In this study, we established a classic model of nerve injury-induced neuropathic pain via chronically constricting the left sciatic nerve of male rats. Similar to what reported by Bennett et al. [37], sciatic constriction induced pain-related behavioral signs of mechanical allodynia and thermal hyperalgesia. The characteristic changes in pain threshold indicated that the model was successfully established for this study.
Here, we demonstrated an important but previously unrecognized role of NF-κB in CCI-induced neuropathic pain. Daily intrathecal infusions of NF-κB inhibitor PDTC (100 and 1000 pmol/d) for 4 consecutive days delayed the onset of neuropathic pain and the activation of spinal microgliosis and inflammation in a dose-dependent manner. Moreover, repeated intrathecal administration of PDTC 3 days after CCI reversed the established mechanical allodynia and thermal hyperalgesia, and reduced activated spinal microglial cells and inflammatory cytokine production in rats. No obvious neurotoxicity was observed after repeated intrathecal infusion of PDTC (1000 pmol/d). The inhibition of the NF-κB signaling pathway was accompanied with decreased injury-induced pain behavior and central nerve system inflammation, suggesting the correlation between spinal NF-κB and neuropathic pain. Therefore, NF-κB is a promising therapeutic target for the prevention of nerve injury-induced neuropathy and neuroinflammation.
NF-κB is a protein complex that controls transcriptional DNA, cytokine production and cell survival and participates in cell responses to stimuli, such as stress, cytokines, free radicals, etc. NF-kappa B also plays a key role in regulating the immune response to nerve injury. Previous research indicated that the incorrect regulation of NF-κB is associated with neuroinflammation and neuropathic pain development in rat model of neuropathic pain [38]. NF-κB is believed to promote the expression of proinflammatory genes and lead to neuronal hypersensitivity in nervous system. In our study, TNF-α expression and microglial activation increased around the primary afferents and second-order neurons in laminae I to III in spinal cord. TNF-α in the dorsal horn may enhance the excitability of sensory neurons and activation of microglia [8–12]. The excitatory signal from the injured nerves to these spinal areas triggered TNF-α production and microglial activation. Furthermore, biochemical changes along the neuronal sensory pathway may be other causes. TNF-α is a member of superfamily of type II proteins containing a full-length membrance TNF-α (mTNF-α) that is cleaved by inducible TNF converted enzyme to release diffusible peptides (soluble TNF-α (sTNF-α)). Each peptide performs a different function. In recent studies, nerve injury-induced pain resulted in an increase in mTNF-α in dorsal horn measured by immunohistochemistry or western blot, while the sTNF-α was unable to be detected in spinal cord by ELISA [39]. This is in agreement with the observation in our study. The mTNF-α might serve to mediate bidirection cell-to-cell crosstalk in neuropathic pain resulting from physical nerve injury. However, sTNF-α is the functional form of TNF-α in neupathic pain caused by paclitaxel and may play a role in initiating inflammatory reactions [40]. Further research might be needed to illuminate the involvement of NF-κB in transcriptional promotion of TNF-α in neuropathic pain. Microglia were also activated in the lateral part of the spinal ventral horn and may play a possible role of phagocytosis around motor neurons [41]. In this study, we observed the dose-dependent suppressive effect of intrathecal PDTC on TNF-α expression and microglial activation induced by CCI, therefore, our data demonstrated that NF-κB activation could mediate TNF-α production and microglial activation. These data suggested that the activation of NF-κB pathway facilitated the development of neuropathic pain via regulating CNS inflammation.
Our results suggest that
However, the activation of NF-κB regulated genes is different in the various conditions and could account for development of neuropathic pain. PDTC was also reported to reduce the levels of CX3CR1, COX–2 , IL–1 and IL–6 in the rats of pain models [22, 32, 38]. Hence, the inhibitory effects of PDTC on these proinflammatory factors may be additional reasons for its analgesic activity observed in this study.
In the in vitro model developed by adding pro-inflammatory factor TNF-α to BV–2 microglial cultures attempted to mimic the signaling cues present in the in vivo neuropathic pain environment. Microglial cells treated with TNF-α presented an activated phagocytic phenotype with morphologic changes and intense phospho-p65 labeling. Activated BV–2 microglia showed bigger soma and few ramifications. Control BV–2 microglia, by contrast, showed smaller soma with distal arborization, characteristic of ramified unactivated microglia. Phospho-p65 is a molecule present in the cell nuclei related to the activation of NF-κB signaling pathway.
In CNS, enhanced spinal microglial CX3CR1 expression was believed to be directly related to neuropathic pain and inflammation, although the factors involved in the regulation of microglial CX3CR1 expression have not been well-characterized. We previously reported that CCI stimulates CX3CR1 expression in spinal cord [32]. In this study, we demonstrated that TNF-α induced an increase in CX3CR1 mRNA and protein expression in vitro in BV–2 microglial cells. Similar observations were reported on CX3CR1 expression in response to TNF-α in rat aortic smooth muscle cells [42]. The inducible CX3CR1 is responsible for the prompt activation of microglia during the inflammatory response. Spinal cord microglia play an important role in the genesis of persistent pain by releasing the proinflammatory cytokines TNF-α and IL–1, and brain derived neurotrophic factor (BDNF) [15, 20–24,43]. Therefore, our results proved that enhanced TNF-α in CNS disorders is associated with neuropathic pain.
However, the molecular mechanism for the increased expression of CX3CR1 following TNF-α treatment remains unclear. NF-κB was reported to be involved in TNF-α-induced CX3CR1 expression in human vascular smooth muscle cells and cryptosporidium parvum infection-induced CX3CR1 expression in epithelial cells [44, 45]. There may be NF-κB consensus sites in the promoter region of rat CX3CR1 gene that regulate CX3CR1 production. Our data showed that TNF-α induced an early and rapid nuclear translocation of p-p65. Therefore, the nuclear translocation of NF-κB p-p65 in BV–2 microglial cells may be the critical step for inflammation in microglia, and is involved in TNF-α-induced CX3CR1 expression.
The CX3CR1 is a novel but not the only target gene of NF-κB signaling pathway in central nervous system. Its target genes are still incompletely understood in the nervous system. Already known regulated genes in neuronal tissue make up inducible NO-synthase (iNOS), μ-opiod receptors, brain-derived neurotrophic factor, calmodulin-dependent protein kinase II, and catechol-o-methyltransferase (COMT) [46, 47]. Previous studies show that increased NF-κB activity enhances transcription of genes that cause pain (e.g., iNOS) and decreases ones that ease pain (e.g., COMT). Therefore, inhibition of NF-κB signaling pathway might exert a useful appraoch for the development of new analgesics.
The activation of NF-κB can be blocked by PDTC, without affecting the DNA binding activity of other transcription factors. In the present study, PDTC prevented NF-κB p65 phosphorylation and inhibited CX3CR1 mRNA and protein expression in BV–2 microglial cells, indicating that NF-κB p65 may be essential for CX3CR1 induction in response to TNF-α in BV–2 microglial cells. These findings further confirmed the results of our rat experiments of neuropathic pain, and proved that NF-κB was essential for microglia activation in neuropathic pain in CNS.
NF-κB may not be the only transcription factor involved in the TNF-α-induced up-regulation of CX3CR1 gene in BV–2 cells, because PDTC pretreatment did not completely inhibit the production of CX3CR1. The results that PDTC inhibited the constitutive expression of CX3CR1 without altering the constitutive p-p65 expression indicated that PDTC might suppress the constitutive expression of CX3CR1 through some pathway other than NF-κB, such as nuclear factor erythroid 2 related factor 2 (Nrf2). PDTC is found to activate Nrf2 signaling and have a neuroprotective and antioxidative function [48]. Recently, several other transcription factors were reported to regulate CX3CR1 gene expression in response to TNF-α in various cells. For example, HIF–1, AP–1 and STAT1/3 are also essential for CX3CR1 expression in various cells. A change on the DNA methylation status of CX3CR1 gene promoter can change its activity and gene expression [49–51]. Therefore, the involvement of those transcription factors in transcriptional regulation of CX3CR1 remains to be evaluated in TNF-α-stimulated BV–2 cells.
There are still some limitations of our study. Although blockade of NF-κB inhibited CCI-induced microglial activation and TNF-α expression by immunohistochemistry assay, we don’t know how these molecules and signals are involved. Some study shows co-localization of microglia CX3CR1 and extracellular signal-regulated protein kinase 5 (ERK5) by immmunofluorescence labelling of them in the dorsal horn and suggests that CX3CR1 enhances nerve injury-induced pain hypersensitivity through the ERK5 signaling pathway [52]. Further study is needed to explore the direct target molecules for NF-κB and CX3CR1 in spinal microglia.
PDTC is a synthetic compound is largely used as an NF-κB inhibitor by inhibiting factor I-κB ubiquitination, however, it can also regulate other cell signaling, such as anti-apoptotic signaling, and we don’t know the relationship between the pain-related NF-κB activation pathway and other effects of PDTC. Some specifical NF-κB inhibitors or siRNA might be the promising approaches to selectively impair NF-κB activation. However, because NF-κB also fulfils a number of physiological functions, complete inhibition may lead to serious effects. Recent studies have shown the embryonic lethality in mice with completely knockout of p65 [53], and another study indicated an inhibitory effect of p65-siRNA on transcription of NF-κB-regulated proteins such as COX–2 [54]. Therefore, drugs that selectively inhibit pain-promoting NF-κB activity while leaving physiological functions unaffected would be beneficial to clinic pain management and be explored in future research.