Despite accumulating evidence on the role of glial cells and their associated chemicals in mechanisms of pain, few studies have addressed the potential role of chemokines in the descending facilitation of chronic pain. We aimed to study the hypothesis that CXCL1/CXCR2 axis in the periaqueductal gray (PAG), a co-restructure of the descending nociceptive system, is involved in descending pain facilitation. Intramedullary injection of Walker 256 mammary gland carcinoma cells of adult female Sprague Dawley rats was used to establish a bone cancer pain (BCP) model. RT-PCR, Western blot, and immunohistochemistry were performed to detect pNfkb, Cxcl1, and Cxcr2 and their protein expression in the ventrolateral PAG (vlPAG). Immunohistochemical co-staining with NeuN, GFAP, and CD11 were used to examine the cellular location of pNFκB, CXCL1, and CXCR2. The effects of NFκB and CXCR2 antagonists and CXCL1 neutralizing antibody on pain hypersensitivity were evaluated by behavioral testing. BCP induced cortical bone damage and persistent mechanical allodynia and increased the expression of pNFκB, CXCL1, and CXCR2 in vlPAG. The induced phosphorylation of NFκB was co-localized with GFAP and NeuN, but not with CD11. Micro-injection of BAY11-7082 attenuated BCP and reduced CXCL1 increase in the spinal cord. The expression level of CXCL1 in vlPAG showed co-localization with GFAP, but not with CD11 and NeuN. Micro-administration of CXCL1 neutralizing antibody from 6 to 9 days after inoculation attenuated mechanical allodynia. Furthermore, vlPAG application of CXCL1 elicited pain hypersensitivity in normal rats. Interestingly, CXCR2 was upregulated in vlPAG neurons (not with CD11 and GFAP) after BCP. CXCR2 antagonist SB225002 completely blocked the CXCL1-induced mechanical allodynia and attenuated BCP-induced pain hypersensitivity. The NFκB-dependent CXCL1-CXCR2 signaling cascade played a role in glial-neuron interactions and in descending facilitation of BCP.

Background: Previous studies have demonstrates that, after nerve injury, extracellular signal-regulated protein kinase (ERK) activation in the spinal cord-initially in neurons, then microglia, and finally astrocytes. In addition, phosphorylation of ERK (p-ERK) contributes to nociceptive responses following inflammation and/or nerve injury. However, the role of spinal cells and the ERK/MAPK pathway in cancer-induced bone pain (CIBP) remains poorly understood. The present study analyzed activation of spinal cells and the ERK/MAPK pathway in a rat model of bone cancer pain. Results: A Sprague Dawley rat model of bone cancer pain was established and the model was evaluated by a series of tests. Moreover, fluorocitrate (reversible glial metabolic inhibitor) and U0126 (a MEK inhibitor) was administered intrathecally. Western blots and double immunofluorescence were used to detect the expression and location of phosphorylation of ERK (p-ERK). Our studies on pain behavior show that the time between day 6 and day 18 is a reasonable period (“time window” as the remaining stages) to investigate bone cancer pain mechanisms and to research analgesic drugs. Double-labeling immunofluorescence revealed that p-ERK was sequentially expressed in neurons, microglia, and astrocytes in the L4-5 superficial spinal cord following inoculation of Walker 256 cells. Phosphorylation of ERK (p-ERK) and the transcription factor cAMP response element-binding protein (p-CREB) increased in the spinal cord of CIBP rats, which was attenuated by intrathecal injection of fluorocitrate or U0126. Conclusions: The ERK inhibitors could have a useful role in CIBP management, because the same target is expressed in various cells at different times.

Bone cancer pain (BCP) is an intractable clinical problem, and lacked effective drugs for treating it. Recent research showed that several chemokines in the spinal cord are involved in the pathogenesis of BCP. In this study, the antinociceptive effects of liquiritin, which is an active component extracted from Glycyrrhizae Radix, were tested and the underlying mechanisms targeting spinal dorsal horn (SDH) were investigated. The BCP group displayed a significant decrease in the mechanical withdrawal threshold on days 6, 12, and 18 when compared with sham groups. Intrathecal administration of different doses of liquiritin alleviated mechanical allodynia in BCP rats. The results of immunofluorescent staining and western blotting showed that liquiritin inhibited BCP-induced activation of astrocytes in the spinal cord. Moreover, intrathecal administration of liquiritin effectively inhibited the activation of CXCL1/CXCR2 signaling pathway and production of IL-1β and IL-17 in BCP rats. In astroglial-enriched cultures, Lipopolysaccharides (LPS) elicited the release of chemokine CXCL1, and the release was decreased in a dose-dependent manner by liquiritin. In primary neurons, liquiritin indirectly reduced the increase of CXCR2 by astroglial-enriched-conditioned medium but not directly on the CXCR2 target site. These results suggested that liquiritin effectively attenuated BCP in rats by inhibiting the activation of spinal astrocytic CXCL1 and neuronal CXCR2 pathway. These findings provided evidence regarding the the antinociceptive effect of liquiritin on BCP.

Background: Chemokine, monocyte chemoattractant protein-1 (MCP-1), is a potential factor to cause cancer-induced bone pain (CIBP). NF-κB signaling is very important in mediating the expression of chemokines and may have a role in CIBP. However, the mechanism is still unclear. This study investigates the role of NF-κB in CIBP by regulating MCP-1/chemokine CC motif receptor-2 (CCR2) signaling pathway. Methods: A rat CIBP model was established by injecting Walker-256 cells into the tibia medullary cavity. Nine days later, animals were intrathecally administrated with MCP-1 neutralizing antibody, CCR2 antagonist (RS504393), or NF-кB inhibitor (BAY11–7081). Mechanical paw withdrawal threshold was used to assess pain behavior and sciatic functional index, and radiographic images were adopted to evaluate the damage of nerve and bone. The spinal cords were harvested for Western blot and quantitative reverse transcription polymerase chain reaction. The distribution of MCP-1, CCR2, and NF-кB was detected by double immunofluorescent staining. Results: CIBP caused remarkable bone destruction, injury of sciatic and femoral nerve, and persistent (>15 days) mechanical allodynia in rats. Tumor cell inoculation upregulate MCP-1 and NF-кB in activated neurons as well as CCR2 in neurons and microglia of the spinal cord. MCP-1 antibody, RS504393, and BAY11–7081 partially reversed CIBP-induced mechanical allodynia, and CIBP regulated the expression levels of pro-inflammatory cytokines, tumor necrosis factor-α and interferon-γ, and anti-inflammatory cytokine, interleukin 4, and BAY11–7081 lowered CIBP-induced MCP-1 and CCR2 expressions in a dose-dependent manner. Conclusion: In conclusion, NF-кB signaling pathway regulates the expressions of MCP-1/CCR2-induced inflammatory factors in the spinal cord of CIBP rats.