Esketamine inhibits the c-Jun N-terminal kinase pathway in the spinal dorsal horn to relieve bone cancer pain in rats

Abstract

Cancer-induced bone pain (CIBP) is one of the most common and feared symptoms in patients with advanced tumors. The X-C motif chemokine ligand 12 (CXCL12) and the CXCR4 receptor have been associated with glial cell activation in bone cancer pain. Moreover, mitogen-activated protein kinases (MAPKs), as downstream CXCL12/CXCR4 signals, and c-Jun, as activator protein AP-1 components, contribute to the development of various types of pain. However, the specific CIBP mechanisms remain unknown. Esketamine is a non-selective N-methyl-d-aspartic acid receptor (NMDA) inhibitor commonly used as an analgesic in the clinic, but its analgesic mechanism in bone cancer pain remains unclear. We used a tumor cell implantation (TCI) model and explored that CXCL12/CXCR4, p-MAPKs, and p-c-Jun were stably up-regulated in the spinal cord. Immunofluorescence images showed activated microglia in the spinal cord on day 14 after TCI and co-expression of CXCL12/CXCR4, p-MAPKs (p-JNK, p-ERK, p-p38 MAPK), and p-c-Jun in microglia. Intrathecal injection of the CXCR4 inhibitor AMD3100 reduced JNK and c-Jun phosphorylations, and intrathecal injection of the JNK inhibitor SP600125 and esketamine also alleviated TCI-induced pain and reduced the expression of p-JNK and p-c-Jun in microglia. Overall, our data suggest that the CXCL12/CXCR4-JNK-c-Jun signaling pathway of microglia in the spinal cord mediates neuronal sensitization and pain hypersensitivity in cancer-induced bone pain and that esketamine exerts its analgesic effect by inhibiting the JNK-c-Jun pathway.

Introduction

Cancer is a leading cause of death worldwide, and the global incidence of cancer has been increasing disturbingly. ¹ The bone is the third largest system affected by metastases after the lungs and the liver. ² Breast, prostate, lung, thyroid, and kidney cancers account for 80% of bone metastases. ³ In particular, 70% of patients with breast and prostate cancers develop bone metastases. ⁴ Cancer-induced bone pain (CIBP) is a serious complication of bone metastases, with up to 85% of patients experiencing high intensity pain that is commonly crippling, persistent, spontaneous, usually accompanied by hyperalgesia, and diminishing the quality of life (QoL). ⁵ The pathophysiology underlying CIBP is complex and multifactorial, and it is mainly associated with traumatic, neuropathic processes, and inflammatory pain, but the mechanisms remain unclear. ⁶ The available treatments involve radiotherapy, chemotherapy, surgery, nerve blocks, and analgesics, all with limited efficacy due to numerous undesirable side effects. ⁶ Therefore, alternative CIBP therapies are needed to ease the suffering of patients.

The X-C motif chemokine ligand 12 (CXCL12) is a CXC chemokine subfamily member, also known as stromal cell-derived factor-1 (SDF-1), and it is extensively expressed in a variety of tissues and cells. CXCL12 exerts its functions upon specifically binding to the X-C motif chemokine receptor 4 (CXCR4), a seven transmembrane G protein-coupled receptor. ⁷ The CXCL12/CXCR4 signaling pathway has been shown to have a key role in different cellular functions, including inflammation, tissue homeostasis, immune surveillance, and tumor growth and migration. ⁸ In addition, studies have demonstrated effects of the CXCL12/CXCR4 signaling pathway in pathological pain, including neuropathic, inflammatory, and bone cancer pains.^9–11 The CXCL12/CXCR4 axis was discovered in connection with a series of pain-related downstream signaling pathways, including phosphatidylinositol 3-kinase (PI3K), nuclear factor of activated T cells (NFAT), nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB), activators of transcription pathways, and the mitogen-activated protein kinases (MAPKs). ¹² The key mechanisms explaining the function of the CXCL12/CXCR4 axis remain unclear.

MAPK is an evolutionarily conserved family of intracellular signaling molecules essential for intracellular signal transduction. ¹³ The MAPK pathways are composed of three major members: the extracellular signal-regulated kinase (ERK) pathway, the p38 signaling pathway, and the c-Jun N-terminal kinase (JNK), all participating in the regulation of cell proliferation, differentiation, apoptosis, and autophagy and with functions in neuroplasticity and inflammatory responses, which are associated with the development of pain. ¹⁴ The JNK and p38 MAPK signaling pathways increase the release of proinflammatory cytokines and result in the concomitant upregulation of autophagy in SNL-induced neuropathic pain. ¹⁵ Moreover, inhibiting JNK and p38/MAPK phosphorylation suppresses the inflammation response in microglia and alleviates the inflammatory pain in mice, and lidocaine inhibition of ERK-NF-κB pathway activation achieves the same effect in rats with CIBP.^16,17 In bone cancer pain, up-regulated MAPK pathways activate astrocytes and microglia to affect pain sensation by an over-expression of inflammatory factors, including IL-1β, IL-6, and TNF-α. ¹⁸ However, how MAPK pathways interact with glial cells to mediate bone cancer pain remains uncertain.

C-Jun is a component of the activator protein 1 (AP-1), which gets activated under physiological stimuli and environmental damage to regulate a wide range of cellular processes and pathogenic developments. ¹⁹ Some studies have found that c-Jun is a key gene of neuropathic pain (NP) and plays a major role in the development of NP by screening differential genes in the model of chronic compression of the DRG (CCD) and the spared nerve injury (SNI).^20,21 Complete Freund’s adjuvant (CFA) induces the activation of p-JNK, and the activated p-JNK further regulates the expression of p-c-Jun to participate in the development of inflammatory pain. ²² Abundant studies have shown that c-Jun is associated with pain; however, to our knowledge, no correlations between c-Jun and bone cancer pain have been reported.

The non-selective N-methyl-d-aspartic acid receptor (NMDA) inhibitor, esketamine, is the S (+)-isomer of ketamine and is twice as effective at alleviating pain as ketamine. ²³ Esketamine is widely used in the clinical treatment of depression, and it has also been used for perioperative analgesia in surgical patients.^23,24 Although esketamine has analgesic effects, its specific action mechanisms are unclear. Animal experiments have shown that ketamine can inhibit NMDAR phosphorylation, regulate the stimulator of interferon genes/TANK binding kinase (STING/TBK) signal channel, reduce the activation of MAPK and the release of pro-inflammatory factors to provide anti-nociceptive effects, and relieve pain.^25,26 However, few reports have mentioned specific analgesic effect mechanisms of esketamine.

In this study, we explored the role of the CXCL12/CXCR4-JNK-c-Jun signaling pathway in bone cancer pain. In addition, we examined the analgesic ability of esketamine in bone cancer pain rats by inhibiting the JNK-c-Jun pathway.

Materials and methods

Animals

The Animal Ethics Committee of the Animal Ethical and Welfare Committee of Nanjing Medical University reviewed and approved the protocol for this animal study. 160–180-g or 60–80-g female Sprague-Dawley (SD) rats were purchased from Suzhou Xinuosai Biotechnology Company (Jiangsu China). We placed rats in standard and individually ventilated cages (four rats per cage) in a set environment at 22 ± 1°C, with 12-h light/dark cycles, and ad libitum water and food pellets. Before the surgical operations, all rats were acclimatized for 7 days. All the rats were divided into different group by completely randomized design. Different researchers conducted CIBP model establishment, intrathecal injection, and pain behavior test to ensure the blinding.

Tumor cell preparation

Walker 256 cells were purchased from Beijing Ding Guo Chang Sheng Biotechnology Company (Beijing, China). The cells were suspended in PBS to an appropriate concentration (2 × 10⁷ cells/mL), and we injected 0.5 mL into 60–80-g female SD rats intraperitoneally. The cancerous ascites was aseptically extracted 1 week after the injection. Next, we washed the cancerous ascites samples three times with sterile PBS by centrifugation at 1000 r/min for 5 min each time, we resuspended the tumor cell pellets in PBS (1 × 10⁵ cells/μL), and kept the suspension tubes on ice until the next injection into the rats to establish the model. ²⁷

CIBP model establishment

We established the CIBP model as described. ²⁷ Briefly, the rats were anesthetized by intraperitoneal injection of tribromoethanol (10 mL/kg); and, then we depilated the right hind limb and disinfected the skin three times with 75% ethanol. To expose the surgical field, we made a 1-cm incision longitudinally in the upper tibia with a surgical blade. A 2-mL syringe needle was used to bore a hole into the marrow cavity in the proximal tibia, and we delivered 10 μL of resuspended Walker 256 cells (1 × 105 cells/μL) into the right bone cavity with a 25-μl micro-syringe. The injection hole was quickly closed with medical glue as soon as the micro-syringe was pulled out. Finally, after disinfecting the wound, the incision was sutured, and all the rats were incubated until they had recovered from the anesthesia. The sham operation group was injected with an equal volume of PBS, and the other procedures were all identical.

Intrathecal injections

The CXCR4 inhibitor, plerixafor (AMD3100), was purchased from Selleck Chemicals (Shanghai, China). The JNK inhibitor SP600125 was purchased from Apexbio (Houston, Texas, USA). Esketamine was purchased from Jiangsu Hengrui Pharmaceutical Company (Jiangsu China). AMD3100 was dissolved and resuspended to 5 µg/10 µL with NS. SP600125 was dissolved in dimethyl sulfoxide (DMSO) and diluted to 5 µg/10 µL with NS. Esketamine was diluted with NS to produce three concentrations at 2 µg/μL, 4 µg/μL, and 6 µg/μL. We depilated the backs of rats and disinfected them after anesthesia induction. Next, we inserted a 25-µL micro-syringe into the L4-5 intervertebral space and injected 10 µL of drug with the needle well positioned, as judged by the brisk swing of the tail or hind limbs upon careful needle tip insertion. The needle was kept in place for 10 s after each injection. ²⁸ We removed animals with signs of motor dysfunction from the experiment.

Pain behavior test

We used a von Frey filament (Danmic Global, California, USA) to measure the 50% paw withdrawal threshold (PWT) following the Dixon up-down method to assess the pain behavior. ⁵ Rats were placed in a transparent plexiglass platform with a grid bottom, and allowed to acclimatize for a period of time until the limbs did not move frequently. Starting at 2.0-g, we applied von Frey filaments to stimulate the plantar area of the right hind limb until it was bent and maintained for 6–8 s. We considered paw licking, shaking, or quick withdrawal as positive responses. We used the next thinner von Frey filament after each positive response, otherwise, we used the next thicker von Frey filament. After the first crossover of negative and positive responses, we obtained four more consecutive measurements to record a series of data, which we converted to the 50% PWT as defined by Dixon. We repeated the measurements three times in all rats and calculated the average of the three results as the ultimate 50% PWT.

X-ray films

We anesthetized rats of each group with tribromoethanol on day 14 following the establishment of the model, and we captured X-ray films of the animals in the supine position to assess the bone destruction resulting from the tumor of the affected limb.

Western blot

We extracted total proteins from L3–L5 spinal cords using radio immunoprecipitation assay buffer (Beyotime Shanghai China) and phenylmethanesulfonyl fluoride (Beyotime Shanghai China). We quantified the protein contents of the samples using a BCA Protein Assay Kit (Beyotime Shanghai China) and used 5X loading buffer to denature the proteins by boiling the samples for 10 min. Proteins of equal quality were loaded on 10% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis and then were transferred to polyvinylidene difluoride (Millipore, Billerica, MA, USA) membranes. Next, we blocked the membranes in 5% nonfat milk for 2 h at room temperature and incubated them overnight at 4°C with the indicated primary antibodies: rabbit anti-CXCR4 (1:1000), CXCL12 (1:1000), JNK (1:1000), p-JNK (1:1000), ERK (1:1000), p-ERK (1:1000), p38 (1:1000), p-p38 (1:1000), c-Jun (1:1000), p-c-Jun (1:1000), and mouse anti-Tubulin (1:2000). The membranes were subsequently washed three times with TBST solution for 10 min each time before a final 2-h incubation at room temperature with horseradish peroxidase conjugated secondary antibodies. After washing the membranes again as before, we visualized the bands using an ECL Prime Western blotting detection and image analysis system, as reported.^29,30 The gray values of the bands were analyzed using the Image J software, and anti-Tubulin as the internal reference for quantification.

Immunofluorescence

We boiled L3-L5 spinal cord paraffin-embedded sections in 10 mM sodium citrate buffer (pH 6.0) for antigen extraction after deparaffinization and rehydration. The sections were sealed with 1% bovine serum albumin (BSA) in PBS for 2 h and incubated overnight with primary antibodies 4°C, and then incubated with fluorescent secondary antibody for 1 h at room temperature. We observed the resulting images of right spinal dorsal horn under a Zeiss laser confocal microscope (LSM 810, Carl Zeiss, Oberkochen, Germany) as described.^31–33 The following antibodies were used: rabbit anti-CXCR4 (1:100), CXCL12 (1:100), p-JNK (1:100), p-ERK (1:100), p-p38 (1:100), and p-c-Jun (1:100). The intensity was analyzed by Zeiss Zen.

Statistical analysis

We conducted all the data analyses using the GraphPad Prism eight and SPSS 26.0 software and expressed results as means ± standard errors (mean ± SEM). We compared the results of the pain behavior tests using a two-way analysis of variance with repeated measures (two-way ANOVA) to analyze significant differences. The results of the Western blot and immunofluorescence were analyzed using a t test or one-way analysis of variance with repeated measures. We compared data from multiple groups by applying Bonferroni post hoc tests. We considered p < .05 as statistically significant.

Results

Establishment of rat cancer-induced bone pain model by intratibial inoculation of walker 256 cancer cells

We used Walker 256 cancer cells to establish a cancer-induced bone pain rat model. We initially produced large numbers of cancerous ascites cells in infant rats 1 week after intraperitoneal injection of Walker 256 cancer cells (Figure 1(a)). The behavioral manifestations in the rats demonstrated that TCI gradually promoted significant pain behaviors, evidenced by the reduction of the paw mechanical withdrawal threshold. After TCI, the paw mechanical withdrawal threshold began to decline on day 3, reached the lowest level on day 11 and was maintained until day 21, the last day of measurement (Figure 1(b)). Moreover, cancer cell inoculation led to significant bone destruction as shown by the X-ray film comparisons between rats in the NC and sham groups on day 14 (Figure 1(c)).

Figure 1.

Mechanical allodynia, bone destruction, and the variation of related proteins in the spinal cord caused by inoculating Walker 256 cells in the tibia. (a) Intraperitoneal injection of Walker 256 cells produced cancerous ascites a week later. (b) PWTs of rats in each group on different days (n = 5). (c) Representative X-ray films of the three groups for comparison. The arrow indicates the bone destruction site. (d) Western blot analysis of CXCR4/CXCL12, P-JNK, P-ERK, P-P38, and P-c-Jun in the spinal cord of NC, sham, and CIBP rats on days 3, 7, 14, and 21 after TCI. (e) Quantitative analysis of relative expressions of CXCR4/CXCL12, P-JNK, P-ERK, P-P38, and P-c-Jun in spinal cords of NC, sham, and CIBP rats on days 3, 7, 14, and 21 after TCI (n = 3). *p < .05, **p < .01 vs. NC group, ^##p < .01 vs. day 3, ^ΔΔP <.01 vs. day 7.

Up-regulation and co-localization of CXCR4/CXCL12, p-MAPKs, and p-c-Jun in the spinal cord of CIBP model rats

Chemokines have a significant role in the development and maintenance of cancer pain in central nervous systems. ³⁴ Chemokine CXCL12 and its receptor CXCR4 were upregulated in the spinal cord of the affected limb, expression began to increase on day 3, rose from 3 to 7 days, reached higher levels around 14 days, declined at day 21 and higher on day 14 and day 21 compared to day 3 or day 7 (Figure 1(d) and (e)). MAPKs are crucial signaling molecules for the development and maintenance of pain. ¹⁸ Based on our results, the upregulation of p-MAPKs (p-JNK, p-ERK, and p-p38 MAPK) followed the same temporal pattern as those of the CXCL12/CXCR4 molecules, and the p-JNK showed the highest increase of all the p-MAPKs (Figure 1(d) and (e)). Therefore, we decided to further test the time-course expression of p-c-Jun, a downstream regulation factor of p-JNK, which has also been associated with all kinds of pain. The protein of p-c-Jun was upregulated from day 3, and achieved its highest level between days 7 and 14, in a time trend similar to that of p-JNK (Figure 1(d) and (e)).

Our double immunofluorescence results show that the activated microglia and astrocytes were more abundant in the spinal cord on day 14 after the TCI (Figures 2 and 3). In addition, we observed the expressions and co-localization of CXCL12/CXCR4, p-JNK, and p-c-Jun in microglia and astrocytes. CXCL12/CXCR4 and p-JNK all colocalized in microglia and astrocytes with enhanced expressions, whereas p-c-Jun was mainly colocalized and expressed in microglia (Figures 2 and 3 and Supplementary Figure 1). These findings suggest a functional association between the CXCL12/CXCR4-JNK-c-Jun pathway in the spinal cord and the development of CIBP.

Figure 2.

Double immunofluorescence images showing the co-localization of CXCR4/CXCL12, P-JNK, and P-c-Jun with microglia in the spinal cord of sham and CIBP rats 14 days after TCI. CXCR4/CXCL12 (green), P-JNK (green), P-c-Jun (green), Iba-1 (red); Scale bars: 100 and 50 µm.

Figure 3.

Double immunofluorescence showing the co-localization of CXCR4/CXCL12, P-JNK, and P-c-Jun with astrocytes in the spinal cords of sham and CIBP rats 14 days after TCI. CXCR4/CXCL12 (green), P-JNK (green), P-c-Jun (green), GFAP (red); Scale bars: 100 and 50 µm.

Antagonizing the activation of CXCR4 relieved TCI-induced cancer pain and inhibited the expression of p-JNK and p-c-Jun in the rat model of CIBP

The above results indicated that CXCL12/CXCR4, p-JNK, and p-c-Jun may be involved in the regulation of bone cancer pain. Therefore, we first explored the specific role of CXCR4 in regulating the bone cancer pain in the spinal cord of rats after intrathecal injection of AMD3100, a classic CXCR4 inhibitor that abrogates the binding of CXCL12 to CXCR4.^9,35,36 The behavioral results of the rats showed that intrathecal injection of AMD3100 once daily from day 12 to day 14 after TCI remarkably attenuated the mechanical allodynia at the maintenance phase of CIBP (Figure 4(a)). We further examined whether inhibition of CXCR4 affected the expression of p-JNK and p-c-Jun in microglia or astrocytes in the CIBP rats. The Western blot results demonstrated that repeated intrathecal injection of AMD3100 once daily for three consecutive days from days 12 to 14 after TCI significantly reduced the upregulation of p-JNK and p-c-Jun in the spinal cord (Figure 4(b)and (c)). The immunofluorescence results further showed that the p-JNK and p-c-Jun co-localization in both microglia and astrocytes was decreased (Figure 5(a) and (b), Supplementary Figure 2(a) and (b)). These results revealed that CXCR4 has a role in the pathogenesis of CIBP and that p-JNK and p-c-Jun may be the downstream effectors of CXCR4 that regulate the development of CIBP.

Figure 4.

Intrathecal injection of CXCR4 inhibitor (AMD3100) attenuated TCI-induced cancer pain and suppressed the expression of P-JNK and P-c-Jun. (a) PWT of rats in each group on different days (n = 6). (b) Western blot analysis of P-JNK, and P-c-Jun in spinal cords after intrathecal injection (n = 3). (c) Quantitative analysis of relative expression of P-JNK and P-c-Jun in spinal cords. AMD3100 (5 μg/10 μL, (i)t.) or saline (vehicle control, 10 μL, i.t.) was administered once daily on days 12, 13, and 14 after TCI. PWTs were measured 4 h after each injection. L3-L5 spinal tissues were collected 4 hours after the last injection. *p < .05, **p < .01 vs. sham + Saline; ^#p < .05, ^##p < .01 vs. CIBP + Saline.

Figure 5.

Double immunofluorescence showing the reduction and co-localization of P-JNK, and P-c-Jun with microglia in spinal cords after intrathecal injection. (a) Fluorescence image of spinal cord after intrathecal injection. (b) Quantitative analysis of fluorescence intensity in microglia of spinal cord. AMD3100 (5 μg/10 μL, (i)t.) or saline (vehicle control, 10 μL, i.t.) were administered once daily on days 12, 13, and 14 after TCI. L3-L5 spinal tissues were collected 4 h after the last injection for double immunofluorescence. P-JNK (green), P-c-Jun (green), Iba-1 (red). Scale bars: 100 and 50 µm. *p < .05, **p < .01 vs. sham + Saline; ^#p < .05, ^##p < .01 vs. CIBP + Saline.

Inhibiting p-JNK activation attenuated the TCI-induced cancer pain and suppressed the expressions of p-JNK and p-c-Jun

The above results indicated that CXCR4 has a significant function in bone cancer pain, and JNK is an important downstream effector of CXCR4. Therefore, we aimed to verify whether JNK also plays an important role in bone cancer pain, we used the JNK inhibitor SP600125 to observe its effects on bone cancer pain. Intrathecal injection of SP600125, once a day for three consecutive days from days 12 to 14 after TCI, alleviated the painful behavior, as evidenced by comparing the recovery PWTs in treated and control rats (Figure 6(a)). These results showed that p-JNK participates in the development of bone cancer pain, and we further explored the specific role of p-JNK in the pain behavior of rats with CIBP. Our Western blot results showed that repeated intrathecal injections of SP600125 significantly reduced the up-regulation of p-JNK and p-c-Jun in the spinal cord (Figure 6(b) and (c)), and immunofluorescence results further showed that p-JNK and p-c-Jun co-localization in both the microglia and astrocytes also decreased (Figure 7(a) and (b), Supplementary Figure 3(a) and (b)). These results definitely indicate that p-JNK in the spinal cord of rats had an effect on the CIBP by regulating the expression of the functional downstream target p-c-Jun.

Figure 6.

Intrathecal injection of JNK inhibitor SP600125 attenuated TCI-induced cancer pain and suppressed the expression of P-JNK and P-c-Jun. (a) PWTs of rats in each group on different days (n = 6). (b) Western blot analysis of P-JNK, and P-c-Jun in spinal cords after intrathecal injection (n = 3). (c) Quantitative analysis of relative expressions of P-JNK and P-c-Jun in spinal cord. SP600125 (5 μg/10 μL, i.t.) or vehicle (vehicle control, 10 μL, i.t.) were administered once daily on days 12, 13, and 14 after TCI. PWTs were measured 4 hours after each injection. L3-L5 spinal tissues were collected 4 hours after the last injection. *p < .05, **p < .01 vs. sham + Vehicle; ^#p < .05, ^##p < .01 vs. CIBP + Vehicle.

Figure 7.

Double immunofluorescence showing the reduction and co-localization of P-JNK, and P-c-Jun with microglia in spinal cords after intrathecal injection. (a) Fluorescence image of spinal cord after intrathecal injection. (b) Quantitative analysis of fluorescence intensity in microglia of spinal cord. SP600125 (5 μg/10 μL, (i)t.) or vehicle (vehicle control, 10 μL, i.t.) were administered once daily on days 12, 13, and 14 after TCI. L3-L5 spinal tissues were collected 4 h after the last injection for double immunofluorescence. P-JNK (green), P-c-Jun (green), Iba-1 (red); Scale bars: 100 and 50 µm. *p < .05, **p < .01 vs. sham + Vehicle; ^#p < .05, ^##p < .01 vs. CIBP + Vehicle.

Intrathecal injection of esketamine attenuated bone cancer pain via inhibition of the expressions of p-JNK and p-c-Jun

On the basis of the prominent analgesic role of esketamine in clinical applications, we explored its analgesic effects in bone cancer pain. Intrathecal injection of esketamine for three consecutive days also reversed the established PWT, which demonstrated the effect of esketamine in attenuating TCI induced pain (Figure 8(a)). In addition, we explored whether esketamine produced its analgesic effects via the CXCL12/CXCR4-JNK-c-Jun signal pathway. Our Western blot results demonstrated that repeated intrathecal injections of esketamine significantly reduced the up-regulations of p-JNK and p-c-Jun in the spinal cord, while resulting in no changes in the expressions of CXCL12 and CXCR4 (Figure 8(b) and (c)). Immunofluorescence results showed that the p-JNK and p-c-Jun co-localization in both microglia and astrocytes also decreased (Figure 9(a) and (b), Supplementary Figure 4(a) and (b)). These results show that esketamine definitely alleviated the existing bone cancer pain, markedly inhibiting the JNK-c-Jun signal pathway without strong effects on the chemokines or chemokine receptors of CXCL12/CXCR4 (Figure 10).

Figure 8.

Intrathecal injections of esketamine attenuated TCI-induced cancer pain and suppressed the expression of P-JNK and P-c-Jun. (a) PWTs of rats in each group on different days (n = 6). (b) Western blot analysis of CXCR4/CXCL12, P-JNK, and P-c-Jun in spinal cord after intrathecal injection (n = 3). (c) Quantitative analysis of relative expression of CXCR4/CXCL12, P-JNK and P-c-Jun in spinal cords. Esketamine (2 µg/µL, 4 µg/µL, and 6 µg/µL, (i)t.) or saline (vehicle control, 10 μL, i.t.) were administered once daily on days 12, 13, and 14 after TCI. PWTs were measured 4 hours after each injection. L3-L5 spinal tissues were collected 4 hours after the last injection. *p < .05, **p < .01 vs. sham + Saline; ^#p < .05, ^##p < .01 vs. CIBP + Saline.

Figure 9.

Double immunofluorescence showing the reduction and co-localization of P-JNK, and P-c-Jun with microglia in the spinal cord after intrathecal injection. (a) Fluorescence image of the spinal cord after intrathecal injection. (b) Quantitative analysis of fluorescence intensity in the spinal cord microglia. Esketamine (5 μg/10 μL, (i)t.) or saline (vehicle control, 10 μL, i.t.) were administered once daily on days 12, 13, and 14 after TCI. L3-L5 spinal tissues were collected 4 h after the last injection for double immunofluorescence. P-JNK (green), P-c-Jun (green), Iba-1 (red); Scale bars: 100 and 50 µm. *p < .05, **p < .01 vs. sham + Saline; ^#p < .05, ^##p < .01 vs. CIBP + Saline.

Figure 10.

Schematic overview of the CXCR4/CXCL12-JNK-c-Jun pathway and the related intracellular signaling mediators in the spinal cord.

Discussion

Bone cancer pain mechanisms are extremely complicated, and include mixed types of pain with overlapping inflammatory and neuropathic pains. ⁶ Central sensitization, glial cell activation, and changes in different signal pathways are associated with occurrence and development of bone cancer pain. ³⁷ The experimental evidence has indicated that chemokines and chemokine receptors contribute to the activation of glial cells in the dorsal horn of the spinal cord and participate in the process of bone cancer pain in rats. ³⁸ Studies have demonstrated that the expression of CXCL12/CXCR4 increases in rats with TCI-induced pain, but the specific mechanisms involved need to be elucidated. ³⁹ Therefore, we focused on the downstream pathways of CXCL12/CXCR4 on glial cell-mediated bone cancer pain. Possible CXCL12/CXCR4 downstream signals include the MAPKs, a family of serine/threonine protein kinases, ⁴⁰ such as ERK, JNK, and p38 MAPK, which can be activated by a wide variety of extracellular stimuli, like inflammatory factors and chemokines. c-Jun is a transcription factor in the nucleus, and a downstream effector of JNK. Studies have shown that c-Jun is involved in pain regulation, but to the best of our knowledge, no reports on its possible function in bone cancer pain exist.^41,42 On the basis of preliminary data, we hypothesized that the JNK-c-Jun signal pathway, functions as a downstream signal for CXCL12/CXCR4 in CIBP. We also explored the specific mechanism of the analgesic effect of esketamine, an NMDA receptor antagonist with analgesic and antidepressant effects, ²³ in bone cancer pain via the CXCL12/CXCR4-JNK-c-Jun signal pathway in the spinal cord.

CXCL12 is a chemokine with a molecular size of 6-14 kDa, and CXCR4 is the unique G protein-coupled receptor of CXCL12. ⁷ The classical CXCL12/CXCR4 signal axis has diverse biological functions and multiple downstream signaling pathways that can be activated to participate in cell proliferation, differentiation, migration, and chemotaxis. ⁸ During CIBP, CXCL12/CXCR4 activates microglia, astrocytes, or neurons in the spinal cord to mediate central sensitization and regulate neuropathic, inflammatory, and bone cancer pain, but the CXCL12/CXCR4 downstream signaling pathway for CIBP had remained unclear. ¹² In our research, the expressions of the CXCL12 and CXCR4 proteins began to increase 3 days after the TCI, reached their peak between days 7 and 14, and remained high in the spinal cord until day 21. CXCR4 and CXCL12 co-located with activated microglia and astrocytes on day 14 after the TCI, and these results are consistent with those of other studies. ³⁹ Moreover, intrathecal injection of AMD3100 also alleviated TCI-induced bone cancer pain. Thus, our results suggest that inhibiting CXCR4 in the microglia and astrocytes of the spinal cord alleviates central sensitization and alleviates TCI-induced bone cancer pain.

Studies have shown that the MAPK signal transmission system can transmit a wide range of extracellular stimulation signals into cells. ¹³ MAPK activation leads to transduction of various signals into the nucleus to promote the phosphorylation of various transcription factors that have roles in different pathophysiological processes such as pain formation, sensitization, and information transmission. ⁴³ In our research, the phosphorylation of MAPKs including ERK, JNK, and p38, began to increase in the spinal cord 3 days after TCI, reached their peak between days 7 and 14, and remained high until day 21. In addition, we showed that p-JNK and p-p38 MAPK were highly expressed in both microglia and astrocytes, while p-ERK was relatively less expressed in these two cells after TCI, which indicates that the activation of MAPKs in spinal glial cells was associated with the development of bone cancer pain. Also, we discovered that the increase in the p-JNK expression level was the largest among the those of the three p-MAPK molecules at the time points detected. Therefore, we made further efforts to explore the expression of the p-JNK-downstream transcription factor c-Jun. c-Jun is a nuclear transcription factor that regulates the expression of inflammatory factors, tumor proliferation, migration, and pain. ⁴⁴ Our results demonstrate that c-Jun was activated after TCI, and the phosphorylation of c-Jun and JNK occurred at similar points in time. In addition, our immunofluorescence results show that p-c-Jun was highly expressed in microglia and intrathecal injection of the JNK inhibitor SP600125 could downregulate the increments of p-JNK and p-c-Jun, which suggests that JNK regulates the expression of the downstream transcription factor c-Jun and is important for bone cancer pain. Moreover, the intrathecal injections of the CXCR4 inhibitor AMD3100 also significantly reduced the expressions of p-JNK and p-c-Jun in microglia, which suggests that JNK is an important downstream of effector of CXCR4 in the CIBP rat model. These results indicate that the released CXCL12 in activated spinal cord glial cells in rats with bone cancer pain binds the CXCR4 receptor to transmit signals to microglia that promote the phosphorylation of JNK, which further transmits signals to activate the c-Jun transcription factor in the nucleus; therefore, the CXCL12/CXCR4-JNK-c-Jun signal axis in the spinal cord seems to regulate the development of bone cancer pain.

NMDA receptor activation in the spinal cord is associated with the activation of microglia and astrocytes.^45,46 Esketamine is a dextral isomer of ketamine with stronger analgesic effects and it strongly blocks the NMDA receptor. ²³ Moreover, esketamine has some affinity for opioid receptors, and it has effects on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and brain derived neurotrophic factor (BDNF) signaling; however, we did not find reports showing inhibition of central glial cell activation and signal pathways to explain esketamine’s analgesic effects. ⁴⁷ Therefore, we explored whether esketamine can inhibit the sensitization of microglia and astrocytes in rats with bone cancer pain to inhibit the expression of signaling pathways and alleviate bone cancer pain. Our results show that intrathecal injections of esketamine relieved the TCI-induced pain and also significantly reduced the expression of p-JNK and p-c-Jun in microglia, without affecting the expressions of CXCL12/CXCR4 in the spinal cord of CIBP rats. CXCL12 exerts its functions upon specifically binding to CXCR4. Esketamine presumably inhibited their binding, making them unable to function and then inhibited p-JNK and p-c-Jun up-regulation. On the basis of these results, we propose that esketamine alleviates bone cancer pain by inhibiting the JNK-c-Jun signaling pathway in microglia.

In summary, our findings show that CXCL12/CXCR4 is a powerful molecular signaler during CIBP and also that the spinal JNK-c-Jun pathway is a downstream target of CXCL12/CXCR4-mediated central sensitization and cancer pain hypersensitivity. Moreover, esketamine significantly alleviates bone cancer pain by inhibiting the analgesic targets of JNK and c-Jun in the spinal cord.

ORCID iD

Lei Wei https://orcid.org/0009-0008-2951-8467