Necrosulfonamide Ameliorates Neurological Impairment in Spinal Cord Injury by Improving Antioxidative Capacity

Abstract

Currently, there is no efficient therapy for spinal cord injury (SCI). Anoxemia after SCI is a key problem, which leads to tissue destruction, while hypoxia after SCI induces cell injury along with inflammation. Mixed-lineage kinase domain-like protein (MLKL) is a critical signal molecule of necroptosis, and mitochondrial dysfunction is regarded as one of the most pivotal events after SCI. Based on the important role of MLKL in cell damage and potential role of mitochondrial dysfunction, our study focuses on the regulation of MLKL by Necrosulfonamide (NSA) in mitochondrial dysfunction of oxygen-glucose deprivation (OGD)-induced cell damage and SCI-mice, which specifically blocks the MLKL. Our results showed that NSA protected against a decrease in the mitochondrial membrane potential, adenosine triphosphate, glutathione, and superoxide dismutase levels and an increase in reactive oxygen species and malonyldialdehyde levels. NSA also improved the locomotor function in SCI-mice and OGD-induced spinal neuron injury through inhibition of MLKL activation independently of receptor-interacting protein kinase 3 (RIP3) phosphorylation. Besides the protective effects, NSA exhibited a therapeutic window. The optimal treatment time was within 12 h after the injury in the SCI-mice model. In conclusion, our data suggest a close association between the NSA level inhibiting p-MLKL independently of RIP3 phosphorylation and induction of neurological impairment by improving antioxidative capacity after SCI. NSA ameliorates neurological impairment in SCI through inhibiting MLKL-dependent necroptosis. It also provides a theoretical basis for further research and application of NSA in the treatment of SCI.

Keywords: antioxidative capacity; mixed-lineage kinase domain-like protein activation; necrosulfonamide; neurological impairment; spinal cord injury.

Figure 1

Cytoprotection of NSA in OGD-induced spinal cord neurons. Cell viability after NSA treatment (1, 3, and 10 μM) (A) and Nec-1 treatment (10, 20, and 50 μM) (B) for 24 h after OGD treatment. Cell viability after 3 μM Nec-1 treatment (C) and 20 μM Nec-1 treatment (D) for 12, 24, 36, and 48 h before OGD treatment, respectively. (E) TUNEL staining was performed for cell damage and DAPI stained all cell nuclei. (F) Histogram analysis of TUNEL positive cell rata. Scale bars: 50 μm. Data are presented as the mean ± SEM, n = 4. *p < 0.05, vs. the control group (CTL). ^# p < 0.05, vs. the OGD group. (A, B and F): Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons tests. (C and D): Data were analyzed using Student’s t-test. OGD, oxygen-glucose deprivation; NSA, necrosulfonamide; Nec-1, necrostatin-1; ATP, adenosine triphosphate;.

Figure 2

NSA suppresses MLKL activation but not p-RIP3. Prevention of MLKL activation by treatment with 3 μM NSA for 24 h after OGD treatment. (A) NeuN and p-MLKL double staining. Cell nuclei were stained with DAPI (blue fluorescence) and neurons were stained with NeuN (red fluorescence). Pictures were taken using a fluorescence microscope (scale bar = 20 μm). (B) WB analysis of the level of p-RIP3 and p-MLKL induced by OGD, p-MLKL level was significantly reversed after NSA treatment. Data are presented as the mean ± SEM, n = 4. *p < 0.05, vs. the control group (CTL). ^# p < 0.05, vs. the OGD group. Data were analyzed using Student’s t-test. RIP3, receptor interacting protein kinase-3; OGD, oxygen-glucose deprivation; NSA, necrosulfonamide; MLKL, mixed-lineage kinase domain-like protein; Nec-1, necrostatin-1; WB, Western blot.

Figure 3

NSA treatment ameliorates mitochondrial dysfunction and neuronal death. After 3 μM NSA treatment for 24 h after OGD treatment, the effect of NSA treatment on ATP level (A), MMP (B), Bax and Bcl-2 expression (C and D), the oxidative stress ROS and MDA levels (E), and antioxidative capacity SOD and GSH levels (F) were detected in OGD-induced spinal cord neuron. Data are presented as the mean ± SEM, n = 4. *p < 0.05, vs. the control group (CTL). ^#p < 0.05, vs. the OGD group. A and B: Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. C: Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. OGD, oxygen-glucose deprivation; NSA, necrosulfonamide; ATP, adenosine triphosphate; MMP, mitochondrial membrane potential; GSH, glutathione; SOD, superoxide dismutase; ROS, reactive oxygen species; MDA, malonyldialdehyde.

Figure 4

NSA improves the motor function and spinal edema of SCI-mice. The behavioral performances in mice with SCI or treatment with NSA (L: 1 mg/kg, M: 5 mg/kg, H: 10 mg/kg) were shown at 1, 2, 3, 7, 14, 21, and 28 days following surgery. (A) pair forepaws were measured. (B) Daily measurement of the BMS scores. (C) Dry and wet specific gravity method was used to measure spinal edema at 3 days post-SCI. Data are reported as the mean ± SEM, n = 9. ^& p < 0.05, vs. the sham group. *p < 0.05, vs. the SCI+Vehicle group. ^# p < 0.05, vs. the NSA-L group. A and B: Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. C: Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. SCI, spinal cord injury; NSA, necrosulfonamide; BMS, basso mouse scale.

Figure 5

The therapeutic window of NSA after SCI. The time of administration of NSA was 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h after surgery in groups, respectively. The behavioral performances conducted by forelimb grip strength test (A) and BMS score system (B) were observed at 1, 3, 7, 14, 21, and 28 days following SCI surgery. (C) Measurement of spinal edema at 3 days post-SCI. Data are reported as the mean ± SEM, n = 9. *p <0.05, vs. the SCI+Vehicle group. ^# p < 0.05, vs. the 24 h group. (A and B): Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. C: Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. SCI, spinal cord injury; NSA, necrosulfonamide; BMS, basso mouse scale.

Figure 6

NSA reverses mitochondrial capacity and antioxidative capacity via inhibiting MLKL activation in spinal cord tissues. After SCI, NSA was treated at different times, such as 15 min, 1 h, 6 h, 12 h, and 24 h. (A) The protein expression of p-MLKL was detected at 3 days post-SCI using WB analysis, histogram analysis of change of p-MLKL. ELISA analysis of mitochondrial dysfunction at 3 days post-SCI, including ATP and MMP levels (B), and the unbalanced antioxidant capacity [the levels of ROS and MDA (C) and the levels of SOD and GSH (D)]. Data are reported as the mean ± SEM, n = 8. *p < 0.05, vs. the sham group. ^# p < 0.05, vs. the SCI+Vehicle group. ^& p < 0.05, vs. the SCI+NSA 15-min group. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. SCI, spinal cord injury; NSA, necrosulfonamide; MLKL, mixed-lineage kinase domain-like protein; ATP, adenosine triphosphate; MMP, mitochondrial membrane potential; GSH, glutathione; SOD, superoxide dismutase; ROS, reactive oxygen species; MDA, malonyldialdehyde; WB, Western blot.

Figure 7

NSA improves the recovery of SCI and the survival of neurons. (A) HE staining results of the sham, SCI and SCI+NSA group. Treated with 5 mg/kg NSA 15 min after SCI, NSA significant protective effect with less necrosis, karyopyknosis, infiltrated macrophages compared with the SCI group, scale bar = 50 μm. (B) NeuN/TUNEL staining results of the sham, SCI and SCI+NSA group, scale bar = 20 μm. Analysis of the positive neurons of the TUNEL staining results. *p < 0.05 vs. the sham group, ^# p  < 0.01 vs. the SCI group. n  =  5. SCI, spinal cord injury; NSA, necrosulfonamide; TUNEL, terminal deoxynucleotidyl transferase-dUTP nick end labeling.