Mouse Model Of Bone Cancer Pain Research Articles

Spinal sirtuin 2 attenuates bone cancer pain by deacetylating FoxO3a

Bone cancer pain (BCP) is refractory to currently used analgesics. Recently, sirtuin 2 (SIRT2) was reported to play a vital role in neuropathic pain but its role in BCP remains unknown. It was hypothesized that spinal SIRT2 attenuates BCP by deacetylating FoxO3a and suppressing oxidative stress. The mouse model of BCP established by injecting tumor cells into the intramedullary space of the femur demonstrated that spinal SIRT2 and FoxO3a were downregulated in BCP development. Intrathecal administration of LV-SIRT2 reduced pain hypersensitivity (mechanical and thermal nociception) in BCP mice. Spinal SIRT2 overexpression upregulated FoxO3a and antioxidant genes (SOD2 and catalase) and inhibited FoxO3a acetylation, phosphorylation, and ubiquitination. Moreover, intrathecal administration of SIRT2 shRNA induced pain hypersensitivity in normal mice. Spinal SIRT2 knockdown downregulated FoxO3a and antioxidant genes and increased FoxO3a acetylation, phosphorylation, and ubiquitination. In summary, spinal SIRT2 increases FoxO3a expression in BCP mice and inhibits oxidative stress by deacetylating FoxO3a and further reducing FoxO3a phosphorylation, ubiquitination, and degradation, leading to BCP relief.

Inhibition of spinal ferroptosis-like cell death alleviates hyperalgesia and spontaneous pain in a mouse model of bone cancer pain

Bone cancer pain (BCP) impairs patients’ quality of life. However, the underlying mechanisms are still unclear. This study investigated the role of spinal interneuron death using a pharmacological inhibitor of ferroptosis in a mouse model of BCP. Lewis lung carcinoma cells were inoculated into the femur, resulting in hyperalgesia and spontaneous pain. Biochemical analysis revealed that spinal levels of reactive oxygen species and malondialdehyde were increased, while those of superoxide dismutase were decreased. Histological analysis showed the loss of spinal GAD65+ interneurons and provided ultrastructural evidence of mitochondrial shrinkage. Pharmacologic inhibition of ferroptosis using ferrostatin-1 (FER-1, 10 mg/kg, intraperitoneal for 20 consecutive days) attenuated ferroptosis-associated iron accumulation and lipid peroxidation and alleviated BCP. Furthermore, FER-1 inhibited the pain-associated activation of ERK1/2 and COX-2 expression and prevented the loss of GABAergic interneurons. Moreover, FER-1 improved analgesia by the COX-2 inhibitor Parecoxib. Taken together, this study shows that pharmacological inhibition of ferroptosis-like cell death of spinal interneurons alleviates BCP in mice. The results suggest that ferroptosis is a potential therapeutic target in patients suffering on BCP and possibly other types of pain.

USP48 alleviates bone cancer pain and regulates MrgC stabilization in spinal cord neurons of male mice.

Ubiquitin-mediated degradation of the Mas-related G protein-coupled receptor C (MrgC) reduces the number of receptors. However, the specific deubiquitinating enzyme antagonize this process has not been reported. In this study, we investigated the effect of ubiquitin-specific protease-48 (USP48) on bone cancer pain (BCP) and its effect on MrgC. A mouse model of BCP was established. BCP behaviours of mice were assessed after intrathecal injection of adeno-associated virus (AAV)-USP48. USP48 and MrgC interactions were studied by immunoprecipitation. Overexpression and knockdown of USP48 were conducted in N2a cells to investigate the effect of USP48 on MrgC receptor number and ubiquitination. Spinal cord level USP48 expression was reduced in mice with BCP. Intrathecal injection of AAV-USP48 increased paw withdrawal mechanical threshold and reduced spontaneous flinching in mice. In N2a cells, there were increased number of MrgC receptors after overexpression of USP48 and decreased number of MrgC receptors after knockdown of USP48. USP48 interacted with MrgC and overexpression of USP48 altered the level of ubiquitination of MrgC. USP48 antagonizes ubiquitin-mediated autophagic degradation of MrgC and alleviates BCP in a murine animal model. Our findings may provide a new perspective for the treatment of BCP. Our finding may provide an important theoretical basis as well as an intervention target for clinical development of drugs for BCP.

MiR-135-5p Alleviates Bone Cancer Pain by Regulating Astrocyte-Mediated Neuroinflammation in Spinal Cord through JAK2/STAT3 Signaling Pathway

Bone cancer pain (BCP) was associated with microRNA dysregulation. In this study, we intended to clarify the potential role of miR-135-5p in a BCP mouse model, which was established by tumor cell implantation (TCI) in the medullary cavity of the mouse femur. The BCP-related behaviors were tested, including the paw withdrawal mechanical threshold (PWMT) and number of spontaneous flinches (NSF). The miRNA expression profiles in astrocytes of the sham and tumor groups were compared, and miRNA microarray and quantitative real-time PCR (qRT-PCR) assays confirmed that the amount of expression of miR-135-5p was significantly decreased in astrocytes of the tumor group. Gain- and loss-of-function studies showed that miR-135-5p could inhibit astrocyte activation and inflammation cytokine (TNF-α and IL-1β) expression. The relation between miR-135-5p and JAK2 was detected by bioinformatic analysis and dual luciferase reporter gene assay. By conducting in vitro experiments, it was shown that the miR-135-5P mimics lowered the level of JAK2/STAT3 proteins and inflammatory factors in astrocytes. Moreover, in vivo analysis on BCP mice model indicated that the miR-135-5p agonist could sufficiently increase PWMT and decrease NSF. Meanwhile, reduced activation of astrocytes in the spinal cord, as well as decreased expression of JAK2/STAT3 and inflammatory mediators, were found after miR-135-5p agonist treatment. Collectively, the results showed that miR-135-5p could potentially reduce BCP in mice through inhibiting astrocyte-mediated neuroinflammation and blocking of the JAK2/STAT3 signaling pathway, indicating that the upregulation of miR-135-5P could be a therapeutic focus in BCP treatment.

Retraction Note to: Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model.

The authors have retracted this article [1] because in further experiments, they found that some experimental data cannot be verified repeatedly.

Spinal cannabinoid receptor 2 activation reduces hypersensitivity associated with bone cancer pain and improves the integrity of the blood–spinal cord barrier

BackgroundDisruption of the blood–spinal cord barrier (BSCB) can facilitate inflammation that results in pain hypersensitivity. Proinflammatory cytokines produced by activated microglia and astrocytes damage the BSCB. This study aims to...

Correction to: Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model.

The original version of this article unfortunately contained a mistake in Fig. 2.

Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model.

Altered expression of circular RNA (circRNA) is recognized as a contributor to malignant pain where microRNA (miRNA) exerts an essential effect. We generated a murine model for bone malignancy pain in which 2472 osteolytic sarcoma cells were injected into the femurs of mice. CircRNA microarray and quantitative PCR (qPCR) and revealed that circ9119 expression was repressed in the spinal cord of bone malignancy pain model mice, which is the first relay site involved in the transmission of nociceptive information to the cerebrum of mice that receive spinal analgesics for malignancy pain. Overexpression of circ9119 by plasmid injection in the model mice reduced progressive thermal hyperalgesia and mechanical hyperalgesia. Bioinformatics prediction and dual-luciferase reporter assay showed that circ9119 functions as a sponge of miR-26a, which targets the TLR3 3'-untranslated region. Furthermore, expression of miR-26a was elevated and TLR3 level was repressed in bone malignancy pain model mice, which were counteracted by circ9119 in the spinal cord of tumor-bearing mice. Moreover, excessive expression of miR-26a was involved in the recovery of mice from progressive thermal hyperalgesia and mechanical hyperalgesia triggered via circ9119. TLR3 knockdown in bone malignancy pain model mice thoroughly impaired pain in the initial stages and reduced the effects of circ9119 on hyperalgesia. Our research findings indicate that targeting the circ9119-miR-26a-TLR3 axis may be a promising analgesic strategy to manage malignancy pain.

Imbalanced spinal infiltration of Th17/Treg cells contributes to bone cancer pain via promoting microglial activation

Increasing evidence suggests that T cells participate in the pathology of neuropathic pain, as well as the activation of microglia. However, whether T cells infiltrate into the spinal cord and contribute to the development of bone cancer pain (BCP) remains unknown. Here, we used a mouse model of BCP to show that numbers of T cells infiltrated into the spinal cord after sarcoma cell implantation with increased BCP, and most infiltrating T cells in the spinal cord were CD3+CD4+ T cells. Both Th17 and Treg subpopulations were analyzed by immunofluorescence. Treg cells in the spinal cord were transiently up-regulated, followed by an imbalance towards Th17 afterwards, and elevated IL-17/IL-17A levels were observed in both blood and spinal cord. Meanwhile, TGF-β, IL-6, and IL-23, the factors which regulate Th17/Treg differentiation, increased their expressions during the development of BCP. Additionally, IL-17A receptor (IL-17AR) was found to be expressed on microglia, and the level of IL-17AR increased with activated microglia during BCP development. Furthermore, BCP was ameliorated when IL-17/IL-17A neutralizing antibodies were intrathecally injected, accompanied with inhibited Th17/Treg infiltration and suppressed microglial activation. In conclusion, T cells infiltrated into the spinal cord with the imbalance of Th17/Treg towards Th17 during the development of BCP, which could promote the microglial activation and further increased BCP, while neutralizing IL-17/IL-17A in the spinal cord could ameliorate BCP. Our results suggest that targeting the imbalanced Th17/Treg infiltration in the spinal cord could be a novel strategy for BCP therapy.

MicroRNA-329 reduces bone cancer pain through the LPAR1-dependent LPAR1/ERK signal transduction pathway in mice.

Background:Bone cancer pain (BCP) is a common symptom occurring among patients with cancer and has a detrimental effect on their quality of life. Growing evidence has implicated microRNA-329 (miR-329) in the progression of bone diseases. In the present study, we aimed to elucidate the potential effects of miR-329 on BCP in a BCP mouse model via binding to lysophosphatidic acid receptor 1 (LPAR1) through the LPAR1/extracellular signal-regulated kinase (ERK) signaling pathway.Methods:Initially, a BCP mouse model was established via injection of 4 × 104 murine breast tumor (4T1 cell) cells (4 μl). The interaction between miR-329 and LPAR1 was identified using a bioinformatics website and dual luciferase reporter gene assay. The modeled mice were subsequently treated with miR-329 mimic, LPAR1 shRNA, or both, in order to examine the effect of miR-329 on the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of mice, the expression of LPAR1/ERK signaling pathway-related genes.Results:The positive expression rate of LPAR1 protein and extent of ERK1/2 phosphorylation were increased in BCP mouse models. LPAR1 is a target gene of miR-329, which can inhibit the expression of LPAR1. In response to miR-329 overexpression and LPAR1 silencing, BCP mice showed increased PWT and PWL, along with decreased LPAR1 expression and ratio of p-ERK/ERK.Conclusions:Altogether, the results obtained indicated that miR-329 can potentially alleviate BCP in mice via the inhibition of LPAR1 and blockade of the LPAR1/ERK signaling pathway, highlighting that upregulation of miR-329 could serve as a therapeutic target for BCP treatment.

Inhibition of Connexin 43 and Phosphorylated NR2B in Spinal Astrocytes Attenuates Bone Cancer Pain in Mice.

Bone cancer pain (BCP) is common in patients with advanced cancers when the tumors are metastasized to bone. The limited understanding of the complex pathogenesis of BCP leads to the poor effectiveness of clinical treatment. Previous studies have shown that astrocyte-specific connexin (Cx) 43, a forming protein of gap junction (GJ) and hemichannel, and N-methyl-D-aspartate receptors (NMDARs), especially the phosphorylated NMDAR 2B subunit (NR2B) phosphorylated NR2B (p-NR2B) subunit are involved in BCP. However, the relationship between Cx43 and p-NR2B in BCP remains unclear. In the present study, we investigated the expressions of Cx43, glial fibrillary acidic protein (GFAP, a marker of astrocytes), and p-NR2B in the spinal dorsal horn (SDH) in a mouse model of BCP established by intra-femural inoculation of Lewis lung carcinoma (LLC) cells via intrathecal (ith) injection of the GJ/hemichannel blocker carbenoxolone (CARB) and the NMDAR antagonist MK801, respectively. We found that the characters of BCP were mimicked by intra-femural inoculation of LLC cells in mice, and the expressions of Cx43, GFAP and p-NR2B in BCP mice were remarkably increased in a time-dependent manner from day 7 to day 21 after cell inoculation with a gradual aggravate in spontaneous pain and mechanical allodynia. Furthermore, Cx43 was predominantly expressed in the spinal astrocytes. Both CARB and MK801 inhibited the expressions of Cx43, GFAP and p-NR2B with attenuated pain hypersensitivity in BCP mice. In addition, Cx43 was co-localized with p-NR2B in the SDH, which further evidenced the presence of functional NR2B in the spinal astrocytes in BCP mice. Our findings demonstrate that inhibition of Cx43 and p-NR2B in spinal astrocytes could attenuate BCP in mice and Cx43 and p-NR2B in the astrocytes of the SDH may play an important role via their combination action in the development and maintenance of BCP in mice. These results may provide a potential therapeutic target in the prevention and/or treatment of BCP.

Blockage of spinal endothelin A receptors attenuates bone cancer pain via regulation of the Akt/ERK signaling pathway in mice

Bone cancer pain (BCP) is a common source of pain in patients with advanced stage and metastatic cancer; however, existing treatment for this kind of pain remains deficient. Being closely related to sensory change and inflammatory pain in both the central and peripheral nervous systems, endothelin A receptor (ETAR) plays an essential role in pain processing. As a result, ETAR antagonist has been reported to alleviate both neuropathic and inflammatory pain. Thus far, the role of ETAR in the process of BCP is still ambiguous. In this study, by using a BCP mouse model, the analgesic effect and molecular mechanism of the ETAR antagonist BQ-123 was investigated. Pain sensation in the BCP mouse model was investigated by the number of spontaneous flinches (NSF) and pain withdrawal threshold (PWT), and the mechanism of BCP was assessed by measuring p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 levels in L4–6 segments of the spinal cord. Our results demonstrated that BCP mice showed a higher NSF and a lower PWT score than Sham mice. In addition to the development of nociceptive sensitization, p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 were up-regulated correspondingly in L4–6 segments of the spinal cord in BCP mice. BQ-123 treatment showed a promising analgesic effect, and the effect was correlated to the down-regulation of p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 in spinal cord cells. The results suggested that intrathecal administration of BQ-123 was able to relieve BCP in mice as a consequence of suppressing the Akt and ERK signalling pathways.

MicroRNA-93-5p may participate in the formation of morphine tolerance in bone cancer pain mouse model by targeting Smad5.

ObjectiveIn this study, we aim to find out the role of microRNA-93-5p (miR-93) and Smad5 in morphine tolerance in mouse models of bone cancer pain (BCP).ResultsAt 7 days after injection of morphine, the PMWT showed no significant difference between the morphine model group and the saline model group (P < 0.05), suggesting that morphine tolerance had formed in the morphine model group. The morphine model group had higher miR-93 expression and lower Smad5 mRNA expression than the saline model group. Smad5 is a downstream target gene of miR-93. At 7, 9 and 14 days after injection of lentiviruses, the L/anti-miR-93 group had the lowest PMWTs, while the Smad5 shRNA group presented the highest PMWTs among these five groups (all P < 0.05).MethodsWe built mouse models of BCP and morphine tolerance and recorded 50% PMWT. After 6 days of modeling, we set saline control group, morphine control, saline model group and morphine model group (morphine tolerance emerged). We performed luciferase reporter gene assay to verify the relation between miR-93 and Smad5. After lentivirus transfection, the mice with morphine tolerance were assigned into L/anti-miR-93 group, Smad5 shRNA group, L/anti-miR-93 + Smad5 shRNA group, blank group and PBS control group. RT-qPCR, Western Blot assay and immumohistochemical staining were performed to observe the changes of miR-93 and Smad5.ConclusionUp-regulation of miR-93 may contribute to the progression of morphine tolerance by targeting Smad5 in mouse model of BCP.

Mas-Related Gene (Mrg) C Activation Attenuates Bone Cancer Pain via Modulating Gi and NR2B.

ObjectiveThis study is to investigate the role of Mas-related gene (Mrg) C in the pathogenesis and treatment of bone cancer pain (BCP).MethodsBCP mouse model was established by osteosarcoma cell inoculation. Pain-related behaviors were assessed with the spontaneous lifting behavior test and mechanical allodynia test. Expression levels of MrgC, Gi, and NR2B in the spinal cord were detected with Western blot analysis and immunohistochemistry.ResultsPain-related behavior tests showed significantly increased spontaneous flinches (NSF) and decreased paw withdrawal mechanical threshold (PWMT) in mouse models of BCP. Western blot analysis showed that, compared with the control group and before modeling, all the expression levels of MrgC, Gi, and NR2B in the spinal cord of BCP mice were dramatically elevated, which were especially increased at day 7 after operation and thereafter, in a time-dependent manner. Moreover, the treatment of MrgC agonist BAM8-22 significantly up-regulated Gi and down-regulated NR2B expression levels, in the spinal cord of BCP mice, in a time-dependent manner. On the other hand, anti-MrgC significantly down-regulated Gi expression, while dramatically up-regulated NR2B expression, in the BCP mice. Similar results were obtained from the immunohistochemical detection. Importantly, BAM8-22 significantly attenuated the nociceptive behaviors in the BCP mice.ConclusionOur results indicated the MrgC-mediated Gi and NR2B expression alterations in the BCP mice, which might contribute to the pain hypersensitivity. These findings may provide a novel strategy for the treatment of BCP in clinic.

Effect of Tanshinone IIA intrathecal injections on pain and spinal inflammation in mice with bone tumors.

The study aimed to investigate the effect of intrathecal injections of Tanshinone IIA on thermal hyperalgesia in a mouse model of bone cancer-pain. Spinal IL-1β, IL-6, TNF-α expression levels were analyzed. C3H/HeNCrlVr male mice were assigned to groups that either received dose-dependent injections of Tanshinone IIA, or the DMSO + Sham, Tanshinone IIA + Sham, DMSO + Tumor, and Control groups. Paw withdrawal thermal latency (PWTL) was measured with a radiant heat stimulus and mRNA expression levels were determined using real-time PCR. Fourteen days post-injection, PWTL in the DMSO + Tumor group was lower than that in the controls (P < 0.05). Twenty-one days post-injection, compared with the Control group, there was no significant difference in PWTL and IL-1β, IL-6, and TNF-α expression levels between the Tanshinone IIA + Sham and DMSO + Sham groups (P > 0.05). PWTL in the DMSO + Tumor group was significantly lower than the Control group (P < 0.05), while the expression levels of IL-1β, IL-6, and TNF-α were significantly higher than controls. Compared with the DMSO + Tumor group, PWTLs were higher in the Tanshinone IIA - 20-μg and 40-μg groups, while expression levels of IL-1β, IL-6, and TNF-α were significantly lower (P < 0.05). These measures were not significantly different between the Tanshinone IIA 10 μg and the DMSO + Tumor groups (P > 0.05). In conclusion, Tanshinone IIA may inhibit the release of inflammatory cytokines, such as, IL-1 β, IL-6 α, TNF-α.

Studies on the analgesic activities of Jia-Yuan-Qing pill and its safety evaluation in mice

The analgesic activity of Porcellio laevis Latreille, Rhizoma Corydalis, and Radix Cynanchi Paniculati have been reported in recent years. A new formula named Jia-Yuan-Qing pill (JYQP) is therefore created by combining the three herbs at 9:7:7 ratio according to traditional Chinese theories. The present study aims to evaluate the effect of JYQP as a novel painkiller in various models. Acute toxicity test was applied to evaluate the safety of JYQP. Acetic-acid-induced writhing, hot plate test, formalin test, and naloxone-pretreated writhing test were employed to elaborate the analgesic activity of JYQP and its possible mechanism. A bone cancer pain mouse model was performed to further assess the effect of JYQP in relieving cancer pain. Test on naloxone-precipitated withdrawal symptoms was conduct to examine the physical dependence of mice on JYQP. Data revealed that JYQP reduced writhing and stretching induced by acetic acid; however, this effect could not be blocked by naloxone. JYQP specifically suppressed the phase II reaction time in formalin-treated mice; meanwhile, no analgesic effect of JYQP in hot plate test was observed, indicating that JYQP exerts analgesic activity against inflammatory pain rather than neurogenic pain. Furthermore, JYQP could successfully relieve bone cancer pain in mice. No physical dependence could be observed upon long-term administration in mice. Collectively, our present results provide experimental evidence in supporting clinical use of JYQP as an effective and safe agent for pain treatment.