily expressed on microglia, has been shown to play a key role in this pathway. Research indicates that Echinacea glycoside (ECH) inhibits p38 MAPK phosphorylation through the P2X7R/CX3CL1/CX3CR1 signaling axis in the spinal cord, suppressing microglial overactivation and the release of inflammatory mediators, thereby alleviating mechanical allodynia, cold allodynia, and thermal hyperalgesia in CCI mice [135].
Fractalkine (CX3CL1) is a unique chemokine predominantly expressed by neurons, and its sole receptor, CX3CR1, is expressed on microglia. An early study demonstrated that the membrane-bound fractalkine in the DRG significantly decreased following SNL, suggesting that after nerve injury, the cleavage and release of fractalkine occurs. This release binds to CX3CR1 on microglia, subsequently activating p38 MAPK in spinal microglia and inducing the production of inflammatory mediators [136]. Additionally, IL-6 induces the expression of CX3CR1 on spinal microglia through p38 MAPK activation. This novel mechanism in NP enhances the responsiveness of spinal microglia to CX3CL1 [137]. Intrathecal injection of anti-CX3CR1 neutralizing antibody inhibited the activation of p38 MAPK in spinal microglia following SNL, alleviating mechanical allodynia [136]. Earlier, it was described that exercise can alleviate mechanical allodynia in SNI mice by inhibiting the BDNF/Akt/mTOR pathway [99]. In a study on morphine-induced hypersensitivity in mice, it was found that daily running at 20 m/min for 30 minutes reduced the development of morphine-induced hyperalgesia and tolerance. Moreover, exercise also decreased the expression of phosphorylated p38 MAPK in the presence of morphine [138]. Recent reviews suggest that exercise may modulate spinal microglial activity through the p38 MAPK signaling pathway. This effect is potentially mediated by the regulation of purinergic receptors and CX3CR1 expression on microglia, leading to reduced microglial activation and lower levels of inflammatory cytokines in the brain, spinal cord, DRG, sciatic nerve, and blood, thereby exerting neuroprotective effects [139,140]. As a non-pharmacological treatment, exercise warrants further investigation in clinical research to explore the precise effects of different types and intensities of exercise in alleviating NP. Additionally, pancreatitis-associated proteins (PAPs) were shown to activate the C-C chemokine receptor 2 (CCR2) and p38 MAPK pathway in spinal microglial cells, enhancing microglial activation and exacerbating neuroinflammatory damage. Gene knockout or antibody treatments that blocked PAP-I alleviated tactile hypersensitivity during the maintenance phase after SNI [141].
(3) Inhibit the upstream gene expression of p38 MAPK
Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, modulates a spectrum of MAPK pathways and is highly expressed in microglia. Recent investigations have demonstrated that l-CDL and rDKK3 can inhibit the ASK1/p38 MAPK pathway in spinal microglial, reducing microglial activation, promoting the shift from the M1 to M2 phenotype, and suppressing neuroinflammation, thereby ameliorating NP symptoms [142,143]. In recent years, miRNA-based therapeutic strategies (such as targeted delivery and combination therapy) have provided a new direction for the management of chronic pain [83]. One study demonstrated that tail vein injection of SHED-derived exosomes (SHED-Exos) delivered miR-24-3p into microglia within the brain of CCI-ION mice, resulting in the inhibition of microglial activation. Further investigations revealed that miR-24-3p, through the IL-1R1/p38 MAPK pathway, alleviated trigeminal neuropathic pain in these mice. This was accompanied by a reduction in the expression of IBA-1 and inflammatory cytokines in the trigeminal spinal nucleus, ultimately relieving the abnormal mechanical pain in the mice [144]. Embryonic lethal abnormal vision (ELAV) proteins, a family of RNA-binding proteins (RBPs) involved in post-transcriptional gene regulation, have been found to play a role in NP regulation. Borgonetti et al. [108] observed increased expression of HuR in the ipsilateral spinal cord of SNI mice. By delivering HuR antisense oligonucleotides intranasally to knock down HuR expression in the spinal cord, they found a reduction in neuropathic pain. The mechanism was partly attributed to the inhibition of the p38 MAPK pathway in microglia, which facilitated the polarization of microglia from the M1 to the M2 phenotype. This novel finding offers a new approach for non-invasive, targeted therapies for NP in the CNS.
Sanguinarine is a natural plant medicine with anti-inflammatory effects. Studies have found that it can inhibit the p38MAPK pathway, reduce spinal microglial cell activation and the release of inflammatory factors, thereby alleviating mechanical allodynia in CCI rats [145]. Paeonol is an anti‐inflammatory and antioxidant compound widely distributed in Paeonia lactiflora, Studies have elucidated that paeonol attenuates thermal hyperalgesia by modulating microglial polarization towards the M2 phenotype via suppression of the RhoA/p38 MAPK signaling cascade in CCI rats [146]. Total glucosides of paeony (TGP), a traditional Chinese medicine, have been shown to alleviate NP symptoms in CIPN rats by inhibiting microglial pyroptosis through the p38MAPK pathway [147].
The aforementioned studies indicate that increased p38 MAPK phosphorylation, microglial activation, and the release of pro-inflammatory cytokines are common features across various NP models. Current research suggests that by interfering with the upstream signaling pathways of p38 MAPK in microglia, such as purinergic receptors (P2Y12, P2X4, P2X7, etc.), the chemokine fractalkine and its receptor CX3CR1, ASK1, TRPC6, and Clec7a, p38 MAPK phosphorylation can be inhibited, leading to the suppression of microglial activation, reduction of inflammatory cytokine release, and alleviation of pain symptoms (Table 3). Additionally, p38 MAPK is not only an upstream signal for the NF-κB pathway but also plays a pivotal role in promoting inflammation. Furthermore, gender differences appear to significantly influence p38 MAPK activation. A study demonstrated that intrathecal administration of minocycline and p38 MAPK inhibitors effectively alleviated abnormal pain in male mice in the CCI model, but was ineffective in female mice [148]. This disparity may be linked to the higher levels of p-p38 MAPK activation observed in female mice compared to males [149]. The differential expression of microglial cells in male and female individuals has long been a focal point in pain research, and this phenomenon may provide mechanistic insights into the observed sex differences. In conclusion, p38 MAPK plays a central role in microglial activation and inflammatory responses. Further studies are needed to elucidate the regulatory role of p38 MAPK signaling in the pathophysiology of pain.
5. JAK2/STAT3 pathway
Recent studies have highlighted that the Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) pathway plays a critical role in the onset and persistence of chronic NP [150-152]. Convincing evidence has demonstrated that the JAK2/STAT3 signaling pathway is aberrantly activated in sensory neurons of the DRG in chronic pain rodent models [153], motor neurons in the ventral spinal cord [105], and in the red nucleus (RN) [154]. However, the specific molecular and cellular mechanisms through which this pathway is activated in microglia remain unknown.
1) Molecular mechanism of JAK2/STAT3 activation
JAK2 is a non-receptor tyrosine kinase that becomes activated through tyrosine phosphorylation, initiating a cytoplasmic signaling cascade that regulates various cellular processes [155]. STAT3, a substrate of JAK2 located on chromosome 17q21, encodes an 89 kDa protein. STAT3 is predominantly expressed in microglia and is considered one of the key marker proteins for CNS injury. A study demonstrated that Sodium tanshinone IIA sulfonate (STS), a derivative of tanshinone IIA, mitigates abnormal mechanical hypersensitivity in SNI rats by upregulating miR-125b-5p expression, thereby inhibiting microglial STAT3 expression and M1 polarization [156]. The signaling cascade of the JAK2/STAT3 pathway is primarily mediated by RTKs, JAK2, and the transcription factor STAT3 [157]. Upon ligand binding, RTKs undergo dimerization on the cell membrane, leading to the phosphorylation of tyrosine residues on the receptor. This phosphorylation event enables the recognition and binding of downstream proteins, such as JAK2, which contains SH2 domains. Activated JAK2 then stimulates the formation of a binding site for STAT3 on the RTK. In the cytoplasm, STAT3 binds to the RTK through its SH2 domain and undergoes phosphorylation under the influence of JAK2. The phosphorylated STAT3 forms a homodimer, translocates into the nucleus, and subsequently activates downstream signal transduction pathways [158]. Several scholars have reviewed the role of the JAK2/STAT3 pathway as a target in chronic pain. Evidence indicates that the translation and expression of cytokines such as IL-6, IL-1β, TNF-α, IL-33, CCL2, and SOCS3 can induce neuroinflammation through the JAK2/STAT3 signaling axis. Conversely, IL-10, TGF-β, and α7nAchR have been shown to inhibit these inflammatory processes [151]. Caveolin-1 (CAV-1), an important target gene of STAT3, regulates neuronal plasticity and receptor trafficking, particularly influencing N-methyl-D-aspartate receptor subunit 2B (NR2B). CAV-1 has been implicated in pathological pain and central sensitization [159].
2) JAK2/STAT3 pathway modulates microglial proliferation and activation, inhibits neuroinflammation, and contributes to the onset and maintenance of NP
(1) Regulation of IL-6 and IL-10 expression
Studies have shown that the JAK2/STAT3 signaling cascade is activated by both pro-nociceptive (IL-6) and anti-nociceptive (IL-10) cytokines. These two forms of activation lead to the transcription of distinct gene sets, which may alter the polarization state of microglia [55,105,150]. Intraperitoneal administration of AG490, a selective JAK2 inhibitor, effectively suppresses the activation of the JAK2/STAT3 signaling pathway and downregulates IL-6 expression, thereby improving the cold pain threshold and alleviating mechanical allodynia in oxaliplatin-induced NP in rats [160]. Furthermore, intranuclear injection of AG490 has been shown to mitigate mechanical allodynia in neuropathic pain models, including CCI and SNI rats, and to decrease the mRNA levels of inflammatory cytokines IL-1β, IL-6, and TNF-α [154,161]. Additional findings suggest that the JAK2/STAT3 signaling pathway contributes to microglial activation through the regulation of the p38 MAPK and ERK pathways, and that AG490 treatment markedly suppresses this effect. Neutralizing IL-6 with specific antibodies has been shown to significantly attenuate both mechanical allodynia and thermal hyperalgesia induced by SCI [105]. Importantly, a recent study demonstrated that minocycline, by inhibiting the IL-6/JAK2/STAT3 axis in spinal microglia, also alleviates chronic mechanical allodynia and thermal hyperalgesia induced by SCI. This represents the first evidence of minocycline’s impact on IL-6 expression within the spinal cord [105].
One study found that Parthenolide (PTL) alleviated abnormal pain and hyperalgesia on day 7 post-CCI, which was associated with upregulation of STAT3 and increased levels of anti-nociceptive factors such as IL-10 and TIMP1 during microglial polarization [55] Kinsenoside, a natural bioactive glycoside extracted from the medicinal plant Anoectochilus roxburghii, have revealed that by activating the IL-10/STAT3/SOCS3 signaling pathway can suppress the expression of TNF-α, IL-1β, and IL-6 in microglia, thereby alleviating the allodynia in SNL rats. NSC74859, a STAT3 inhibitor, significantly reduced analgesic effects [162].
(2) Activate the negative feedback regulators
Suppressor of cytokine signaling 3 (SOCS3), a target gene of the STAT3 transcription factor, has been identified as a key feedback regulator of the JAK2/STAT3 pathway, playing a significant role in chronic pain mechanisms. Liver cancer stem cells (DILC) are expressed at higher levels in the spinal cord than in the brain of adult rats, with predominant expression in microglia. A study demonstrated that intrathecal administration of DILC siRNA significantly alleviated mechanical allodynia and thermal hyperalgesia in bCCI male rats. This effect was likely mediated by downregulation of DILC, induction of SOCS3 expression, and inhibition of the JAK2/STAT3 pathway, which subsequently reduced the production of IL-1β, IL-6, and TNF-α in microglia [163]. In contrast, another study found that recombinant human milk fat globule epidermal growth factor-8 (rhMFG-E8) promoted M2 microglial polarization in the spinal cord of mice and alleviated ITGβ3/SOCS3/STAT3 pathway-mediated neuroinflammation, leading to improvements in mechanical allodynia and thermal hyperalgesia in SNI mice. Notably, these effects were reversed by treatment with either ITGβ3 siRNA or SOCS3 siRNA [164].
The α-7 nicotinic acetylcholine receptor (α7nAChR) is widely expressed in the spinal cord and DRG, where it modulates chronic pain by inhibiting the phosphorylation of JAK2/STAT3, and the release of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. A study demonstrated that in the SNI rat model, 2 Hz EA treatment significantly activated α7nAChR, suppressed JAK2/STAT3 signaling, and restored the balance between pro-inflammatory and anti-inflammatory cytokines in the DRG [165]. Other studies have revealed that activation of α7nAChR induces an analgesic effect in neuropathic pain rat models by increasing the expression of IL-10 and β-endorphin in spinal microglia. This mechanical anti-allodynic effect is blocked by methyllycaconitine (an α7-nAChR antagonist) [166,167]. It is clear that activation of α7nAChR exerts an analgesic effect through microglial cells, making it a critical target for the treatment of NP.
(3) Downregulation of other upstream/downstream factors
CSF1R is a critical signaling pathway essential for the development and maintenance of microglial cells [12]. Studies have found that the expression of CSF1R and microglia in the spinal dorsal horn (SDH) of SNI mice was significantly increased. Treatment with PLX3397 (a CSF1R inhibitor) reduces the expression of CSF1R and microglial cells in the dorsal horn of the spinal cord post-SNI, significantly alleviating hyperalgesia in SNI mice [168]. A recent study further demonstrated that (+)-catechin reduced the expression levels of IBA1 and CSF1R in the DRG of CCI rats, alleviating mechanical allodynia. This effect was mediated by the suppression of microglial proliferation in the DRG and the inhibition of the CSF1R/JAK2/STAT3 pathway [153].
The P2Y13 receptor is a neurotransmitter receptor found on spinal microglia. A study showed that intrathecal administration of the MRS2211 (P2Y13 receptor antagonist) reduced the expression of IL-6 and IL-1β in the spinal cord and inhibited microglial activation in the early stage (4 weeks post-STZ injection) of the DNP rat model. The mechanism appears to involve the suppression of the JAK2/STAT3 signaling pathway. Additionally, MRS2211 treatment reduced the phosphorylation of NMDAR via its downstream target gene NR2B, thereby delaying central sensitization [169]. Another study indicates that JAK2 and STAT3 are predominantly expressed in activated microglia within the dorsal horn of the spinal cord in diabetic neuropathy (DNP) rats, where they may exacerbate pain symptoms via the modulation of downstream CAV-1 and NR2B. The application of AG490 leads to a reduction in the expression of phosphorylated JAK2 (p-JAK2) and STAT3 (p-STAT3), resulting in pain relief [150].
In recent years, epigenetic regulation has emerged as a novel therapeutic approach for NP [170]. Valproic acid (VPA), a histone deacetylase inhibitor, has been shown to alleviate mechanical allodynia in SNL rats by inhibiting the JAK2/STAT3 signaling pathway. This effect is mediated by promoting M2 polarization of microglia and suppressing spinal neuroinflammation, offering a potential strategy for pain management [171]. Furthermore, the combination of HDAC inhibitors with anticancer drugs to prevent CIPN is a novel approach to the treatment of this condition [172].
As previously mentioned, JAK2/STAT3 cascade activation can be induced by IL-6 and IL-10, suggesting that the activation of this pathway has dual effects on microglial activation, including both promoting and resolving inflammation, as well as driving M1/M2 polarization of microglia. SOCS3 and α7nAChR act as key feedback regulators of the JAK2/STAT3 pathway, influencing the phosphorylation of its molecular components and should be considered important therapeutic targets in the development of treatments for NP. Modulating the expression of upstream molecules such as IL-10, IL-6, CSF1R, P2Y13 receptors, or downstream target genes such as CAV-1/NR2B can alleviate NP, and research shows that the JAK2/STAT3 signaling pathway, to some extent, activates p38 MAPK and ERK pathways in microglia, contributing to the maintenance of NP (Table 4).
6. Nrf2/HO-1 pathway
Oxidative stress (OS) arises from an imbalance between the production of normal metabolic byproducts and the body's cellular defense mechanisms [25]. Previous studies have shown that OS plays a crucial role in inflammation and chronic pain [173]. The nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO-1) signaling pathway is an essential component of the oxidative stress response, involved in processes such as inflammation, antioxidant defense, and apoptosis. Early studies have demonstrated that activation of the Nrf2/HO-1 pathway can alleviate oxidative stress and neuroinflammation in the spinal cord following CCI [174-176]. Nrf2 pathway activation and HO-1 induction have been considered underlying therapeutic strategies for NP [177]. However, the underlying mechanisms of interaction between these pathways and microglia remain unclear. Studies have indicated that activation of the Nrf2/HO-1 pathway can regulate microglial polarization, thereby alleviating neuroinflammation [178,179].
1) Molecular mechanism of Nrf2/HO-1 activation
Nrf2, encoded by the NFE2L2 gene, is a basic leucine zipper transcription factor consisting of 605 amino acids and divided into seven highly conserved functional domains (Neh1-Neh7) [180]. The activity of Nrf2 is primarily regulated by Kelch-like ECH-associated protein 1 (Keap1), which directs the proteasomal degradation of Nrf2 through its interaction with Keap1. Keap1 is composed of five structural domains: an N-terminal region (NTR), a tramtrack and bric-à-brac (BTB) domain, a central intervening region (IVR) with a nuclear export signal (NES) that mediates Keap1's cytoplasmic localization, six Kelch repeats, and a C-terminal region (CTR) [181]. Under non-stress conditions, Keap1 and Cullin 3 (Cul3) form a ubiquitin E3 ligase complex in the cytoplasm, primarily driven by the BTB domain, which polyubiquitinates Nrf2 for rapid degradation through the proteasomal system [182]. Under stress conditions, Nrf2 is released from the Keap1-Cul3-RBX1 complex and translocates to the nucleus. There, Nrf2 dimerizes with small Maf proteins and binds to antioxidant response elements (ARE), driving the transcription of protective genes. This activation results in the expression of a range of antioxidant enzymes and phase II detoxifying enzymes, including HO-1, NAD(P)H, quinone oxidoreductase (NQO1), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), which remove harmful ROS and promote antioxidant stress, anti-inflammatory, and anti-apoptotic cellular protective mechanisms [183].
HO-1 is a phase II antioxidant enzyme regulated by Nrf2 and is a critical stress-response protein. It catalyzes the conversion of hemoglobin into carbon monoxide (CO), ferrous iron (Fe2+), and biliverdin, which are subsequently reduced to bilirubin. This process exerts antioxidant, anti-inflammatory, and anti-apoptotic effects [184]. Studies have shown that intrathecal injection of recombinant lentivirus overexpressing HO-1 can suppress NP symptoms induced by SNL [185].
2) Activation of the Nrf2/HO-1 signaling alleviates NP through its anti-inflammatory, antioxidant, and neuroprotective properties
In recent years, the Nrf2/HO-1 signaling pathway has garnered significant attention as a key regulator of intracellular antioxidant stress, and it is extensively studied as a target for alleviating NP. A study has shown that the compound NaHS can alleviate abnormal pain behaviors in CCI rats by activating the Nrf2/HO-1 pathway, thereby reducing the excessive release of TNF-α, IL-1β, IL-6, and HMGB1 from spinal microglia [175]. BA, a naturally occurring pentacyclic triterpenoid lupane compound, has been shown to alleviate abnormal pain behaviors in CCI mice by activating the Nrf2/HO-1 pathway. This mechanism reduces glial cell activation and oxidative stress, while attenuating the release of pro-inflammatory cytokines from the spinal cord [176]. Mitsugumin 53 (MG 53), a member of the TRIM protein family, has been shown to improve neuropathic pain, neuroinflammation, and oxidative stress in CCI rats by activating the Nrf2/HO-1 signaling pathway in the spinal cord [186]. Additionally, a study has shown that preconditioning with dexmedetomidine can alleviate CCI-induced mechanical allodynia by activating the Keap-1/Nrf2/HO-1 pathway in CCI rats. This mechanism suppresses the release of inflammatory cytokines, inhibits microglial apoptosis, and enhances antioxidant effects [187].
(1) CORMs and CoPP
Studies have demonstrated the analgesic effects of both carbon monoxide releasing molecules (CORMs) and HO-1 inducers in models of nerve injury, primarily through their anti-inflammatory and protective properties [188]. Experiments conducted in various preclinical pain models have shown that the spinal cord, amygdala, and hippocampal regions of animals with nerve injury-induced NP exhibit increased activation of microglia and astrocyte expression [189-191]. Systemic administration of CORM-2 or HO-1 inducers (cobalt protoporphyrin IX, CoPP) reduces spinal microglial activation, enhances HO-1 protein expression, and induces overexpression of inducible nitric oxide synthase (NOS1, NOS2), thus alleviating NP symptoms [191]. Notably, these treatments did not affect Nrf2 protein expression in the observed regions [190]. This supports the hypothesis that the peripheral and central protective effects of CORM-2 and CoPP in chronic neuropathic pain are primarily mediated through HO-1 activation [188]. Nanocarrier-mediated delivery of CORM-2, owing to its slow CO release and prolonged duration of action, exhibits superior antiallodynic effects compared to free CORM-2 [192]. CoPP has also been shown to block microglial activation induced by diabetes [193], as well as the enhanced expression of astrocytes and microglia caused by chemotherapy agents [194]. Furthermore, CoPP has been shown to reverse paclitaxel (PTX)-induced mechanical and cold allodynia, while also normalizing the associated anxiety and depressive-like behaviors [195]. These findings underscore the promising analgesic and anti-inflammatory effects of systemic administration of CoPP and CORMs in the spinal cord and specific brain regions.
(2) Nrf2 activators
Multiple studies suggest that the regulation of chronic pain induced by Nrf2 transcription factor activators may, in part, be explained by the activation of the Nrf2/HO-1/NQO1 signaling pathway, along with the inhibition of microglial activation and phosphorylation of NF-κB, PI3K/Akt, and MAPK. It has been confirmed that the administration of various Nrf2 activators, such as sulforaphane, oltipraz, quercetin, dimethyl fumarate, and TAT-14 exerts antinociceptive effects in chronic pain models [196-200]. Studies have shown that oral administration of dimethyl fumarate can activate Nrf2 antioxidant signaling in microglia of the DRG, reducing the gene expression and protein levels of IL-1β, TNF-α, and CCL2, thereby alleviating SNI-induced hyperalgesia [197,199]. This effect is inhibited by the trigonelline (Nrf2 inhibitor). Regardless of whether peripheral or central nervous system injuries induce widespread oxidative stress, research has found decreased expression of Nrf2 and HO-1 in the spinal cord, amygdala, prefrontal cortex, and hippocampus of SNI animal models, which can be restored by treatment with sulforaphane or oltipraz [190]. Interestingly, both inducers were also shown to suppress depression-like and/or anxiety-like behaviors associated with NP, potentially through the inhibition of microglial activation in the hippocampus and selective activation of the Nrf2/HO-1/NQO1 signaling pathway [198,199]. Activation of this pathway is a critical target for treating mood disorders related to chronic NP.
It is noteworthy that pharmacological and genetic studies have confirmed the functional crosstalk between Nrf2 and NF-κB. One study demonstrated that treatment with diosmetin, a compound derived from traditional herbs, reduced the expression of Keap1 and NF-κB p65 proteins in the DRG of SNL mice, while increasing the expression of Nrf2 and HO-1 proteins. This treatment also inhibited the activation of inflammatory cytokines and microglia in the spinal cord, leading to a reduction in abnormal mechanical pain in SNL mice [201]. In another study, NF-κB levels were significantly elevated in the sciatic nerve and DRG of rats in a CCI model, while Nrf2 levels were reduced. Treatment with plumbagin increased Nrf2 levels, enhancing antioxidant defenses, and reduced NF-κB levels in the sciatic nerve and DRG, thereby alleviating neuroinflammation and hyperalgesia induced by CCI [196]. As previously mentioned, (+)-catechin can alleviate CCI-induced NP in rats by triggering the Nrf2-mediated antioxidant system, inhibiting the TLR4/NF-κB pathway, reducing ROS production, and preventing the activation of microglia in the dorsal horn of the spinal cord and NLRP3 inflammasomes [202]. Probucol exhibits similar effects [174]. Furthermore, studies have shown an interaction between the Nrf2/HO-1 pathway and the PI3K/Akt and MAPK pathways [203,204].
In summary, the activation of the Nrf2/HO-1 pathway in microglia alleviates NP symptoms through its anti-inflammatory, antioxidant, and neuroprotective properties, as demonstrated in various neuropathic pain models. Additionally, this pathway also contributes to the modulation of NP-associated depressive-like or anxiety-like behaviors. Activation of the Nrf2/HO-1 pathway can regulate other signaling pathways such as NF-κB, PI3K/Akt, and MAPKs, suppressing microglial activation and reducing the release of inflammatory cytokines, thereby mitigating NP. Various Nrf2/HO-1 activators and CORMs have been shown to relieve NP (Table 5), although further studies are required to validate the potential analgesic effects of these novel compounds in other preclinical pain models and assess their safety for more effective clinical application.
CONCLUSIONS
A substantial body of evidence underscores the critical role of activated microglia in the development of pain hypersensitivity. As a microglial inhibitor, minocycline demonstrates limited efficacy, primarily due to its non-specific effects on neurons other than microglia, thereby complicating the interpretation of its therapeutic potential [205]. Given the current limitations in the present understanding of the mechanisms underlying chronic pain and its treatment, this review delineates the molecular mechanisms governing microglia-derived signaling pathways (Fig. 1), and provides an update on the latest mechanisms of action of traditional clinical first-line drugs/herbal medicine/physiotherapy, etc. The objective is to explore how modulating intracellular pathways can influence microglial activation, thereby mitigating neuroinflammation and alleviating both hypersensitivity and abnormal pain states. This work aims to provide a theoretical framework for the development of novel therapeutic compounds targeting the pathophysiology of NP. Future research should continue to investigate the intricate activation mechanisms of microglial pathways, with particular emphasis on the key molecular drivers involved in microglial polarization transitions. It is noteworthy that specific blockade of the novel target AT1R has been confirmed to alleviate NP, and the angiotensin converting enzyme/angiotensin II/angiotensin receptor-1 (ACE/Ang II/AT1R) axis, as well as the glucocorticoid receptor/dynorphin A pathway, will be new targets for intervention.
Furthermore, significant differences exist in the translation from rodent-based research to human clinical studies. For instance, TLR4 is highly expressed in the microglia of rodent models, yet it is only minimally expressed in human microglia [206]. Therefore, integrating human-derived cell or tissue models in conjunction with rodent studies is essential for more accurate and valuable insights. Currently, most studies focus on the activation of microglia in the spinal cord and DRG, with limited exploration of pain-related brain regions in the central nervous system, such as the anterior cingulate cortex (ACC), medial prefrontal cortex (mPFC), and hippocampus. Further investigation into the distribution and functional roles of microglia in these areas is warranted. Additionally, it is important to note that intracellular signaling pathways underlie various physiological functions, and their non-selective or complete inhibition may lead to severe side effects. Thus, identifying more specific targets for intervention is crucial, ensuring that therapeutic efficacy is balanced with the minimization of adverse reactions and toxicity.
Regarding novel strategies for the treatment of NP, the authors believe the focus should be on stimulating endogenous anti-nociceptive factors, as this approach is more physiologically aligned than the complete abolition of the nociceptive pathway. Maintaining a balance between neuroprotective and neurotoxic microglial phenotypes, while minimizing excessive or prolonged M1 polarization and promoting enhanced M2 microglial polarization, could represent an ideal therapeutic target for NP management.
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