Abstract
BackgroundThe pathological process of traumatic spinal cord injury (SCI) involves excessive activation of microglia leading to the overproduction of proinflammatory cytokines and causing neuronal injury. Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in mediating inflammation, cell proliferation, survival, and immunity.MethodsWe aim to investigate the mechanism and pathway of the Sphk1-mediated neuroinflammatory response in a rodent model of SCI. Sixty Sprague-Dawley rats were randomly assigned to sham surgery, SCI, or PF543 (a specific Sphk1 inhibitor) groups. Functional outcomes included blinded hindlimb locomotor rating and inclined plane test.ResultsWe discovered that Sphk1 is upregulated in injured spinal cord tissue of rats after SCI and is associated with production of S1P and subsequent NF-κB p65 activation. PF543 attenuated p65 activation, reduced inflammatory response, and relieved neuronal damage, leading to improved functional recovery. Western blot analysis confirmed that expression of S1P receptor 3 (S1PR3) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) are activated in microglia of SCI rats and mitigated by PF543. In vitro, we demonstrated that Bay11-7085 suppressed NF-κB p65 and inhibited amplification of the inflammation cascade by S1P, reducing the release of proinflammatory TNF-α. We further confirmed that phosphorylation of p38 MAPK and activation of NF-κB p65 is inhibited by PF543 and CAY10444. p38 MAPK phosphorylation and NF-κB p65 activation were enhanced by exogenous S1P and inhibited by the specific inhibitor SB204580, ultimately indicating that the S1P/S1PR3/p38 MAPK pathway contributes to the NF-κB p65 inflammatory response.ConclusionOur results demonstrate a critical role of Sphk1 in the post-traumatic SCI inflammatory cascade and present the Sphk1/S1P/S1PR3 axis as a potential target for therapeutic intervention to control neuroinflammation, relieve neuronal damage, and improve functional outcomes in SCI.
Highlights
Spinal cord injury (SCI) affects approximately 282,000 people in the USA, with 17,000 new cases each year [1]
To investigate the role of Sphingosine kinase 1 (Sphk1) following spinal cord injury (SCI), we examined the effects of PF543, a highly selective and efficient inhibitor of Sphk1 leading to reduced intracellular S1P [30], on microglial M1 polarization and neuronal apoptosis 3 days after surgery
Western blot assays for additional markers showed that expression of M1 phenotypic markers Inducible nitric oxide synthase (iNOS) and COX-2 and proinflammatory cytokines tumor necrosis factor (TNF)-α and IL-6 were significantly increased after SCI (Fig. 1b, Sham group vs. SCI group: 1.00 ± 0.07 vs. 2.21 ± 0.16, P = 0.001 for iNOS, 1.00 ± 0.11 vs. 2.14 ± 0.10, P = 0.002 for COX-2, 1.00 ± 0.10 vs. 3.16 ± 0.28, P < 0.001 for tumor necrosis factor-α (TNF-α), 1.00 ± 0.12 vs. 2.02 ± 0.16, P = 0.004 for IL-6)
Summary
Spinal cord injury (SCI) affects approximately 282,000 people in the USA, with 17,000 new cases each year [1]. Less than 1% of patients recover full neurological function at the time of discharge, and despite advances in science, life expectancy for SCI patients is significantly lower than the general population, and there has been minimal change in mortality rate for these patients over the last 40 years [1]. Current clinical interventions such as surgical decompression, drug therapies, and hypothermia are aimed at reduction of secondary injury and remain non-curative [2], and the care costs of this disease remain immense [3]. Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in mediating inflammation, cell proliferation, survival, and immunity
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