Role of Smad3 inhibitor and the pyroptosis pathway in spinal cord injury

Abstract

The aim of the present study was to investigate the role of Smad3 inhibitors and the pyroptosis pathway in spinal cord injury, and to determine the underlying mechanism. The pyroptosis signaling pathway may be involved in spinal cord injury during the recovery period. Smad3 inhibitor may serve a role in alleviating spinal cord injury by reducing the pyroptosis of neurons, which is induced by caspase-1, absent in melanoma-2 or NOD-like receptors protein-1 during the recovery period of spinal cord injury. In the present study, spinal cord injury was alleviated by caspase-1 and Smad3 inhibitors. Therefore, a Smad3 inhibitor could relieve spinal cord injury in mice by directly downregulating caspase-1 and reducing neuron pyroptosis following spinal cord injury during the recovery period.

Keywords: Smad3; caspase-1; growth differentiation factor-11; pyroptosis; spinal cord injury.

Figure 1

Pyroptosis signaling pathway may be involved in SCI during the recovery period. In the sci group, SCI injury was established by artery clamp through clamping of the T10 level spinal cord for a minute after laminectomy. In the con group, mice received a sham operation. (A) Survival curve of SCI mice 14 days after operation. (B) BMS score of Sci mice. (C) Expression of pyroptosis pathway markers. Mice were sacrificed after the operation and the spinal cords (6 mm including the injured level) were obtained for western blot analysis. The molecular weight of caspase-1 is ~51 kDa and mature form caspase-1 (p20) is ~20 kDa. (D) Statistical analysis of ASC western blot results. (E) Statistical analysis of caspase-1 western blot results. (F) Statistical analysis of p20 western blot results. (G) Statistical analysis of IL-1β western blot results. (H) Injured spinal cord tissue was harvested for immunohistochemistry at day 14 after surgery. Magnification, x400. ^*P<0.05 vs. respective con group. SCI/sci/S, spinal cord injury; Con/C, control; ASC, apoptosis-associated speck-like protein containing a caspase-activating recruitment domain; IL, interleukin; BMS, Basso Mouse Scale.

Figure 2

Survival rate and BMS scores of SCI mice during the first 2 weeks. SCI mice were treated with 20 µl normal saline (N+sci) caspase-1 inhibitor (C+sci) or Smad3 inhibitor (S+sci) during operation. (A) Survival curve 14 days after operation. (B) BMS scores of sci mice. ^*P<0.05 vs. S+sci group; ^#P<0.05 vs. C+sci group. SCI/sci, spinal cord injury; C, caspase-1 inhibitor; S, Smad3 inhibitor; N, normal saline; BMS, Basso Mouse Scale.

Figure 3

Association between the GDF-11/Smads pathway and the pyroptosis pathway in SCI during the recovery period. Mice were sacrificed on day 14 after operation, and injured tissue from spinal cords was harvested for western blot analysis and immunofluorescence. (A) Expressions of pyroptosis pathway markers and GDF-11/Smad4 were detected by western blotting. (B) Statistical analysis of ASC expression. (C) Statistical analysis of caspase-1 expression. (D) Statistical analysis of IL-1β expression. (E) Statistical analysis of GDF-11 expression. (F) Statistical analysis of Smad4 expression. (G) Immunofluorescence co-staining of caspase-1 and Smad2/3 in spinal cord. Magnification, x400. Scale bars represent 50 µm. Positive expression is indicated by the white arrows in merged images. ^#P<0.05. GDF-11, growth differentiation factor-11; SCI/sci; spinal cord injury; Con, control; C, caspase-1 inhibitor; S, Smad3 inhibitor; N, normal saline; ASC, apoptosis-associated speck-like protein containing a caspase-activating recruitment domain; IL, interleukin.

Figure 4

AIM2 and NLRP1 expression in spinal cord. Mice were sacrificed on day 14 day after the operation and the spinal cord was harvested for western blot analysis or immunofluorescence co-staining. (A) AIM2 and NLRP1 were detected by western blotting. (B) Statistical analysis of NLRP1 western blots. (C) Statistical analysis of AIM2 western blots. (D) Immunofluorescence co-staining of GDF-11 and AIM-2. (E) Immunofluorescence co-staining of GDF-11 and NLRP1. Magnification, x400. Scale bars represent 50 µm. Positive expression is indicated by the white arrows in merged images. AIM-2, absent in melanoma-2; NLRP1, NOD-like receptor protein-1; GDF-11, growth differentiation factor-11; S, Smad3 inhibitor; N, normal saline; SCI/sci, spinalcord injury; con, control.