Abstract
Background
Microglial activation is one of the causative factors of neuroinflammation in cerebral ischemia/reperfusion (IR). Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in the regulation of proinflammatory cytokines in activated microglia. Recent research demonstrated that S1P increased IL-17A-secretion and then worsened CNS (central nervous system) inflammation. Thus, in the present study, we sought to use microglial cells as the object of study to discuss the molecular mechanisms in Sphk1/S1P-regulated IL-17A-secretion in IR.
Methods
We used immunofluorescence and confocal microscopy to detect whether Sphk1 is expressed in microglia after cerebral IR or oxygen-glucose deprivation (OGDR). Western blot analysis was used to estimate the total Sphk1 protein level at different time points after OGDR. To detect cytokine secretion in microglial supernatants in response to OGDR, we measured the concentration of IL-17A in the culture supernatants using an enzyme-linked immunosorbent assay (ELISA). To evaluate whether microglia subjected to OGDR exhibited neuronal injury, we used a commercially available terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) kit to detect apoptotic neurons.
Results
Sphk1 was expressed in microglia in response to cerebral IR or OGDR at appointed time. Pre-injection with PF-543, an inhibitor of Sphk1, before IR clearly reduced the expression of Sphk1 in microglia relative to brain IR alone. The number of TUNEL-positive neurons was also decreased in the PF-543-pretreated animals before IR compared to the animals with IR alone. When S1P was administered in OGDR microglia, IL-17A expression and neuronal apoptosis were increased compared to OGDR alone and the administration of S1P alone. ELISA further confirmed the above results. Moreover, the inhibition of Sphk1 by siRNA reduced IL-17A production and relieved neuronal apoptosis in OGDR microglia.
Conclusion
These results indicated that Sphk1/S1P regulates the expression of IL-17A in activated microglia, inducing neuronal apoptosis in cerebral ischemia/reperfusion. The microglial Sphk1/S1P pathway may thus be a potential therapeutic target to control neuroinflammation in brain IR.
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Abbreviations
- Sphk1:
-
Sphingosine kinase 1
- S1P:
-
Sphingosine-1-phosphate
- IL-17A:
-
Interleukin-17A
- IRI:
-
Ischemia/reperfusion injury
- CNS:
-
Central nervous system
- OGDR:
-
Oxygen-glucose deprivation reperfusion
- NO:
-
Nitric oxide
- TNF-α:
-
Tumor necrosis factor-α
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Acknowledgments
This research was supported by the fund of the First Affiliated Hospital of Harbin Medical University (2012BS003). We are grateful for the technical assistance of all members of the Department of Pharmacology of Harbin Medical University.
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Responsible Editor: Ji Zhang.
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11_2016_939_MOESM1_ESM.jpg
Supplement 1. Identification of the purity of microglia cells. (a) Primary cultured microglia. (b) After purification, the microglial growth factor was added to cultured medium. (c) In addition the slide plate, microglia growth on glass slides. (d-f) Immunofluorescence images showed microglia was double-stained with Iba1 (red) and DAPI (blue). Confocal microscopy, scale bar: 100 μm (JPEG 1953 kb)
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Supplement 2. Identification of the purity of neurons. Primary cultured cells were double-stained with Neun (green) and DAPI (blue). Confocal microscopy, scale bar: 100 μm (JPEG 2163 kb)
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Supplement 3. Expression of EGFP on microglia subjected to SphK1 siRNA. Primary microglia transfected with SphK1 siRNA were culture for 48 h and stained with antibody against EGFP (green) and microglia (red). Confocal microscopy, scale bar: 100 μm (JPEG 745 kb)
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Lv, M., Zhang, D., Dai, D. et al. Sphingosine kinase 1/sphingosine-1-phosphate regulates the expression of interleukin-17A in activated microglia in cerebral ischemia/reperfusion. Inflamm. Res. 65, 551–562 (2016). https://doi.org/10.1007/s00011-016-0939-9
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DOI: https://doi.org/10.1007/s00011-016-0939-9