Botulinum toxin type A-targeted SPP1 contributes to neuropathic pain by the activation of microglia pyroptosis

Abstract

Background: Neuropathic pain (NP) is the primary symptom of various neurological conditions. Patients with NP often experience mood disorders, particularly depression and anxiety, that can severely affect their normal lives. Microglial cells are associated with NP. Excessive inflammatory responses, especially the secretion of large amounts of pro-inflammatory cytokines, ultimately lead to neuroinflammation. Microglial pyroptosis is a newly discovered form of inflammatory cell death associated with immune responses and inflammation-related diseases of the central nervous system.

Aim: To investigate the effects of botulinum toxin type A (BTX-A) on microglial pyroptosis in terms of NP and associated mechanisms.

Methods: Two models, an in vitro lipopolysaccharide (LPS)-stimulated microglial cell model and a selective nerve injury model using BTX-A and SPP1 knockdown treatments, were used. Key proteins in the pyroptosis signaling pathway, NLRP3-GSDMD, were assessed using western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence. Inflammatory factors [interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α] were assessed using enzyme-linked immunosorbent assay. We also evaluated microglial cell proliferation and apoptosis. Furthermore, we measured pain sensation by assessing the delayed hind paw withdrawal latency using thermal stimulation.

Results: The expression levels of ACS and GSDMD-N and the mRNA expression of TNF-α, IL-6, and IL-1β were enhanced in LPS-treated microglia. Furthermore, SPP1 expression was also induced in LPS-treated microglia. Notably, BTX-A inhibited SPP1 mRNA and protein expression in the LPS-treated microglia. Additionally, depletion of SPP1 or BTX-A inhibited cell viability and induced apoptosis in LPS-treated microglia, whereas co-treatment with BTX-A enhanced the effect of SPP1 short hairpin (sh)RNA in LPS-treated microglia. Finally, SPP1 depletion or BTX-A treatment reduced the levels of GSDMD-N, NLPRP3, and ASC and suppressed the production of inflammatory factors.

Conclusion: Notably, BTX-A therapy and SPP1 shRNA enhance microglial proliferation and apoptosis and inhibit microglial death. It improves pain perception and inhibits microglial activation in rats with selective nerve pain.

Keywords: Botulinum toxin A; Microglia; Neuropathic pain; Pyroptosis; SPP1.

Figure 1

SPP1 and pyroptosis are activated in lipopolysaccharide-induced microglia. A-E: The microglia were treated with lipopolysaccharide. The expression of ACS, GSDMD, and GSDMD-N was measured by western blot (A). The levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β were detected by quantitative real-time polymerase chain reaction (B-D). The expression of SPP1 was analyzed by quantitative real-time polymerase chain reaction (E). ^aP < 0.001. LPS: Lipopolysaccharide; TNF: Tumor necrosis factor; IL: Interleukin; NC: Negative control.

Figure 2

SPP1 targeted by botulinum toxin type A promotes proliferation and represses apoptosis of lipopolysaccharide-induced microglia. A-E: Lipopolysaccharide-treated microglia were treated with control short hairpin RNA (shRNA) or SPP1 shRNA, or co-treated with botulinum toxin type A. The expression of SPP1 was analyzed by quantitative real-time polymerase chain reaction (A). The expression of SPP1 was measured by western blot (B). Cell viability was detected by cell counting kit-8 assay (C). Cell apoptosis was examined by flow cytometry (D and E). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. BTX-A: Botulinum toxin type A.

Figure 3

SPP1 targeted by botulinum toxin type A contributes to pyroptosis of lipopolysaccharide-induced microglia. A-F: Lipopolysaccharide-treated rat microglia were treated with control short hairpin RNA (shRNA) or SPP1 shRNA, or co-treated with botulinum toxin type A. The expression of GSDMD-N was measured by western blot (A). The levels of NLRP3 and ASC were detected by immunofluorescence (B and C). The levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β were analyzed by enzyme-linked immunosorbent assay (D-F). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. TNF: Tumor necrosis factor; IL: Interleukin; BTX-A: Botulinum toxin type A.

Figure 4

SPP1 targeted by botulinum toxin type A enhances microglia pyroptosis during neuropathic pain induced by spared nerve injury in rat. A-G: The spared nerve injury model was established in SD rats and the rats were treated with control short hairpin RNA (shRNA) or SPP1 shRNA, or co-treated with botulinum toxin type A. The mechanical withdrawal threshold and thermal withdrawal latency were analyzed in the rats (A and B). The expression of SPP1, ACS, and GSDMD-N was measured by western blot in spinal cord of the rats (C). The levels of IBA1 and SPP1 were detected by immunofluorescence in spinal cord of the rats (D). The levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β were analyzed by enzyme-linked immunosorbent assay (E-G). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. TNF: Tumor necrosis factor; IL: Interleukin; BTX-A: Botulinum toxin type A.