Nanoparticulate MgH 2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain

Abstract

Background:

Bone cancer metastases are the third most common site of cancer spread after lungs and liver. This condition often causes severe pain that impairs patients’ physical, psychological, and social well-being. We aimed to explore the potential therapeutic benefits of magnesium hydride (MgH₂) on bone cancer pain (BCP).

Methods:

A BCP model was established in Wistar rats. Daily oral dosing of 0.5% w/w MgH₂ was administered. Assessment included pain sensitivity, motor coordination, and emotional behaviors. Hippocampal samples underwent RNA sequencing, Western blotting, immunofluorescence, and quantitative RT-PCR.

Results:

MgH₂ markedly reduced mechanical hypersensitivity and depressive behaviors in rats with BCP. These effects were linked to suppression of the TRPM2–NLRP3 signaling axis in hippocampal microglia. Additionally, MgH₂ served as an adjuvant to reduce opioid tolerance during fentanyl co-treatment, enabling lower opioid dosages. Collectively, MgH₂ inhibited TRPM2 activation, microglial activation, oxidative stress, and NLRP3 inflammasome formation, which together reduced neuroinflammation and improved therapeutic outcomes.

Conclusion:

MgH₂ nanoparticles may relieve BCP and comorbid depressive symptoms by inhibiting TRPM2-mediated NLRP3 inflammasome activation in hippocampal microglia.

Introduction

With advances in cancer therapy extending patient survival, the proportion of individuals developing bone metastases has risen sharply, with bone lesions now accounting for roughly 80% of all metastatic sites. ¹ These patients often experience severe bone cancer pain (BCP) and depressive symptoms. Clinical data demonstrate that approximately one-third of cancer patients suffer from BCP, ² and severe depression affects 15% to 20% or more of cancer patients.^{3 –5} Current pharmacological treatments for BCP include systemic cancer therapies (such as chemotherapy, hormone therapy, and targeted therapy), opioid-based analgesics, osteoclast inhibitors, and radioactive agents.^{6 –8} However, these treatments yield suboptimal analgesia and fail to ameliorate associated depression. Moreover, long-term opioid use carries risks of tolerance, dependence, and even opioid-induced hyperalgesia. ⁹ These shortcomings underscore the urgent need to elucidate the neural underpinnings of BCP to develop more effective and safer therapies.

Growing evidence implicates neuroinflammation as a driver of the pathophysiology of BCP.^10,11 Therapies targeting neuroinflammation include non-steroidal anti-inflammatory drugs (NSAIDs), systemic corticosteroids, cytokine-specific biologics, opioids, and other potential agents.^{12 –14} NSAIDs exhibit limited analgesic efficacy and poor blood-brain barrier (BBB) permeability. ¹⁵ Corticosteroids cause adverse side effects including prolonged immunosuppression, increased infection risk and gastric bleeding. ¹² Systemic tumor necrosis factor-α blockade elevates severe infection risk, including latent tuberculosis reactivation. ¹⁶ Opioid receptors are implicated in neuroinflammation; however, their precise biological mechanisms and associated addiction risks remain concerns. ¹⁷ Other novel neuroinflammatory targets, such as anti-TRPV1, are also implicated.^18,19 Therefore, current neuroinflammation therapies are limited by poor BBB penetration, off-target effects, and potentially severe long-term systemic immunosuppression.

Previous studies have spotlighted the hippocampus as a critical hub for both nociceptive modulation^{20 –22} and affective regulation, with hippocampal neuroinflammation as a key underlying mechanism.^3,21 For instance, Dai et al. ³ showed that minocycline prevented depressive behavior and hyperalgesia in rats with BCP by modulating hippocampal microglia. Their BCP model indicated a microglial shift from the anti-inflammatory M2 state to the pro-inflammatory M1 state in the hippocampus, contributing to both pain and emotional disturbances. ³

Our previous work demonstrated that the novel nanoparticulate drug magnesium hydride (MgH₂) alleviated systemic inflammation, reduced axon damage in the hippocampus, and relieved depressive and anxiety-like behaviors in a mouse model of multiple sclerosis (MS). ²³ In particular, experimental autoimmune encephalomyelitis (EAE) is the most widely used animal model for MS and replicates key pathological features such as leukocyte-mediated demyelination and neuroinflammation.^23,24 In our EAE model, low-toxic MgH₂ administration reduced reactive oxygen species (ROS) via microglial polarization and alleviated depressive behaviors. ²³ Taken together, we hypothesize that MgH₂ releases hydrogen, penetrates BBB, and causes fewer systemic side effects. The study aims to investigate hippocampal microglial inflammation in BCP, confirm MgH₂’s capacity to alleviate neuroinflammatory responses, and explore novel mechanisms underlying BCP.

Materials and methods

Animals and study design

All surgical procedures and experimental protocols conformed to the criteria of the National Institutes of Health and the ethical standards of the International Association. For real clinical scenarios, our study focused on breast cancer-induced BCP using Walker 256 rat mammary gland cancer cells, as nearly all breast cancer patients were female. The design aligned with clinical pathology and enhanced translational relevance. Walker 256 cells were suitable for BCP as they exhibited the capability for bone resorption and skeletal fragility, consistent with the phenotype observed in breast cancer patients with bone metastasis.^25,26 Therefore, mature female Wistar rats (220–250 g) were housed under controlled conditions with a 12-h light/dark cycle and unrestricted access to food and water.

Twenty-four rats were allocated to sham (n = 12) and BCP (n = 12) groups for behavioral testing. Molecular experiments included 4 rats per group for Western blot, 8 for immunofluorescence, and 5 for RNA sequencing. For MgH₂ treatment, 40 rats were divided into four groups: sham (n = 10), sham + MgH₂ (n = 10), BCP (n = 10), and BCP + MgH₂ (n = 10), with subsets allocated to Western blot/Q-PCR (4 per group) and immunofluorescence (6 per group).

An additional 20 rats were selected for studies involving fentanyl, with an assignment of either the BCP (n = 10) or the BCP + MgH₂ (n = 10) group. The BCP + MgH₂ group received an MgH₂-enriched diet for 7 days before surgery, followed by daily low-dose fentanyl treatment initiated on the day of surgery. Skin mechanical sensitivity was assessed every 2 days for 2 weeks post-surgery. Sucrose preference tests were conducted on postoperative days 1, 3, 5, 7, 9, 11, 13, and 15. On day 15, forced swim and rotarod tests were conducted before euthanasia, and hippocampal tissue was then collected for quantitative real‑time PCR (Q-PCR) and Western blotting. Blinded researchers performed behavioral tests.

Procedure for BCP model and quality control

The BCP model used in the study is a well-established and reproducible model, with the prior publication of our team supporting its robustness and translational relevance. ²⁷ Briefly, the rats were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium. An incision was made along the left patellar ligament to expose the tibia. A 7 mm long, 23-gauge needle was carefully inserted into the left tibial medullary cavity near the intercondylar eminence. A microinjection syringe was used to precisely administer 10 μL of a suspension containing 1 × 10⁷ Walker 256 rat mammary gland cancer cells into the cavity.

To minimize potential heterogeneity, we adhered strictly to previously validated protocols ²⁷ and ensured that the same trained personnel performed all procedures. We also took specific precautions to prevent cell leakage during inoculation, including the use of fine-gauge needles and careful monitoring of injection pressure and volume.

Oral administration of nanoparticulate MgH₂

Rats in the Sham group were fed a standard rat diet (AIN93G). In contrast, rats in the experimental groups were provided with a diet enriched with Nanoparticulate MgH₂ (AIN93G + 0.5% w/w of MgH₂) starting 7 days before the experiments and continuing until the study’s end. Food consumption was monitored to account for any differences in dietary preference.

Pain sensitivity

Rats’ sensitivity to mechanical stimuli was assessed using von-Frey filaments, with the up-down method determining the force needed for paw withdrawal. Thermal hyperalgesia was measured via thermal withdrawal latency using a radiant heat instrument (IITC Life Science Instruments, USA) (52 ± 0.2°C; 50 W, 8 V bulb) on the plantar surface of the hind paw. The latency period until paw retraction included raising, licking, flicking, shaking, and jumping (cut-off: 60 s, measured 5 times with 5-min intervals).

Emotional behaviors

The sucrose preference test measured anhedonia, calculating the ratio of sucrose intake to total fluid consumption, where a lower preference indicated depressive-like traits. On day 15, the tail suspension test (TST) recorded immobility over 6 min, with blinded observers noting behavior in the final 4 min.

Motor coordination

The rotarod test was used to evaluate motor skills and balance. Rats were placed on a rotating rod, and the speed was gradually increased. The test measured the latency to fall time, defined as the duration the rats could maintain their balance on the rod before falling.

RNA sequencing

Total RNA from hippocampal samples was extracted using the RNAmini kit (Qiagen, Germany), and its quality was evaluated using gel electrophoresis and a Qubit fluorometer (Thermo, Waltham, MA, USA). Strand-specific libraries were created using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), and sequencing was done on the Illumina Novaseq 6000 instrument. Skewer processed raw data, and FastQC (v0.11.2) ensured quality. Clean reads were aligned to the Human genome (hg38) with STAR, and Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were calculated using Perl. Differential expressed transcripts/genes (DETs/DEGs) were identified using the MA-plot-based approach with a random sampling model in the DEGseq package, with cut-off values of p < 0.05 and an absolute fold change of ≥2. Enrichment analysis was performed via Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment (GSEA).

Protein-to-protein interaction (PPI) analysis

The STRING database (http://string-db.org/) was used to construct a PPI network for the hub genes with a minimum interaction score threshold of 0.15. The network was analyzed and visualized using Cytoscape (version 3.8.2) with the CytoHubba plugin, which identifies hub genes.

RNA isolation and Q-PCR

RNA was extracted from primary hippocampal tissue using Trizol (Invitrogen), and cDNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Q-PCR was conducted on a LightCycler 96 instrument (Roche) using TOYOBO’s SYBR Green Real-time PCR Master Mix. Gene expression was normalized to the housekeeping gene (GAPDH) using the ∆∆CT method. Gene-specific primer sequences are provided in Supplemental Table 1.

Western blotting

Brain tissue lysates were prepared in cold buffer with protease inhibitors and centrifugated at 12,000 rpm for 15 min at 4°C. Protein concentrations were measured using a BSA assay kit (P0006, Beyotime, Jiangsu, China). Samples underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%), transferred to PVDF membranes, and incubated overnight at 4°C with primary antibodies (Supplemental Table 2), followed by an incubation of goat anti-mouse or anti-rabbit secondary antibodies (1:10,000; Santa Cruz Biotechnology, USA) for 1 h at room temperature. Detection was performed using the BeyoECL kit (Beyotime, China) and visualized with the Tanon 5200 system.

Immunofluorescence

Frozen tissue sections were rinsed with PBS for 15 min, then blocked in PBS containing 3% BSA and 0.1% Triton X-100 (Bio-Rad) for 30 min at room temperature. The samples were incubated overnight at 4°C with a primary antibody (Supplemental Table 2), followed by incubation for 1 h at room temperature with a goat anti-rabbit IgG secondary antibody conjugated with fluorophore (Sigma F9887 1:160). DAPI was used to counterstain the nuclei. Images were captured using a Leica TCS SP8 confocal microscope.

Statistical analysis

GraphPad Prism 9 (GraphPad Software, Inc., CA) was used to analyze the data. Quantitative results were presented as mean ± SD, with tests for homogeneity and normality of variance conducted before analysis. A Student’s t-test was used for comparisons between two groups. For comparisons involving more than two groups, one-way ANOVA was applied, followed by Bonferroni’s post hoc test. Two-way ANOVA was applied to time-dependent comparisons. Statistical significance was set at p < 0.05. For bioinformatic analysis, the packages of clusterProfiler, msigdbr, org.Rn.eg.db, GOplot and gglot2 were used in R 4.2.1.

Results

Behavioral alterations in BCP rats

Walker 256 rat mammary cancer cells were injected into the tibias of experimental rats to model BCP. The right hind paw’s mechanical withdrawal threshold and thermal withdrawal latency were assessed on days 1, 3, 5, 7, 9, 11, 13, and 15. Beginning on day 3, BCP rats exhibited a significant reduction in mechanical withdrawal threshold (Figure 1(a)) and thermal withdrawal latency (Figure 1(b)), while no notable decreases were observed in sham-operated rats. In the TST, BCP rats displayed considerably prolonged immobility compared to sham controls (Figure 1(c)). The sucrose preference test further revealed that BCP rats consumed significantly less sucrose solution than sham controls on days 7 (p < 0.05), 11 (p < 0.01), and 15 (p < 0.001) (Figure 1(d)), despite total liquid intake being comparable between the groups (Figure 1(e)), suggesting depression-like behaviors in BCP rats. Both groups performed similarly in the rotarod test, indicating preserved motor function (Figure 1(f)). These findings indicate the significant manifestation of bone cancer-associated pain and depressive behaviors in BCP rats.

Figure 1.

Behavioral changes in the walker 256 tibial injection-induced bone cancer pain model (n = 12 per group): (a) withdrawal threshold of the right hind paw measured on postoperative days, (b) thermal hyperalgesia measured on postoperative days, (c) increased immobility time observed in TST on postoperative day 15 in BCP rats, (d) sucrose preference test conducted from postoperative days 1 to 15, revealing a decreased preference for sucrose in the BCP group compared to the sham-operated group, (e) total fluid consumption in both the BCP and sham groups, and (f) motor ability accessed by rotarod test on postoperative day 15 in both the BCP and sham-operated groups.

RNA sequencing reveals robust neuroinflammatory response and inflammasome activation in the BCP hippocampus

To investigate hippocampal transcriptomic alterations following bone pain induction, bulk RNA sequencing was performed on rat hippocampus tissues from BCP and sham-operation controls at day 14 post-model induction. Differential expression analysis revealed 21,670 genes with detectable expression in both groups, with 592 genes differentially expressed in BCP rats compared to sham controls (506 upregulated and 93 downregulated genes; cutoff set as adjusted p-value < 0.05, absolute fold change >2) (Figure 2(a)). The volcano plot highlighted the upregulated DEGs in different colors, showing immune or inflammatory genes accounting for approximately 60% (303/506), with the top 20 upregulated DEGs presented in Figure 2(b).

Figure 2.

RNA sequencing reveals an intensive neuroinflammatory response in the hippocampus associated with BCP: (a) volcano plot displaying DEGs, with the upregulated DEGs highlighted and classified into immune/inflammatory associated, non-inflammatory and unnamed genes, (b) violin plot of the top 20 immune/inflammatory DEGs, (c) GO and KEGG analysis with representative pathways, and (d)–(f) analyses of inflammasome-associated pathways, protein network and gene expression alterations visualized by GSEA (d), PPI (e) and FPKM (f).

Enrichment analysis using GO terms identified significant biological process (BP) items, including leukocyte-mediated immunity, lymphocyte-mediated immunity, and regulation of innate immune response (Figure 2(c)). In addition, the top-ranked items of cell component (CC), molecular function (MF), and KEGG were associated with antigen-presenting cells (e.g. macrophages) (Figure 2(c)). Specifically, we identified enriched macrophage-linked and inflammasome-linked pathways, such as interleukin 1 (IL-1) signaling and NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasome in GSEA (Figure 2(d)). The PPI network showed hub genes concerning inflammasome activation in BCP models (Figure 2(e)), with an elevated transcriptional profile, including upregulation of Trpm2 (Transient Receptor Potential Cation Channel Subfamily M Member 2), Iba1 (Ionized Calcium-Binding Adapter Molecule 1), and Nlrp3 in the hippocampus of BCP rats (Figure 2(f)). Collectively, these data indicate a robust neuroinflammatory response and inflammasome activation in the hippocampus of BCP rats.

Activation of microglia, TRPM2 channel, and NLRP3 inflammasome in the BCP hippocampus

Based on previous RNA sequencing analysis, we aimed to further validate and investigate the inflammasome-driven neuroinflammatory response. Immunofluorescence staining was conducted on the hippocampal region of both sham-operated and BCP rats on day 15, revealing that TRPM2, NLRP3, and IBA1 protein expression levels increased significantly in the hippocampus of BCP rats compared to sham controls (Figure 3(a)–(c)). The staining also showed enhanced colocalization of TRPM2 and IBA1 proteins (Figure 3(a) and (b)). Furthermore, the hippocampal region of BCP rats showed a notable increase in microglial activation, evidenced by the rise in IBA1 and morphological changes of microglial cells (Figure 3(d)). Western blot analysis confirmed a time-dependent escalation in TRPM2, NLRP3, CASP1, ASC (Pycard), and IBA1 protein levels within the hippocampus of BCP rats (Figures 3(e) and (f)). On day 15, Q-PCR demonstrated a significant increase in mRNA levels of Trpm2, Nlrp3, Casp1, Asc, and Iba1 (Figures 3(g)). These findings suggest that activation of microglia, the TRPM2 channel, and the NLRP3 inflammasome in the hippocampus contributes to pain and depressive behaviors in BCP rats.

Figure 3.

BCP induces neuroinflammation in the rat hippocampus: (a) immunofluorescence micrographs for BCP and sham groups, with TRPM2 and NLRP3-positive cells shown in red, and microglial, stained with Iba1 and shown in green, (b) and (c) quantitative analysis of merged TRPM2-positive and NLRP3-positive cells between the two groups, (d) representative images showing the effect of BCP on cell number and morphology of the microglia in the hippocampus. The white arrows indicate changed morphology of microglial (right panel), (e) and (f) western blot images of TRPM2, NLRP3, CASP1, ASC, IBA1 protein levels (e) and their quantitative intensity (f) in sham, 1-, 7- and 15-day BCP groups. Comparisons to the sham group are marked as *, and (g) DIFFERENCES in mRNA expression levels of Trpm2, Nlrp3, Casp1, Asc, and Iba1 across groups.

Nano MgH₂ administration alleviates mechanical pain and depressive-like behaviors in BCP

Nano MgH₂, an antioxidant, was administered to rats through MgH₂-enriched feed. Compared to the BCP group, BCP rats treated with MgH₂ showed significantly reduced mechanical allodynia (Figure 4(a)) and thermal hyperalgesia from the early stages after surgery (Figure 4(b)). For evaluation of depressive-like behaviors, we found that BCP rats receiving MgH₂-enriched feed exhibited reduced immobility time in the TST compared to the BCP group (Figure 4(c)). In addition, on the seventh postoperative day, the sucrose preference test indicated that BCP rats consuming MgH₂-enriched feed showed increased sucrose intake (Figure 4(d)). Notably, total water consumption (Figure 4(e)) and motor coordination performance in the rotarod test (Figure 4(f)) were comparable among all groups. These quality control parameters indicate that the improvements of depressive-like behaviors observed in TST and sucrose preference were not confounded by baseline differences in locomotor function and fluid intake, thereby reinforcing their validity as reliable indicators. Collectively, our findings indicate that MgH₂-enriched feed can mitigate mechanical allodynia and depressive-like behaviors in BCP rats.

Figure 4.

MgH₂ treatment alleviates mechanical pain and depressive behaviors (n = 10 per group): (a) and (b) MgH₂ administration enhanced mechanical pain thresholds (a) and alleviates thermal hyperalgesia (b) in rats with BCP. Comparisons to the sham group or the BCP group are marked as * or ^#, (c) rats receiving MgH₂ showed reduced immobility duration in the TST 15 days after surgery, (d) and (e) the MgH₂ group exhibited increased sucrose consumption in the sucrose preference test, despite that total water consumption remained consistent across groups, and (f) no significant difference was observed between groups in the rotarod test.

Nano MgH₂ mitigates tumor-induced bone pain by modulating microglia activation, TRPM2 channels, and NLRP3 inflammasome in the hippocampus

Immunofluorescence staining assessed TRPM2, NLRP3, and IBA1 protein expression levels in the hippocampal region of sham-operated, BCP, and BCP with MgH₂ intervention groups on day 15, revealing that MgH₂ reduced expression of TRPM2, NLRP3, and IBA1 proteins compared to the BCP group (Figure 5(a)–(c)). In addition, lower levels of TRPM2, NLRP3, IBA1, CASP1, and ASC proteins were found in the hippocampus of the MgH₂-treated group relative to the BCP group (Figure 5(d) and (e)). On day 15, Q-PCR analysis showed that Trpm2, Nlrp3, Iba1, Casp1, and Asc transcriptional expression in the hippocampus was lower in the MgH₂-treated group compared to the BCP group (Figure 5(f)). These results indicate that MgH₂-enriched feed reduced microglial activation, TRPM2 channel activity, and NLRP3 inflammasome upregulation in the hippocampus of BCP rats. Furthermore, we observed increased oxidative stress in the brain of BCP rats, which was mitigated by MgH₂ administration (Supplemental Figure 1). These findings suggest that MgH₂ attenuates activation of microglia, the TRPM2 channel, and the NLRP3 inflammasome in the hippocampus of BCP rats.

Figure 5.

MgH₂ treatment reduces neuroinflammation in the hippocampus: (a)–(c) immunofluorescence micrographs showing TRPM2-, NLRP3-positive cells (red), and IBA1-positive microglia (green) (a), with quantitative analysis of merged TRPM2⁺ IBA1⁺ positive cells (b) and NLRP3⁺ IBA1⁺ cells (c) across groups, (d) and (e) western blot image of TRPM2, NLRP3, CASP1, ASC, IBA1 protein levels (d) and quantitative intensity (e) in each group. Comparisons to the sham group or the BCP + MgH₂ group are marked as * or ^#, and (f) transcriptional differences of Trpm2, Nlrp3, Casp1, Asc, and Iba1 across groups.

Nano MgH₂ functions as an effective adjuvant for pain and depression relief and reduces opioid tolerance in BCP

To assess the efficacy of nano MgH₂ as an adjuvant to opioid therapy, we administered a small dose of fentanyl to simulate real clinical pain management scenarios. The BCP + MgH₂ group received an MgH₂-enriched diet 7 days before surgery. On the day of surgery, both groups (the BCP and BCP + MgH₂ group) intraperitoneally received small-dose fentanyl (0.1 mg/kg) every day (Figure 6(a)). We observed that the MgH2-treated rats had a significantly higher pain threshold than the fentanyl-only group from the 7th day after surgery (Figure 6(b)). Compared to baseline on the day of surgery, the increase in TST immobility time 15 days post-surgery was less pronounced in the MgH₂ group than in the BCP-only group, indicating milder depressive symptoms after MgH₂ treatment (Figure 6(c)). In addition, mechanical pain measurements taken before and after fentanyl treatment revealed that MgH₂ started to serve as an effective adjuvant in improving pain threshold approximately the fifth day post-surgery (Figure 6(d)). After the ninth day after surgery, the MgH₂-treated group showed a reduced tolerance to fentanyl compared to the BCP-only group (Figure 6(d)). Notably, on day 15, fentanyl alone failed to diminish depressive behaviors, while a combination of fentanyl and MgH₂ resulted in reduced depressive behaviors (Figure 6(e)). These findings indicate that MgH₂, when administered alongside a small-dose fentanyl, may effectively alleviate pain and mitigate emotional deterioration in BCP rats, which could help manage opioid resistance and reduce overall opioid dosage.

Figure 6.

MgH₂ enhances analgesic effect and reduces opioid resistance in fentanyl treatment: (a) workflow for small-dose fentanyl treatment with MgH₂ intervention, (b) right hind paw withdrawal thresholds 2-h post-fentanyl treatment across all postoperative days, (c) TST conducted on BCP rats before surgery and on day 15 post-surgery, (d) right hind paw withdrawal thresholds measured before and 2 h after fentanyl treatment on postoperative days 1, 3, 5,7, 9, 11, 13, and 15, and (e) TST performed on day 15 before and 2 h after fentanyl treatment with or without MgH₂.

Discussion

In this study, we investigated the neuroprotective effects of nano MgH₂ in a rat model of BCP, focusing on its regulatory role in hippocampal microglia. Our results demonstrated that MgH₂ inhibited TRPM2 channel activation, which is implicated in calcium influx, mitochondrial ROS (mtROS) production, and subsequent NLRP3 inflammasome activation. This cascade likely contributes to microglial activation in the hippocampus. These molecular changes were associated with improved mechanical pain sensitivity and depressive-like behaviors in BCP rats (Figure 7).

Figure 7.

Proposed mechanism of MgH₂ in relieving mechanical pain and depressive-like behaviors via hippocampal microglial modulation. This schematic plot summarizing both experimentally validated and literature-supported mechanisms through which MgH₂ may exert its effects. The potential TRPM2–NLRP3 pathways (1–3) corresponding to three possible routes of action: (1) calcium-associated pathways, (2) mitochondrial ROS-dependent mechanisms, and (3) direct or indirect signaling modulation via other pathways.

The proposed mechanism suggests that MgH₂ may exert its effects through three interrelated pathways: (1) calcium-associated signaling downstream of TRPM2, (2) mtROS-mediated activation of NLRP3, and (3) additional direct or indirect regulatory effects on the TRPM2–NLRP3 axis. While partial mechanisms were experimentally validated, others remain hypothetical and warrant further investigation (Figure 7). Moreover, our results highlighted the potential clinical use of MgH₂ in managing neuroinflammation and chronic pain, and we found that MgH₂ served as an adjuvant therapy in lowering both the required dose of fentanyl and tolerance in BCP management. It holds considerable significance, especially given global concerns about opioid misuse and resistance.

Neurological mechanisms in BCP remain to be fully elucidated. BCP is recognized as a severe form of chronic pain, combining characteristics of neuropathic pain and inflammatory pain affecting the nervous system. Microglia act as both neural and immune cells for sensory transmission and key targets in chronic pain research, and rapidly respond to external stimuli and injuries by polarizing and modulating local immune function.^28,29 Recent studies have highlighted the critical role of microglia in BCP pathophysiology, contributing to central sensitization and alterations in dorsal horn neurons following nerve injury.^{30 –32} Specifically, increased astrocyte and microglia activity in the dorsal horn of the ipsilateral spinal cord has been observed in BCP rodent models.^{33 –36} Several studies have shown that inhibiting glial activation or their pro-inflammatory cytokine production reduces BCP-related mechanical allodynia, thermal hyperalgesia, and spontaneous pain.^{37 –39} However, most studies have mainly focused on the tissue of spinal cord or dorsal root ganglion, yet BCP broadly impacts patients’ health. Moreover, cancer patients frequently experience intense emotional disturbances, which can reduce the efficacy of opioids in managing chronic cancer pain. Thus, the study chose the hippocampus as the primary region of interest due to its well-established role in affective processing, memory, and central pain modulation, especially in chronic pain states. ⁴⁰ Also, hippocampal neuroinflammation contributes to emotional disturbances and the sensitization of pain pathways. ⁴¹ Our transcriptomic and histological analyses focused on this region to explore the potential central mechanisms underlying MgH₂’s antidepressant and analgesic effects.

Clinically, BCP is managed primarily through a three-step analgesic approach. However, managing BCP is challenging due to the prolonged nature of BCP and the adverse effects associated with opioid use, such as addiction and tolerance. Previous research has concluded that the hippocampus may bridge between chronic pain and its comorbidities.^42,43 To illustrate, the dorsal hippocampus modulates functional connectivity by mitigating neuropathic pain through local excitatory and opioid-related mechanisms. Our research, focusing on the hippocampal region in rats, found that a novel nano drug MgH₂ administration alleviated neuropathic pain symptoms and ameliorated depressive behaviors in BCP rats. Histological analysis showed that MgH₂ reduced microglia activation and Nlrp3 inflammasome formation in the hippocampus. Further experiments revealed that MgH₂ reduced opioid consumption and tolerance during fentanyl treatment in BCP rats. As a low-toxicity anti-inflammatory agent, MgH₂ holds substantial potential for use in analgesia or as an adjunctive therapy for BCP.

In the stomach, MgH₂ nanoparticles generate significant amounts of hydrogen (H₂) through a sustainable reaction: MgH₂ + 2H₂O → Mg (OH)₂ + 2H₂. H₂ is biologically inert, and growing evidence indicates that it suppresses neuroinflammation and oxidative stress, key contributors to anxiety and depression. Compared to traditional methods of H₂ delivery, such as hydrogen-rich water, MgH₂ offers a more sustained release of H₂ while simultaneously providing magnesium ions (Mg²⁺), known as an agent for blocking calcium ion channels involved in various biological processes. MgH₂ has also demonstrated ROS-suppressing properties in the brain. Our previous studies indicated that MgH₂ reduced depressive behaviors in mice with EAE by modulating microglia polarization and reducing ROS. ²³ Of note, we previously reported that MgH₂ was a low-toxic agent. ²³ In our BCP study, we did not observe significant adverse effects including reduced food intake, weight loss, or behavioral abnormalities, although more comprehensive side effect evaluations in both animal models and humans would be necessary in future studies. Additionally, MgH₂ was found to increase survival rates and reduce lung injury in lipopolysaccharide (LPS)-induced murine models by diminishing oxidative stress and cell apoptosis via NF-κB/NLRP3/IL-1β pathways, resulting in reduced inflammatory cell infiltration and alveolar thickening. ⁴⁴ However, a research gap exists regarding MgH₂’s function and underlying mechanisms in BCP.

The role of Mg²⁺ as a calcium antagonist is well-established, with studies showing that Mg²⁺ enhances opioid efficacy and reduces required dosages, thereby minimizing side effects.^{45 –49} Kulik et al. ⁵⁰ demonstrated that Mg²⁺ is a physiological antagonist of N-methyl-D-aspartate receptors (NMDAR) and thus enhances opioid efficacy in chronic pain patients. Also, Koc et al. ⁵¹ showed that mice treated with magnesium citrate exhibited increased pain thresholds and lowered TLR4 concentrations in the brain. In two animal models of neuropathic pain, including sciatic nerve ligation and diabetic neuropathy, Mg²⁺ and non-competitive NMDAR antagonists effectively attenuated hyperalgesia by curbing the opening of NMDAR channels. ⁵² We utilized similar methodologies to conclude that MgH₂ might exert its analgesic through Mg²⁺ directly antagonizing the Trpm2 calcium channel. Of note, TRP channels were reported to be key contributors to neuropathic pain,^53,54 consistent with our findings.

Besides Mg²⁺, H₂ is another byproduct of MgH₂ after its reaction with water. H₂, known for its potent reducing properties, inhibits oxidative stress and supports cellular function within mitochondrial compartments.^55,56 It is related to the observed efficacy of MgH₂ in our experiment. Yuan et al. ³⁴ suggested that H₂-rich water provided neuroprotection in traumatic brain injury by diminishing oxidative stress and activating the Nrf2 pathway. Imai et al. ⁵⁷ indicated that H₂ could suppress LPS-induced pro-inflammatory cytokine expression, oxidative damage, and microglial activation in fetal brains. In primary cultured microglia, H₂ inhibited ROS production induced by LPS or cytokines, thereby reducing LPS-induced microglial neurotoxicity. Another study by Imai et al. ⁵⁷ demonstrated that inhaling 2% H₂ ameliorated trimethyltin-induced neurotoxicity and cognitive impairment in C57BL/6 mice, and also decreased Alzheimer’s Disease (AD)-related biomarkers (such as Apo-E, a-β-40, p-tau, and Bax), oxidative stress markers (such as ROS, nitric oxide, and calcium), and inflammatory factors (such as interleukins and tumor necrosis factors) in both serum and brain. ⁵⁸ Our research suggests that BCP elevates Trpm2, leading to mitochondrial ROS, a process that H₂ could potentially inhibit, thereby contributing to the alleviation of BCP.

Several limitations should be noted. First, our findings did not establish definitive causal relationships among TRPM2–NLRP3-induced neuroinflammation, microglial modulation, and behavioral outcomes. Instead, the results supported the possibility that MgH₂ exerted its therapeutic effects, at least partially, by regulating neuroinflammatory processes. Second, the tibial injection of Walker 256 cells in the BCP model, though widely used for studying BCP, has limitations and notable differences from clinical settings. The model only replicates certain aspects of bone cancer pain and does not fully represent the pain experienced by patients, including pain in other bone regions and soft tissue. Also, Walker 256 cells are breast cancer-derived cells primarily used in female models. Estrogen fluctuations in female rodents may influence neuroinflammation, glial activity, and pain signaling. ⁵⁹ Future studies may include male rats to explore sex-specific mechanisms. Third, due to constraints in our laboratory resources, we could not perform a detailed dosage curve for MgH₂ or evaluate its pharmacokinetics, which warrant further investigation. Fourth, our assessment of depressive-like behaviors was not comprehensive, as we did not include measures such as sociability, burrowing behavior, or other locomotor activity (e.g. distance travelled). Last, although MgH₂ shows promise as an adjunctive treatment, current clinical practice remains predominantly reliant on opioid medications, which highlights the urgent need for further research into novel mechanisms and therapeutic agents to improve treatment strategies and reduce opioid reliance.

Collectively, our findings suggest that modulation of TRPM2 channel activity in the hippocampus may represent a potential strategy for alleviating pain and depression in the context of bone cancer. Treatment with the newly developed nano MgH₂ was associated with suppression of TRPM2 channel activation and downstream NLRP3 inflammasome formation, along with reduced microglial activation and apoptosis in the hippocampal region of BCP rats. Improvements in pain-related and depressive-like behaviors accompanied these molecular changes. Furthermore, nano MgH₂ appeared to delay the onset of opioid tolerance and enhance the analgesic effects of fentanyl, highlighting its potential translational value.

Conclusion

Nanoparticulate MgH₂ alleviates mechanical hypersensitivity, depressive-like behaviors, and fentanyl tolerance in a rat model of BCP by inhibiting TRPM2-mediated NLRP3 inflammasome activation in hippocampal microglia. It reduces microglial activation, mitochondrial ROS, and neuroinflammatory cytokines, thereby improving pain thresholds, mood, and opioid efficacy. Our study positions the hippocampal TRPM2–NLRP3 axis as a promising target in cancer-related pain management and validates MgH₂’s function. Further work will define MgH₂’s pharmacokinetics, optimal dosing, and long-term safety to facilitate translation into clinical trials.

Supplemental Material

sj-docx-1-mpx-10.1177_17448069251348770 – Supplemental material for Nanoparticulate MgH2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain

Supplemental material, sj-docx-1-mpx-10.1177_17448069251348770 for Nanoparticulate MgH2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain by Hang Xu, Hongtao Lu, Lu Lu, Zhenghao Li, Zhisheng Piao, Yi Jia, Xiaoyan Meng and Feixiang Wu in Molecular Pain

Supplemental Material

sj-docx-2-mpx-10.1177_17448069251348770 – Supplemental material for Nanoparticulate MgH2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain

Supplemental material, sj-docx-2-mpx-10.1177_17448069251348770 for Nanoparticulate MgH2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain by Hang Xu, Hongtao Lu, Lu Lu, Zhenghao Li, Zhisheng Piao, Yi Jia, Xiaoyan Meng and Feixiang Wu in Molecular Pain

Supplemental Material

sj-docx-3-mpx-10.1177_17448069251348770 – Supplemental material for Nanoparticulate MgH2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain

Supplemental material, sj-docx-3-mpx-10.1177_17448069251348770 for Nanoparticulate MgH2 suppresses TRPM2-mediated NLRP3 inflammasome to relieve bone cancer pain by Hang Xu, Hongtao Lu, Lu Lu, Zhenghao Li, Zhisheng Piao, Yi Jia, Xiaoyan Meng and Feixiang Wu in Molecular Pain