The proteasome inhibitor bortezomib (BTZ) is a potent first-line anticancer drug for multiple myeloma; nonetheless, it induced peripheral neuropathy. It has been suggested that many cytokines may play a role in mediating neuropathic pain, but the underlying molecular mechanism is not fully understood. Recent studies have shown that neuropathic pain is closely related to the purinergic ligand-gated ion channel 7 receptor (P2X7R), one of the P2X receptors, which is richly expressed in glial cells. P2X7-p38 pathway is correlated with microglia- and satellite glial cell- (SGC-) mediated neuropathic pain. However, the association of P2X7R and p38MAPK in mediating BTZ-induced neuropathic pain remains unclear. In this study, the relationship between P2X7R activation and p38 phosphorylation in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) in the development and maintenance of BTZ-induced neuropathic pain was elucidated. The results showed that BTZ increased mechanical thresholds in rats, accompanied with upregulation of P2X7R expression and p38MAPK phosphorylation, indicating that P2X7R and p38MAPK are key molecules in the development and maintenance of BTZ-induced neuropathic pain. Inhibiting p38MAPK phosphorylation with SB203580 resulted in downregulation of P2X7R expression levels. Inhibition of P2X7R with Brilliant Blue G (BBG) reversed neuropathic pain might decrease through the expression of tumor necrosis factor-
Proteasome inhibitors used for treating cancers have been identified to result in a significant higher risk for inducing sensory peripheral neuropathy [
Neuroinflammation is one of the major contributors to the initiation of BIPN; as most recently, it has been shown that persistent neuroinflammation and progressive structural damage of the dorsal root ganglion (DRG) in addition to neuroinflammation-related spinal cord sensitization were associated with development of BIPN in mice [
Primary DRG neurons transmit sensory information about pain, in which ATP-dependent P2X receptors are richly expressed [
The role of P2X7R in pain has been demonstrated in animal models of neuropathic and inflammatory pain [
Although by increasing surveillance and earlier dose adjustment of the proteasome inhibitor could minimize the risk of sensory peripheral neuropathy in clinical trials [
The Wistar rats were from Shandong University Experimental Animal Center. All animal experimental procedures were in accordance with the 8th Edition of the
A total of 40 male Wistar rats were used in this study. Twenty rats were randomly divided into the following 4 groups (each group consisted of 5 rats). (1) Control group: rats received peritoneal injection (i.p.) of 0.9% NaCl at the equivalent volume the other groups. (2) Brilliant Blue G (BBG) group: rats received BBG (50 mg/kg body weight, i.p.) at a concentration of 10 mg/ml. BBG is a P2X7R inhibitor which was used in this experiment aimed to clarify whether this inhibitor alone affects the experimental results. (3) BTZ group: rats received BTZ (0.2 mg/kg body weight, i.p.) at a concentration of 0.5 mg/ml for 5 successive days. (4) BTZ+BBG group: rats received BBG (50 mg/kg body weight, i.p.) 30 min before BTZ (0.2 mg/kg body weight, i.p.) administration with the corresponding concentration. Another 20 rats were randomly divided into the following 4 groups (each group of 5 rats). (1) Control group: rats received intrathecal injection (i.t.) of 0.9% NaCl at the equivalent volume the other groups. (2) SB203580 group: rats received SB203580 (10
BTZ (0.2 mg/kg, 0.5 mg/ml) and P2X7R antagonist BBG (50 mg/kg, 10 mg/ml) were administered via peritoneal injection (i.p.) according to the above experimental design. p38MAPK inhibitor SB203580 (10
The pain thresholds of mechanical stimulation were measured with von Frey filaments before (day 0) and after (days 2, 4, 6, 8, and 10) BTZ treatment. The protocol of the mechanical allodynia behavior test was similar to our previous reports [
After treatment with different reagents and the last mechanical simulation behavior test finished, fresh L4-6 DRG and the corresponding segment SDH tissue were removed from each animal under anesthesia. Total RNA of DRG and SDH tissue was extracted with TRIzol. cDNA was synthesized with a cDNA synthesis kit (Thermo Scientific). Quantitative PCR was performed with SYBR Green master mixes (Cwbiotech), and amplification was performed with synthetic oligonucleotide primers. The PCR reaction was performed at 55°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles at 94°C for 5 seconds, 58°C for 30 seconds, and 72°C for 45 seconds and the final results carried out by using the 2-
The sequences of oligonucleotide primers.
Genes | Primer sequences |
---|---|
P2X7R | 5 |
5 | |
IL-1 | 5 |
5 | |
IL-6 | 5 |
5 | |
TNF- | 5 |
5 | |
p38 | 5 |
5 | |
GAPDH | 5 |
5 |
Upon L4-6 DRG and the corresponding segment SDH tissue removal, the DRG and SDH tissue was prepared for Western blot assay for detecting the variation of the targeting proteins. DRG and SDH tissue was lysed in RIPA buffer (Beyotime Biotechnology) containing protease and phosphatase inhibitors (Roche) on ice for 20 minutes and removed to a refrigerated centrifuge at 12000 rpm for 15 minutes to collect supernatant. The protein samples (50
The antibodies for immunoblotting.
Category | Antibodies | Concentration | Source |
---|---|---|---|
Primary | Rabbit anti-p-p38 monoclonal IgG | 1 : 1000 | Cell Signaling Technology, Danvers, MA |
Primary | Rabbit anti-P2X7R monoclonal IgG | 1 : 1000 | Alomone Labs, Jerusalem, Israel |
Primary | Rabbit anti-GAPDH monoclonal IgG | 1 : 1000 | Cell Signaling Technology, Danvers, MA |
Primary | Mouse anti- | 1 : 5000 | Cell Signaling Technology, Danvers, MA |
Secondary | Goat anti-rabbit IgG-HRP | 1 : 5000 | Beijing Sequoia Jinqiao Biological Technology Co., Ltd., Beijing, China |
Secondary | Goat anti-mouse IgG-HRP | 1 : 5000 | Beijing Sequoia Jinqiao Biological Technology Co., Ltd., Beijing, China |
After treatment with different reagents and the last mechanical simulation behavior test finished, the animals were anaesthetized and perfused with cold (4°C) 4% paraformaldehyde (pH 7.4). L4-6 DRG and the corresponding segment SDH tissue were removed from each animal. DRG and SDH tissue slide of 15
The antibodies for fluorescence labeling.
Category | Antibodies | Concentration | Source |
---|---|---|---|
Primary | Rabbit anti-P2X7R monoclonal IgG | 1 : 400 | Alomone Labs, Jerusalem, Israel |
Primary | Rabbit anti-p-p38 monoclonal IgG | 1 : 100 | Cell Signaling Technology, Danvers, MA |
Primary | Chicken anti-NF-200 monoclonal IgG | 1 : 1000 | Cell Signaling Technology, Danvers, MA |
Primary | Mouse anti-F4/80 monoclonal IgG | 1 : 100 | Santa Cruz Biotechnology, Santa Cruz, CA |
Primary | Mouse anti-Iba-1 monoclonal IgG | 1 : 200 | Abcam, Cambridge, MA |
Primary | Mouse anti-GFAP monoclonal IgG | 1 : 500 | Abcam, Cambridge, MA |
Secondary | Goat anti-rabbit IgG-TRITC | 1 : 200 | Beijing Sequoia Jinqiao Biological Technology Co., Ltd., Beijing, China |
Secondary | Goat anti-chicken IgG-FITC | 1 : 200 | Beijing Sequoia Jinqiao Biological Technology Co., Ltd., Beijing, China |
Secondary | Goat anti-mouse IgG-TRITC | 1 : 200 | Beijing Sequoia Jinqiao Biological Technology Co., Ltd., Beijing, China |
Secondary | Goat anti-mouse IgG-FITC | 1 : 500 | Beijing Sequoia Jinqiao Biological Technology Co., Ltd., Beijing, China |
To clarify BTZ injection on mechanical allodynia behavior in rats, the mechanical thresholds of the bilateral plantar surface were measured by using the von Frey filament test before (0 day) and after 2, 4, 6, 8, and 10 days from the first BTZ injection. The mechanical threshold was decreased to a much lower point after 10 days from the first BTZ injection (Figure
Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells.
To investigate the successive expression pattern of P2X7R in DRG after BTZ administration, P2X7R expression in DRG tissue from rats after 2, 4, 6, 8, and 10 days from the first BTZ injection was analyzed with Western blot. P2X7R protein elevation was observed from 2 days after BTZ injection and gradually upregulated to the highest levels between 8 and 10 days after BTZ injection (Figures
To determine what cell types express P2X7R and p-p38 in DRG and SDH, we carried out a series of experiments by using DRG and SDH tissue sections with double immunofluorescence labeling technique to approach these questions. In DRG tissue sections, P2X7R and GFAP (a satellite glial cell marker) double fluorescence labeling showed that P2X7R was coexpressed with GFAP, whereas P2X7R and NF-200 (a neuronal marker) double fluorescence labeling showed that P2X7R was not coexpressed with NF-200 (Figure
To clarify p38 phosphorylation in DRG and SDH after BTZ treatment with P2X7R inhibition, BTZ-treated rats received P2X7R inhibitor BBG. After 10 days of the first BTZ injection, DRG and SDH tissue was removed from each animal. The p38 mRNA, p-p38 protein levels, and p-p38 expression in situ in DRG or SDH tissue were analyzed with quantitative real-time PCR, Western blot, and fluorescence labeling, respectively.
In DRG, BTZ treatment triggered a significant higher upregulation of p38 mRNA levels (Figure
p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density.
In SDH, p38 mRNA levels (Figure
p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density.
Inhibition of P2X7R with a P2X7R inhibitor BBG in BTZ-treated rats significantly decreased p38 mRNA and p-p38 expression in SDH tissue. Administration of BBG alone in naïve rats did not affect p38 mRNA and p-p38 expression in SDH tissue. These results implied that the activation of P2X7R in microglia of SDH is closely related to p38 phosphorylation in microglia of SDH. This might be another novel mechanism clarified in this experiment for the first time in BTZ-induced painful peripheral neuropathy.
It has been shown that inflammatory cytokines such as IL-1
IL-1
To determine whether P2X7R is expressed in DRG and SDH after BTZ treatment with p38 inhibition, the p38 selective inhibitor SB203580 was injected into BTZ-treated rats. The P2X7R expression in DRG or SDH tissue was analyzed with quantitative real-time PCR, Western blot, and fluorescence labeling, respectively.
In DRG, BTZ treatment triggered a significant higher upregulation of P2X7R mRNA levels (Figure
P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density.
In SDH, BTZ treatment induced a significant upregulation of P2X7R mRNA levels (Figure
P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density.
In this study, IL-1
IL-1
In the present study, a model of BTZ was used to stimulate a state of neuropathic pain. Applying behavioral measurements, we found that, compared with the BTZ group, intraperitoneal administration of the P2X7R selective inhibitor BBG before BTZ application could significantly improve the paw withdrawal thresholds for mechanical stimuli (Figure
Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38.
Peripheral neuropathy is an obvious side effect of BTZ and a key factor leading to neurotoxicity. Although hyperalgesia was initially thought to be caused by changes in the activity of primary sensory neurons or neurons in the spinal cord, evidence has indicated that glial cells may also play a vital role in the development of pain. Nerve injury can cause spinal microglia P2X7R activation; as a result, the expression of inflammatory factors can be downregulated to reduce the pain-relevant behavioral hypersensitivity after P2X7R-specific inhibitor BBG interference [
The key step of p38MAPK phosphorylation in the central nervous system [
This study confirmed that BTZ can significantly activate P2X7R in SGCs of DRG, and P2X7R expression is consistent with the change of mechanical threshold in neuropathic pain rats.
Inhibition of P2X7R by intraperitoneal injection of BBG can result in downregulation of p-p38MAPK expression levels. Intrathecal administration of the p38MAPK inhibitor SB203580 significantly reduced the release of inflammatory factors, but had no effect on P2X7R expression. P2X7R association with p38MAPK in mediating inflammatory cytokine upregulation in DRG contributes to BTZ-induced mechanical allodynia represents a unique pharmacological property of P2X7R in mediating BTZ-induced neuropathic pain. Our results also showed that P2X7R is expressed specifically in microglial cells of SDH. Selective P2X7R antagonist BBG interference can obviously reduce the phosphorylation of p38MAPK in SDH. Inhibition of p38MAPK phosphorylation with SB203580 can result in downregulation of P2X7R expression levels in microglial cells of SDH. The association of P2X7R activation and p38MAPK phosphorylation in microglial cells of SDH also contributes to the development and progression of BTZ-induced neuropathic pain.
The evidence from clinical trial of patients with BTZ treatment [
BTZ increased mechanical thresholds in rats, accompanied with upregulation of P2X7R expression and p38MAPK phosphorylation, indicating P2X7R and p38MAPK are key molecules in the development and maintenance of BTZ-induced neuropathic pain. Blocking p38MAPK phosphorylation with SB203580 can result in downregulating P2X7R expression levels. The application of P2X7R inhibitor BBG reversed neuropathic pain might decrease through the expression of TNF-
The data used to support the findings in this study are available from the corresponding author upon reasonable request.
The experimental procedures were approved by the Animal Experimentation Ethical Committee of Shandong University (Document No. ECSBMSSDU-2018-2-007).
The authors declare no conflict of interest.
Yan Guo, Zhenzhong Li, and Zhen Liu conceived this study, designed the experiments, and wrote the manuscript. Yan Guo carried out the behavior test, PCR, Western blot assay, and immunofluorescence staining. Xiaobo Xu and Jingyi Huang helped to carry out PCR and Western blot assay and revised the manuscript. Zhen Wang helped to carry out the behavior tests and immunofluorescence staining and prepared the reagents and solutions used in the experiments. All authors read and approved the final manuscript.
This research was supported by the Shandong Provincial Key Research and Development Project in China (No. 2019GSF107062).