Receptor-Interacting Protein Kinase 3 Inhibition Relieves Mechanical Allodynia and Suppresses NLRP3 Inflammasome and NF-κB in a Rat Model of Spinal Cord Injury

Abstract

Background: Neuroinflammation is critical in developing and maintaining neuropathic pain after spinal cord injury (SCI). The receptor-interacting protein kinase 3 (RIPK3) has been shown to promote inflammatory response by exerting its non-necroptotic functions. In this study, we explored the involvement of RIPK3 in neuropathic pain after SCI.

Methods: Thoracic (T10) SCI rat model was conducted, and the mechanical threshold in rats was measured. The expressions of RIPK3, nod-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, and nuclear factor-κB (NF-κB) were measured with western blotting analysis or quantitative real-time polymerase chain reaction (qRT-PCR). Double immunofluorescence staining was used to explore the colabeled NLRP3 with NeuN, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (IBA1). In addition, enzyme-linked immunosorbent assay (ELISA) was applied to analyze the levels of proinflammatory factors interleukin 1 beta (IL-1β), interleukin 18 (IL-18), and tumor necrosis factor alpha (TNF-α).

Results: The expression of RIPK3 was elevated from postoperative days 7-21, which was consistent with the development of mechanical allodynia. Intrathecal administration of RIPK3 inhibitor GSK872 could alleviate the mechanical allodynia in SCI rats and reduce the expression levels of RIPK3. The activation of NLRP3 inflammasome and NF-κB was attenuated by GSK872 treatment. Furthermore, immunofluorescence suggested that NLRP3 had colocalization with glial cells and neurons in the L4-L6 spinal dorsal horns. In addition, GSK872 treatment reduced the production of inflammatory cytokines.

Conclusion: Our findings indicated that RIPK3 was an important facilitated factor for SCI-induced mechanical allodynia. RIPK3 inhibition might relieve mechanical allodynia by inhibiting NLRP3 inflammasome, NF-κB, and the associated inflammation.

Keywords: GSK872; NLRP3 inflammasome; neuropathic pain after spinal cord injury; nuclear factor-kappa B (NF-κB); receptor-interacting protein kinase 3.

Figure 1

Intrathecal administration of GSK872 (10 μl, 25 mmol/L) attenuated mechanical hypersensitivity induced by SCI (n = 7 for each group). All data were calculated as mean ± SD. *p < 0.05 vs. the sham group; ^#p < 0.05 the vehicle group. Comparisons between multiple groups were conducted by repeated-measures analysis of variance and Turkey's post-hoc analysis SCI: spinal cord injury. 50% PWT, fifty percentage paw withdrawal threshold.

Figure 2

RIPK3 was up-regulated in the dorsal horns after thoracic SCI. (A) The expression of RIPK3 in the SCI group was measured by western blot analysis on the 7th, 14th, 21th postoperative days (n = 5 for each group). (B) Quantitative analysis of the mRNA expression of RIPK3 in the sham group and SCI group on the 7th, 14th, 21th postoperative days (n = 4 for each group). All data were calculated as mean ± SD. *p < 0.05, vs. the sham group. Comparisons between multiple groups were carried out by one-way ANOVA and Turkey's post-hoc analysis. RIPK3, receptor-interacting protein kinase 3.

Figure 3

GSK872 (10 μl, 25 mmol/L) reduced the expression levels of RIPK3 after SCI. (A) The expression of RIPK3 in different groups, including sham, SCI, vehicle and GSK872 group, was measured by western blot analysis (n = 5 for each group). (B) Quantitative analysis of the RIPK3 mRNA expressions in different groups including sham, SCI, vehicle and GSK872 group (n = 4 for each group). All data were calculated as mean ± SD. *p < 0.05 vs. the sham group. ^#p < 0.05 vs. the vehicle group. Comparisons between multiple groups were carried out by one-way ANOVA and Turkey's post-hoc analysis.

Figure 4

Intrathecal injection of GSK872 (10 μl, 25 mmol/L) inhibited neuroinflammation in the lumbar spinal dorsal horns (n = 4 for each group). (A–C) The levels of TNF-α, IL-1β, and IL-18 in different groups, including sham, vehicle and GSK872 group, were investigated by ELISA. The protein level of TNF-α, IL-1β, and IL-18 were reduced after GSK872 treatment. All data were calculated as mean ± SD. *p < 0.05 vs. the sham group. ^#p < 0.05 vs. the vehicle group. Comparisons between multiple groups were carried out by one-way ANOVA and Turkey's post-hoc analysis. IL, interleukin; TNF-α, tumor necrosis factor-α.

Figure 5

GSK872 (10 μl, 25 mmol/L) suppressed the activation of NLRP3 inflammasome in glia and neurons. (A,B) The expressions of NLRP3 and caspase-1 in different groups, including sham, SCI, vehicle and GSK872 group, were measured by western blot analysis (n = 5 for each group). (C,D) Quantitative analysis of NLRP3 and caspase-1 mRNA expressions in different groups including sham, SCI, vehicle and GSK872 group (n = 4 for each group). (E–P) Immunofluorescence staining of NLRP3 (red) with NeuN, a neuronal marker (green); GFAP, an astrocyte marker (green); and Iba-1, a microglial marker (green) in the lumbar dorsal horns of SCI rats. Scale bar, 50 μm (E–G,I–K). Scale bar, 25 μm (M–O). Scale bar, 20 μm (H,L,P). All data were calculated as mean ± SD. *p < 0.05 vs. the sham group. ^#p < 0.05 vs. the vehicle group. Comparisons between multiple groups were carried out by one-way ANOVA and Turkey's post-hoc analysis.

Figure 6

Intrathecal injection of GSK872 (10 μl, 25 mmol/L) inhibited NF-κB p65 in the lumbar spinal dorsal horns (n = 5 for each group). The expression of NF-κB p65 in different groups, including sham, SCI, vehicle and GSK872 group, was measured by western blot analysis. All data were calculated as mean ± SD. *p < 0.05 vs. the sham group. ^#p < 0.05 vs. the vehicle group. Comparisons between multiple groups were carried out by one-way ANOVA and Turkey's post-hoc analysis. NF-κB, nuclear factor-kappa B.