Bone Marrow-Derived Mesenchymal Stromal Cells Alleviate Bone Cancer Pain by Modulating Microglial Polarization: Involvement of the Nrf2/HO-1 Pathway.

BACKGROUND

Dysregulated polarization of spinal microglia is a key contributor to the pathophysiology of bone cancer pain (BCP). Bone marrow-derived mesenchymal stromal cells (BMSCs) have demonstrated significant analgesic and microglial-modulatory effects in various pain models. The nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway is implicated in the regulation of microglial polarization. However, the specific role and mechanistic basis of intrathecally administered BMSCs in influencing spinal microglial polarization during BCP remain to be fully clarified. In this study, we investigated the impact of intrathecal administration of BMSCs on spinal microglial polarization in a rat model of BCP, and assessed the potential contribution of the Nrf2/HO-1 signaling pathway to this process.

METHODS

In a rat model of BCP, animals received three intrathecal injections of 2 × 106 BMSCs at every other day. Pain behavioral tests, including measurements of the 50% paw withdrawal threshold (PWT) and the number of spontaneous flinches (NSF), were assessed before and after BMSCs administration. The CatWalk automated gait analysis system was employed to quantify BMSC-mediated amelioration of BCP-associated locomotor deficits. Protein expression levels of the pro-inflammatory microglial markers cluster of differentiation 86 (CD86) and inducible nitric oxide synthase (iNOS), along with the anti-inflammatory markers CD206 and Arginase-1 (Arg-1), as well as key components of the Nrf2/HO-1 signaling pathway, were measured in spinal cord tissues using Western blot and immunofluorescence staining. In addition, a non-contact co-culture system of BMSCs and lipopolysaccharide (LPS)-activated BV2 cells was established. The expression of CD86, iNOS, CD206, Arg-1, and pathway components, together with the mRNA levels of the inflammatory cytokines interleukin (IL)-1β and IL-10, were analyzed via Western blot, immunofluorescence, and quantitative real-time PCR (RT-qPCR).

RESULTS

Intrathecal injection of BMSCs significantly alleviated established pain behaviors in BCP rats, as evidenced by increased 50% PWT, reduced NSF, and normalized gait parameters. At the molecular level, BMSC treatment downregulated the spinal cord expression of the pro-inflammatory markers CD86 and iNOS while concurrently upregulating the anti-inflammatory markers CD206 and Arg-1. In the co-culture system, BMSC enhanced the activation of the Nrf2/HO-1 signaling pathway within LPS-stimulated BV2 cells. This enhancement was associated with a reduction in the expression of CD86 and iNOS, an increase in CD206 and Arg-1, decreased levels of IL-1β, and elevated levels of IL-10.

CONCLUSIONS

Intrathecal administration of BMSCs effectively attenuates pain in BCP rats by shifting spinal microglial polarization from a pro-inflammatory toward an anti-inflammatory phenotype. The observed modulation of microglial polarization by BMSCs is consistent with an involvement of the Nrf2/HO-1 signaling pathway.