pf-543 and Brain-Ischemia

To investigate the influence of dexmedetomidine (Dex) on cerebral ischemia/reperfusion (I/R)-injured rat neuronal cells by regulating the Sphk1/S1P pathway.. The rats were divided into the following groups, with 18 rats in each group categorized on the basis of random number tables: sham (Sham), I/R (I/R), Dex, Sphk1 inhibitor (PF-543), and Dex together with the Sphk1 agonist phorbol-12-myristate-13-acetate (Dex+PMA). The neurological functions of the rats were assessed by the Longa scoring system at 24 h post reperfusion. The area of brain infarction was inspected using 2,3,5-triphenyltetrazolium chloride staining, and the water content of brain tissue was determined by the dry-wet weight method. The morphology of neurons in the CA1 region of the rat hippocampus was inspected using Nissl staining, while the apoptosis of neurons in this region was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling staining. The Sphk1 and S1P protein levels were determined by immunofluorescence and western blotting, respectively.. Compared to the I/R group, rats in the Dex, PF-543, and Dex+PMA groups had a significantly lower neurological function score, as well as lower brain water content and a decreased infarction area. Moreover, the apoptotic index of the neurons and the Sphk1 and S1P levels in the hippocampal CA1 region were significantly lower in these groups (p<0.05). PMA, an agonist of Sphk1, was able to reverse the protective effects of Dex on I/R-induced neuronal cell injury.. Dex could protect cerebral I/R-induced neuronal cell injury by suppressing the Sphk1/S1P signaling pathway.

Topics: Animals; Apoptosis; Brain Ischemia; CA1 Region, Hippocampal; Cerebral Infarction; Dexmedetomidine; Neurons; Rats; Rats, Sprague-Dawley; Reperfusion; Reperfusion Injury; Signal Transduction

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