sphingosine-kinase and Brain-Ischemia

Sphingosine 1-phosphate (S1P) is an important lipid biomolecule that exerts pleiotropic cellular actions as it binds to and activates its five G-protein-coupled receptors, S1P

Topics: Animals; Brain Damage, Chronic; Brain Ischemia; Clinical Trials as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Fingolimod Hydrochloride; Humans; Infarction, Middle Cerebral Artery; Inflammation; Ischemic Stroke; Lysophospholipids; Neovascularization, Physiologic; Nerve Tissue Proteins; Neuroprotective Agents; Phosphotransferases (Alcohol Group Acceptor); Rats; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Microglial hyperactivation mediated by sphingosine kinase 1/sphingosine-1-phosphate (SphK1/S1P) signalling and the consequent inflammatory mediator production serve as the key drivers of cerebral ischaemia-reperfusion injury (CIRI). Although SphK1 reportedly controls autophagy and microglial activation, it remains uncertain as to whether SphK1 is similarly capable of regulating damage mediated by CIRI-activated microglia. In the current study, we adopted both in vitro oxygen-glucose deprivation reperfusion (OGDR) models and in vivo rat models of focal CIRI to ascertain this possibility. It was found that CIRI upregulated SphK1 and induced autophagy in microglia, while inhibiting these changes significantly impaired to prevented neuronal apoptosis. Results of mechanistic investigation revealed that SphK1 promoted autophagy via the tumour necrosis factor receptor associated factor 2 (TRAF2) pathway. Altogether, our findings unfolded to reveal a novel mechanism, whereby SphK1-induced autophagy in microglia contributed to the pathogenesis of CIRI, potentially highlighting novel avenues for future therapeutic intervention in ischaemic stroke patients.

Topics: Animals; Autophagy; Brain Ischemia; Microglia; Phosphotransferases (Alcohol Group Acceptor); Rats; Reperfusion; Reperfusion Injury; Stroke

Sphingosine kinase 1 (Sphk1) and the signaling molecule sphingosine-1-phosphate (S1P) are known to be key regulators of a variety of important biological processes, such as neovascularization. Nitric oxide (NO) is also known to play a role in vasoactive properties, whether Sphk1/S1P signaling is able to alter angiogenesis in the context of cerebral ischemia-reperfusion injury (IRI), and whether such activity is linked with NO production, however, remains uncertain.. We used immunofluorescence to detect the expression of Sphk1 and NOS in cerebral epithelial cells (EC) after IR or oxygen-glucose deprivation (OGDR). Western blotting was used to detect the Sphk1 and NOS protein levels in brain tissues or HBMECs. Adenovirus transfection was used to inhibit Sphk1 and NOS. An NO kit was used to detect NO contents in brain tissues and epithelial cells. Tube formation assays were conducted to measure angiogenesis.. We determined that EC used in a model of cerebral IRI expressed Sphk1, and that inhibiting this expression led to decreased expression of two isoforms of NO synthase (eNOS and iNOS), as well as to decrease neovascularization density and NO production following injury. In HBMECs, knocking down Sphk1 markedly reduced NO production owing to reduced eNOS activity, and inhibiting eNOS directly similarly decreased NO production in a manner which could be reversed via exogenously treating cells with S1P. We further found that knocking down Sphk1 reduced HBMEC eNOS expression, in addition to decreasing the adhesion, migration, and tube formation abilities of these cells under OGDR conditions.. Based on these results, we therefore postulate that Sphk1/S1P signaling is able to mediate angiogenesis following cerebral IRI via the regulation of eNOS activity and NO production. As such, targeting these pathways may potentially represent a novel means of improving patient prognosis in those suffering from cerebral IRI.

Topics: Animals; Brain Ischemia; Cells, Cultured; Humans; Lysophospholipids; Male; Neovascularization, Pathologic; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Reperfusion Injury; Sphingosine

Endoplasm reticulum stress and inflammation response have been found to be linked to cerebral ischemia-reperfusion (IR) injury. Sphingosine kinase 1 (SPHK1) has been reported to be a novel endoplasm reticulum regulator. The aim of our study is to figure out the role of SPHK1 in cerebral IR injury and verify whether it has an ability to regulate inflammation and endoplasm reticulum stress. Hydrogen peroxide was used to induce cerebral IR injury. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, western blots, and immunofluorescence were used to measure the alterations of cell viability, inflammation response, and endoplasm reticulum stress. The results demonstrated that after exposure to hydrogen peroxide, cell viability was reduced whereas SPHK1 expression was significantly elevated. Knockdown of SPHK1 attenuated hydrogen peroxide-mediated cell death and reversed cell viability. Our data also demonstrated that SPHK1 deletion reduced endoplasm reticulum stress and alleviated inflammation response in hydrogen peroxide-treated cells. In addition, we also found that SHPK1 modulated endoplasm reticulum stress and inflammation response to through the NF-κB signaling pathway. Inhibition of NF-κB signaling pathway has similar results when compared with the cells with SPHK1 deletion. Altogether, our results demonstrated that SPHK1 upregulation, induced by hydrogen peroxide, is responsible for cerebral IR injury through inducing endoplasm reticulum stress and inflammation response in a manner working through the NF-κB signaling pathway. This finding provides new insight into the molecular mechanism to explain the neuron death induced by cerebral IR injury.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Stress; Inflammation; Mice; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Signal Transduction

There is thought to be a strong relationship between sphingosine-1-phosphate (S1P) signaling and pathophysiolosy of cerebral ischemia. We examined the change of expression and distribution of S1P receptors (S1PRs) and sphingosine kinases (SphKs) after cerebral ischemia in male C57BL6/J mice using immunohistochemical analysis at 1, 5, 14, and 28 days after 30 min of transient middle cerebral artery occlusion (tMCAO). S1PR1, 3, and 5 were transiently induced in the cells, which were morphologically similar to neurons in the peri-infarct lesion with a peak seen at 1 day after tMCAO (p < 0.01 vs. sham control). S1PR2 appeared in the inner layer of vessels in the ischemic core (p < 0.01 vs. sham control) and the peri-infarct lesion (p < 0.01 vs. sham control) at the acute phase after tMCAO. However, SphK1 was strongly induced at 1 and 5 days after tMCAO (p < 0.01 vs. sham control) in the peri-infarct lesion, whereas SphK2 expression did not change. Western blot analysis at 1 and 5 days after 30 min of tMCAO revealed that the expression of S1PRs were transiently enhanced at the acute phase, which was consistent with the immunohistochemical results. Double immunofluorescent analysis revealed S1PR2/NG2- and S1PR2/CD31-, S1PR3/CD31-, and S1PR5/CD31-double positive cells in the peri-infarct lesion 1 day after tMCAO. The present results suggest that S1PRs and SphK1 may be important therapeutic targets for rescuing the peri-infarct lesion.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Neurons; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Transcriptional Activation

Isoflurane preconditioning could reduce different kinds of brain injury via sphingosine kinase (SPK). Both sphingosine kinase 1 and sphingosine kinase 2 play important roles in brain protection. However, the effects of isoflurane preconditioning on SPK expression in hypertension have not been investigated before.. To verify whether the neuroprotective effects of the anesthetic isoflurane after an ischemic injury are altered in hypertension and to identify its possible mechanisms involving SPK.. Wistar rats (control) and spontaneous hypertension rats (SHR) were exposed to isoflurane preconditioning before transient middle cerebral artery occlusion. The infarct volumes of cortical and subcortical brain areas were measured. The expression levels of SPK1 and SPK2 were measured before and after isoflurane preconditioning.. In the SHR group, isoflurane preconditioning significantly reduced only the infarct volumes of the subcortical brain (p < 0.05), not of the cortical brain. After 3 h of isoflurane exposure and preconditioning, SPK2 levels in the SHR group increased in the cortical brain (p < 0.05), but not in the subcortical brain area, Unlike in the control group, isoflurane exposure and preconditioning could significantly increase SPK2 levels in both cortical and subcortical brain area.. The brain protection effects induced by isoflurane preconditioning after an ischemic injury are mainly mediated by the SPK2 isoform and are somewhat impaired in hypertension. Attention should be paid to ischemic injury patients with hypertension.

Topics: Anesthetics, Inhalation; Animals; Brain Ischemia; Humans; Hypertension; Ischemic Preconditioning; Isoflurane; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingosine

Topics: Animals; Brain; Brain Ischemia; Enzyme Inhibitors; Hemodynamics; Humans; Lysophospholipids; Male; Mice; Microscopy; Neuroprotection; Neuroprotective Agents; Oxygen; Phosphotransferases (Alcohol Group Acceptor); Photoacoustic Techniques; Sphingosine; Stroke

Stroke is associated with high morbidity and mortality, and much remains unknown about the injury-related mechanisms that occur following reperfusion. This study aimed to explore the roles of Toll-like receptor 2 (TLR2) and sphingosine kinase 1 (Sphk1) in microglial cells in inflammatory responses induced by cerebral ischemia/reperfusion (I/R). For this purpose, C57BL/6 mice were randomly divided into 4 groups as follows: the sham-operated group, the I/R group, the I/R group treated with TLR2 antibody, and the I/R group treated with N,N-dimethylsphingosine. Focal cerebral I/R was induced by middle cerebral artery occlusion. Double-labeling immunofluorescence was used to observe the protein expression of TLR2 and Sphk1 in the ischemic brain tissue. Quantitative polymerase chain reaction was performed to determine the mRNA levels of TLR2 and Sphkl in ischemic brain tissue. Enzyme-linked immunosorbent assay was carried out to detect the protein contents of interleukin (IL)-1β, tumor necrosis factor-α (TNF‑α), IL-17 and IL-23 in ischemic brain tissue. The results revealed that I/R upregulated TLR2 and Sphk1 expression in microglial cells, and the inhibition of either TLR2 or Sphk1 inhibited the expression of the pro-inflammatory cytokines, IL-1β, TNF-α, IL-17 and IL-23. Notably, the inhibition of TLR2 activity also decreased Sphk1 expression. These results thus indicate that the activation of microglial cells, via a TLR2→Sphk1→pro-inflammatory cytokine (IL-1β, TNF-α, IL-17 and IL-23) pathway, may participate in I/R injury.

Topics: Animals; Brain; Brain Ischemia; Cytokines; Inflammation; Male; Mice; Mice, Inbred C57BL; Microglia; Phosphotransferases (Alcohol Group Acceptor); Reperfusion Injury; Toll-Like Receptor 2

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.. 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.. 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.. 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.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cells, Cultured; Glucose; Hypoxia, Brain; Infarction, Middle Cerebral Artery; Interleukin-17; Lysophospholipids; Male; Methanol; Microglia; Neurons; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Small Interfering; Sphingosine; Sulfones

Allicin, one of the main biologically active compounds derived from garlic, has been shown to exert various pharmacological activities and is considered to have therapeutic potential for many pathologic conditions. In the present study, we investigated the potential post-ischemic neuroprotective effects of allicin and its underlying mechanisms. Using a rat middle cerebral artery occlusion (MCAO) model, we found that intraperitoneal treatment with 50 mg/kg allicin significantly reduced brain infarct volume, attenuated cerebral edema and decreased the neurological deficit score. Allicin treatment also diminished TUNEL positive cells and inhibited the activation of caspase-3 after MCAO. These protective effects could be observed even if the administration was delayed to 6 h after injury. In addition, we evaluated the in vitro protective effects of allicin against oxygen glucose deprivation (OGD) induced neuronal injury in primary cultured cortical neurons. Allicin (50 μM) increased neuronal viability, decreased lactate dehydrogenase (LDH) release and inhibited apoptotic neuronal death after OGD. These protective effects could be observed even if the administration was delayed to 4 h after injury. Furthermore, allicin significantly increased the expression of sphingosine kinases 2 (Sphk2) both in vivo and in vitro. Pretreatment with the Sphk2 inhibitor ABC294640 partially reversed the protective effects of allicin against MCAO and OGD injury, indicating that an Sphk2-mediated mechanism was involved in allicin-induced protection in our models. The combination of findings suggests that post-injury administration of allicin has potential as a neuroprotective strategy for ischemic stroke.

Topics: Animals; Brain Injuries; Brain Ischemia; Cell Survival; Cells, Cultured; Cerebral Cortex; Disulfides; Drug Administration Schedule; Enzyme Activation; Male; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Sulfinic Acids

Initial and recurrent stroke produces central nervous system (CNS) damage, involving neuroinflammation. Receptor-mediated S1P signaling can influence neuroinflammation and has been implicated in cerebral ischemia through effects on the immune system. However, S1P-mediated events also occur within the brain itself where its roles during stroke have been less well studied. Here we investigated the involvement of S1P signaling in initial and recurrent stroke by using a transient middle cerebral artery occlusion/reperfusion (M/R) model combined with analyses of S1P signaling. Gene expression for S1P receptors and involved enzymes was altered during M/R, supporting changes in S1P signaling. Direct S1P microinjection into the normal CNS induced neuroglial activation, implicating S1P-initiated neuroinflammatory responses that resembled CNS changes seen during initial M/R challenge. Moreover, S1P microinjection combined with M/R potentiated brain damage, approximating a model for recurrent stroke dependent on S1P and suggesting that reduction in S1P signaling could ameliorate stroke damage. Delivery of FTY720 that removes S1P signaling with chronic exposure reduced damage in both initial and S1P-potentiated M/R-challenged brain, while reducing stroke markers like TNF-α. These results implicate direct S1P CNS signaling in the etiology of initial and recurrent stroke that can be therapeutically accessed by S1P modulators acting within the brain.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Fingolimod Hydrochloride; Lysophospholipids; Male; Mice; Mice, Inbred ICR; Microinjections; Neuroglia; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Stroke; Tumor Necrosis Factor-alpha

Although there is evidence that sphingosine-1-phosphate receptor-1 (S1P1) activation occurs following experimental brain injury, there is little information about its metabolic pathway in cerebral ischemia. The purpose of this study was to evaluate the role of the sphingosine metabolic pathway including S1P1, sphingosine kinases 1 (SphK1), and 2 (SphK2) in transient middle cerebral artery occlusion (MCAO). Fifty-eight male Sprague-Dawley rats were used to asses temporal profiles of S1P1, SphK1 and 2 on neurons in infarct and periinfarct cortices at pre-infarct state, 6, and 24 hours after MCAO. The animals were then treated with vehicle and 0.25 mg/kg FTY720, which is an agonist of S1P receptors, and evaluated regarding neurological function, infarct volume, and S1P1 expression on neurons at 24 hours after MCAO. The expressions of S1P1, SphK1, and SphK2 were significantly decreased after MCAO. Labeling of all markers were reduced in the infarct cortex but remained present in the periinfarct cortex, and some were found to be on neurons. Significant improvements of neurological function and brain injury were observed in the FTY720 group compared with the vehicle and untreated groups, although S1P1 expression on neurons was reduced in the FTY720 group compared with the vehicle group. We demonstrated that S1P1, SphK1, and SphK2 were downregulated in the infarct cortex, whereas they were preserved in the periinfarct cortex where FTY720 reduced neuronal injury possibly via S1P1 activation. Our findings suggest that activation of the sphingosine metabolic pathway may be neuroprotective in cerebral ischemia.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Fingolimod Hydrochloride; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Male; Neurons; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Rats; Rats, Sprague-Dawley; Sphingosine

Cerebral preconditioning provides insights into endogenous mechanisms that protect the brain from ischemic injury. Hypoxia and the anesthetic isoflurane are powerful preconditioning agents. Recent data show that sphingosine 1-phosphate receptor stimulation improves outcome in rodent models of stroke. Endogenous sphingosine 1-phosphate levels are controlled by the expression and activity of sphingosine kinases (SPK). We hypothesize that SPK upregulation mediates preconditioning induced by isoflurane and hypoxia and reduces ischemic injury.. Male wild-type C57BL/J, SPK1(-/-) and SPK2(-/-) mice were exposed to isoflurane or hypoxia preconditioning before transient middle cerebral artery occlusion. Infarct volume and neurological outcome were measured 24 hours later. SPK inhibitors (SKI-II and ABC294640) were used to test the involvement of SPK2. Expressions of SPK1, SPK2, and hypoxia-inducible factor 1α were determined. Primary cultures of mouse cortical neurons were exposed to isoflurane before glutamate- or hydrogen peroxide-induced cell death.. Isoflurane preconditioning and hypoxia preconditioning significantly reduced infarct volume and improved neurological outcome in wild-type and SPK1(-/-) mice but not in SPK2(-/-) mice. Pretreatment with SKI-II or ABC294640 abolished the isoflurane preconditioning-induced tolerance. Western blot showed a rapid and sustained increase in SPK2 level, whereas SPK1 level was similar between preconditioned mice and controls. Hypoxia-inducible factor 1α was upregulated in wild-type isoflurane-preconditioned mice but not in SPK2(-/-). Isoflurane preconditioning protected primary neurons against cell death, which was abolished in ABC294640-treated cells.. Applying genetic and pharmacological approaches, we demonstrate that neuronal SPK2 isoform plays an important role in cerebral preconditioning.

Topics: Animals; Brain; Brain Ischemia; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Male; Mice; Mice, Knockout; Neurons; Phosphotransferases (Alcohol Group Acceptor)

Protection of the blood-brain barrier (BBB) is correlated with improved outcome in stroke. Sphingosine kinase (SphK)-directed production of sphingosine-1-phosphate, which we previously documented as being vital to preconditioning-induced stroke protection, mediates peripheral vascular integrity via junctional protein regulation. We used a hypoxic preconditioning (HPC) model in adult wild-type and SphK2-null mice to examine the isoform-specific role of SphK2 signaling for ischemic tolerance to transient middle cerebral artery occlusion and attendant BBB protection. Reductions in infarct volume and BBB permeability in HPC-treated mice were completely lost in SphK2-null mice. Hypoxic preconditioning-induced attenuation of postischemic BBB disruption in wild types, evidenced by reduced extravascular immunoglobulin G intensity, suggests direct protection of BBB integrity. Measurement of BBB junctional protein status in response to HPC revealed SphK2-dependent increases in triton-insoluble claudin-5 and VE-cadherin, which may serve to strengthen the BBB before stroke. Postischemic loss of VE-cadherin, occludin, and zona occludens-1 in SphK2-null mice with prior HPC suggests that SphK2-dependent protection of these adherens and tight junction proteins is compulsory for HPC to establish a vasculoprotective phenotype. Further elucidation of the mediators of this endogenous, HPC-activated lipid signaling pathway, and their role in protecting the ischemic BBB, may provide new therapeutic targets for cerebrovascular protection in stroke patients.

Topics: Animals; Antigens, CD; Blood-Brain Barrier; Brain Ischemia; Cadherins; Claudin-5; Claudins; Humans; Ischemic Preconditioning; Isoenzymes; Male; Membrane Proteins; Mice; Mice, Knockout; Nerve Tissue Proteins; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Stroke; Zonula Occludens-1 Protein

The two ubiquitously expressed sphingosine kinases (SphK) 1 and 2 are key regulators of the sphingolipid signaling pathway. Despite the formation of an identical messenger, i.e. sphingosine 1-phosphate (S1P), they exert strikingly different functions. Particularly, SphK2 is necessary for the phosphorylation of the sphingosine analog fingolimod (FTY720), which is protective in rodent stroke models. Using gene deficient mice lacking either SphK1 or SphK2, we investigated the role of the two lipid kinases in experimental stroke. We performed 2h transient middle cerebral artery occlusion (tMCAO) and analyzed lesion size and neurological function after 24h. Treatment groups received 1mg/kg FTY720. Neutrophil infiltration, microglia activation, mRNA and protein expression of SphK1, SphK2 and the S1P(1) receptor after tMCAO were studied. Genetic deletion of SphK2 but not SphK1 increased ischemic lesion size and worsened neurological function after tMCAO. The protective effect of FTY720 was conserved in SphK1(-/-) mice but not in SphK2(-/-) mice. This suggests that SphK2 activity is an important endogenous protective mechanism in cerebral ischemia and corroborates that the protective effect of FTY720 is mediated via phospho-FTY720.

Topics: Animals; Brain Ischemia; Enzyme Activation; Fingolimod Hydrochloride; Gene Deletion; Infarction, Middle Cerebral Artery; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Sphingosine