Differential Proteomic Analysis of the Spinal Cord in Bone Cancer Pain Rats by Two-Dimensional Gel Electrophoresis

Background: Bone cancer pain (BCP) is a common chronic pain that is caused by a primary or metastatic bone tumor. It is refractory to currently available clinical treatment owing to its complicated underlying mechanisms. Methods: In this study, we used proteomics approaches to investigate expressional changes of the rat spinal cord proteome from 7 to 21 d after inoculation. Proteins from the rat L4-6 spinal cord homogenates of BCP and Sham animals were fractionated by two-dimensional (2-DE) gel electrophoresis to produce a high-resolution map of the spinal cord soluble proteins. Proteins showing altered expression levels between BCP and Sham were selected. Results: A total of 60 spots were obtained, and isolated proteins were in-gel trypsin-digested and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Using the mass spectrometric data, 34 differentially expressed proteins (DEPs) were identified. GO analysis of the identified proteins allowed us to explore the function of the represented proteins. Conclusions: Based on these results, the identified proteins may contribute to the maintenance of BCP, and may provided new or valuable information in the discovery of new therapeutic targets for BCP.

Neuroligins Facilitate The Development of Bone Cancer Pain via Regulating Synaptic Transmission

Background The underlying mechanism of chronic pain involves the plasticity in synaptic receptors and neurotransmitters. This study aimed to investigate potential roles of neuroligins (NLs) within the spinal dorsal horn of rats in a newly established bone cancer pain (BCP) model. Methods Using our rat BCP model, we assessed pain hypersensitivity over time. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to investigate NL expression, and NLs were overexpressed in the rat spinal cord using lentiviral vectors. Immunofluorescence staining and whole-cell patch-clamp recordings were deployed to investigate the role of NLs in the development of BCP. Results We observed reduced expression levels of NL1 and NL2, but not NL3, within the rat spinal cord, which were found to be associated with and essential for the development of BCP in our model. Accordingly, NL1 or NL2 overexpression in the spinal cord alleviated mechanical hypersensitivity of rats. Electrophysiological experiments indicated that NL1 and NL2 are involved in BCP via regulating γ-aminobutyric acid-ergic interneuronal synapses and the activity of glutamatergic interneuronal synapses, respectively. Conclusions Our observations unravel the role of NLs in cancer-related chronic pain and further suggest that inhibitory mechanisms are central features of BCP in the spinal dorsal horn. These results provide a new perspective and basis for subsequent studies elucidating the onset and progression of BCP.

Curcumin Exerts Antinociceptive Effects in Cancer-Induced Bone Pain via an Endogenous Opioid Mechanism

Cancer pain is one of the main complications in advanced cancer patients, and its management is still challenging. Therefore, there is an urgent need to develop novel pharmacotherapy for cancer pain. Several natural products have attracted the interest of researchers. In previous studies, curcumin has proved to exhibit antitumor, antiviral, antioxidant, anti-inflammatory, and analgesic effects. However, the analgesic mechanism of curcumin has not been elucidated. Thus, in this study, we aimed to elucidate the antinociceptive potency and analgesic mechanism of curcumin in cancer-induced bone pain. Our results showed that consecutive curcumin treatment (30, 60, 120 mg/kg, i.p., twice daily for 11 days) produced significant analgesic activity, but had no effect on the progress of the bone cancer pain. Notably, pretreatment with naloxone, a non-selective opioid receptor antagonist, markedly reversed the antinociceptive effect induced by curcumin. Moreover, in primary cultured rat dorsal root ganglion (DRG) neurons, curcumin significantly up-regulated the expression of proopiomelanocortin (Pomc) and promoted the release of β-endorphin and enkephalin. Furthermore, pretreatment with the antiserum of β-endorphin or enkephalin markedly attenuated curcumin-induced analgesia in cancer-induced bone pain. Our present study, for the first time, showed that curcumin attenuates cancer-induced bone pain. The results also suggested that stimulation of expression of DRG neurons β-endorphin and enkephalin mediates the antinociceptive effect of curcumin in pain hypersensitivity conditions.

Rosiglitazone Alleviates Mechanical Allodynia of Rats with Bone Cancer Pain through the Activation of PPAR-γ to Inhibit the NF-κB/NLRP3 Inflammatory Axis in Spinal Cord Neurons

Bone cancer pain (BCP) is a serious clinical problem that affects the quality of life of cancer patients. However, the current treatment methods for this condition are still unsatisfactory. This study investigated whether intrathecal injection of rosiglitazone modulates the noxious behaviors associated with BCP, and the possible mechanisms related to this effect were explored. We found that rosiglitazone treatment relieved bone cancer-induced mechanical hyperalgesia in a dose-dependent manner, promoted the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in spinal cord neurons, and inhibited the activation of the nuclear factor-kappa B (NF-κB)/nod-like receptor protein 3 (NLRP3) inflammatory axis induced by BCP. However, concurrent administration of the PPAR-γ antagonist GW9662 reversed these effects. The results show that rosiglitazone inhibits the NF-κB/NLRP3 inflammation axis by activating PPAR-γ in spinal neurons, thereby alleviating BCP. Therefore, the PPAR-γ/NF-κB/NLRP3 signaling pathway may be a potential target for the treatment of BCP in the future.

Transcriptomic Analysis of Long Noncoding RNA and mRNA Expression Profiles in the Amygdala of Rats with Bone Cancer Pain-Depression Comorbidity

Bone cancer pain (BCP)–depression comorbidity has become a complex clinical problem during cancer treatment; however, its underlying molecular mechanisms have not been clarified. Several long noncoding RNAs (lncRNAs) have been demonstrated to be promising therapeutic targets in depression, but research on the role of lncRNAs in BCP–depression comorbidity has been limited. Therefore, high-throughput RNA sequencing was performed to detect differentially expressed profiles in the amygdala of a BCP–depression rat model in this study. We detected 330 differentially expressed mRNAs (DEmRNAs) and 78 differentially expressed lncRNAs (DElncRNAs) in the BCP–depression comorbidity model and then verified the expression of six DEmRNAs and six DElncRNAs with the greatest degrees of difference by RT-qPCR. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that differentially expressed genes were strongly enriched in inflammatory and immunologic systemic responses. Then the nuclear factor kappa B (NF-κB) signaling pathway and the Th17 differentiation pathway showed significant differences, as determined by Western blot analysis. Finally, we constructed a protein–protein interaction (PPI) network to explore the potential regulatory mechanism of DEmRNAs. In conclusion, our study reveals a new resource for the understanding of dysregulated lncRNAs and mRNAs in BCP–depression comorbidity and provides novel potential therapeutic targets for further approaches.

Metformin Attenuates Bone Cancer Pain by Reducing TRPV1 and ASIC3 Expression

Bone cancer pain (BCP) is a common pathologic pain associated with destruction of bone and pathological reconstruction of nervous system. Current treatment strategies in clinical is inadequate and have unacceptable side effects due to the unclear pathology mechanism. In the present study, we showed that transplantation of Walker 256 cells aggravated mechanical allodynia of BCP rats (**p < 0.01 vs. Sham), and the expression of ASIC3 (Acid-sensitive ion channel 3) and TRPV1 was obviously enhanced in L4-6 dorsal root ganglions (DRGs) of BCP rats (**p < 0.01 vs. Sham). ASIC3 and TRPV1 was mainly expressed in CGRP and IB4 positive neurons of L4-6 DRGs. While, TRPV1 but not ASIC3 was markedly upregulated in L4-6 spinal dorsal horn (SDH) of BCP rats (**p < 0.01 vs. Sham). Importantly, intrathecal injection of CPZ (a TRPV1 inhibitor) or Amiloride (an ASICs antagonist) markedly increased the paw withdraw threshold (PWT) of BCP rats response to Von Frey filaments (**p < 0.01 vs. BCP + NS). What’s more, intraperitoneally injection of Metformin or Vinorelbine markedly elevated the PWT of BCP rats, but reduced the expression of TRPV1 and ASIC3 in L4-6 DRGs and decreased the TRPV1 expression in SDH (*p < 0.05, **p < 0.01 vs. BCP + NS). Collectively, these results suggest an effective analgesic effect of Metformin on mechanical allodynia of BCP rats, which may be mediated by the downregulation of ASIC3 and TRPV1.