sphingosine-kinase and dihydrosphingosine-1-phosphate

Cardiac fibrosis and myocyte hypertrophy are hallmarks of the cardiac remodelling process in cardiomyopathies such as heart failure (HF). Dyslipidemia or dysregulation of lipids contribute to HF. The dysregulation of high density lipoproteins (HDL) could lead to altered levels of other lipid metabolites that are bound to it such as sphingosine-1- phosphate (S1P). Recently, it has been shown that S1P and its analogue dihydrosphingosine-1-phosphate (dhS1P) are bound to HDL in plasma. The effects of dhS1P on cardiac cells have been obscure. In this study, we show that extracellular dhS1P is able to increase collagen synthesis in neonatal rat cardiac fibroblasts (NCFs) and cause hypertrophy of neonatal cardiac myocytes (NCMs). The janus kinase/signal transducer and activator (JAK/STAT) signalling pathway was involved in the increased collagen synthesis by dhS1P, through sustained increase of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). Extracellular dhS1P increased phosphorylation levels of STAT1 and STAT3 proteins, also caused an early increase in gene expression of transforming growth factor-β (TGFβ), and sustained increase in TIMP1. Inhibition of JAKs led to inhibition of TIMP1 and TGFβ gene and protein expression. We also show that dhS1P is able to cause NCM hypertrophy through S1P-receptor-1 (S1PR1) signalling which is opposite to that of its analogue, S1P. Taken together, our results show that dhS1P increases collagen synthesis in cardiac fibroblasts causing fibrosis through dhS1P-JAK/STAT-TIMP1 signalling.

Topics: Animals; Animals, Newborn; Biomarkers; Cell Differentiation; Collagen; Fibroblasts; Gene Expression Regulation; Hypertrophy; Janus Kinases; Lysophospholipids; Matrix Metalloproteinase 2; Models, Biological; Myocardium; Myocytes, Cardiac; Oxadiazoles; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Smad2 Protein; Sphingosine; STAT Transcription Factors; Thiophenes; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Topics: Antimicrobial Cationic Peptides; Cathelicidins; Cell Differentiation; Cells, Cultured; Endoplasmic Reticulum Stress; Humans; Keratinocytes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Primary Cell Culture; Sphingosine

Preanalytical standardization is required for a reliable quantification of the signaling molecules sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and sphingosine (SPH).. Methanolic protein precipitation of 15μL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3min by hydrophilic interaction liquid chromatography (HILIC, SeQuant™ ZIC®-HILIC column) followed by tandem mass spectrometry. Stability of analytes in whole blood and plasma was investigated. Sphingolipid concentrations were determined in human plasma (n=50) and mice deficient in sphingosine kinase 1 (SK1) and 2 (SK2) (n=5).. Storing EDTA whole blood >60min after blood withdrawal at room temperature resulted in an increase in S1P and SPH concentrations of ≥25%. Significant changes in SPH levels of +37% were observed after 60min of storage of EDTA plasma at room temperature. Repeated freeze-thaw cycles of EDTA plasma resulted in increased S1P and SPH levels. Concentrations in human EDTA plasma were between 55.5 and 145.2ng/mL for S1P and between 8.9 and 35.3ng/mL for SA1P. Concentrations of S1P were 36% lower and 96% higher in EDTA-plasma from SK1- and SK2-deficient mice, respectively, compared to the wild type.. Preanalytical standardization is a precondition for the analysis of sphingolipids in human blood.

Topics: Animals; Blood Chemical Analysis; Chromatography, Liquid; Female; Healthy Volunteers; Humans; Lysophospholipids; Male; Mice; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Reference Standards; Reproducibility of Results; Sphingosine; Tandem Mass Spectrometry; Time Factors

Tumor-associated inflammation mediates the development of a systemic immunosuppressive milieu that is a major obstacle to effective treatment of cancer. Inflammation has been shown to promote the systemic expansion of immature myeloid cells which have been shown to exert immunosuppressive activity in laboratory models of cancer as well as cancer patients. Consequentially, significant effort is underway toward the development of therapies that decrease tumor-associated inflammation and immunosuppressive cells. The current study demonstrated that a previously described deep tissue imaging modality, which utilized indocyanine green-loaded calcium phosphosilicate nanoparticles (ICG-CPSNPs), could be utilized as an immunoregulatory agent. The theranostic application of ICG-CPSNPs as photosensitizers for photodynamic therapy was shown to block tumor growth in murine models of breast cancer, pancreatic cancer, and metastatic osteosarcoma by decreasing inflammation-expanded immature myeloid cells. Therefore, this therapeutic modality was termed PhotoImmunoNanoTherapy. As phosphorylated sphingolipid metabolites have been shown to have immunomodulatory roles, it was hypothesized that the reduction of immature myeloid cells by PhotoImmunoNanoTherapy was dependent upon bioactive sphingolipids. Mechanistically, PhotoImmunoNanoTherapy induced a sphingosine kinase 2-dependent increase in sphingosine-1-phosphate and dihydrosphingosine-1-phosphate. Furthermore, dihydrosphingosine-1-phosphate was shown to selectively abrogate myeloid lineage cells while concomitantly allowing the expansion of lymphocytes that exerted an antitumor effect. Collectively, these findings revealed that PhotoImmunoNanoTherapy, utilizing the novel nontoxic theranostic agent ICG-CPSNP, can decrease tumor-associated inflammation and immature myeloid cells in a sphingosine kinase 2-dependent manner. These findings further defined a novel myeloid regulatory role for dihydrosphingosine-1-phosphate. PhotoImmunoNanoTherapy holds the potential to be a revolutionary treatment for cancers with inflammatory and immunosuppressive phenotypes.

Topics: Animals; Cell Line, Tumor; Combined Modality Therapy; Female; Humans; Immunotherapy; Indocyanine Green; Lymphocytes; Mice; Mice, Nude; Myeloid Cells; Nanoparticles; Nanotechnology; Neoplasms, Experimental; Phosphotransferases (Alcohol Group Acceptor); Photochemotherapy; Silicates; Sphingosine; Xenograft Model Antitumor Assays

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.

Topics: Animals; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Female; Gene Expression Regulation, Enzymologic; Humans; Mice; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Fibrosis; Radiation Injuries, Experimental; Serine C-Palmitoyltransferase; Signal Transduction; Sphingosine; Thorax; Time Factors; Transforming Growth Factor beta; Up-Regulation

Three bioactive sphingolipids, namely sphingosine-1-phosphate (S1P), ceramide (CER) and sphingosine (SPH) were shown to be involved in ischemia/reperfusion injury of the heart. S1P is a powerful cardioprotectant, CER activates apoptosis and SPH in a low dose is cardioprotective whereas in a high dose is cardiotoxic. The aim of the present study was to examine effects of experimental myocardial infarction on the level of selected sphingolipids in plasma, erythrocytes and platelets in the rat. Myocardial infarction was produced in male Wistar rats by ligation of the left coronary artery. Blood was taken from the abdominal aorta at 1, 6 and 24 h after the ligation. Plasma, erythrocytes and platelets were isolated and S1P, dihydrosphingosine-1-phosphate (DHS1P), SPH, dihydrosphingosine (DHS) and CER were quantified by means of an Agilent 6460 triple quadrupole mass spectrometer using positive ion electrospray ionization source with multiple reaction monitoring. The infarction reduced the plasma level of S1P, DHS1P, SPH and DHS but increased the level of total CER. In erythrocytes, there was a sharp elevation in the level of SPH and DHS early after the infarction and a reduction after 24 h whereas the level of S1P, DHS1P and total CER gradually increased. In platelets, the level of each of the examined compounds profoundly decreased 1 and 6 h after the infarction and partially normalized in 24 h. The results obtained clearly show that experimental heart infarction in rats produces deep changes in metabolism of sphingolipids in the plasma, platelets and erythrocytes.

Topics: Anesthesia; Animals; Ceramides; Coronary Vessels; Erythrocyte Count; Femoral Artery; Ligation; Lysophospholipids; Male; Myocardial Infarction; Phosphotransferases (Alcohol Group Acceptor); Platelet Count; Rats; Rats, Wistar; Sphingolipids; Sphingosine; Troponin T

Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein-coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP.

Topics: Animals; Drosophila melanogaster; Drosophila Proteins; Endosomes; Lipid Metabolism; Lysophospholipids; Lysosomes; Phosphotransferases (Alcohol Group Acceptor); Photoreceptor Cells; Protein Transport; Sphingosine; Transient Receptor Potential Channels

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P(1,3-5) receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-(13)C, (15)N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent K(i) of 2.15 microm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.

Topics: Cells, Cultured; Ceramides; Chromatography, Liquid; Endothelium, Vascular; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lung; Lysophospholipids; Oxidoreductases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Serine C-Palmitoyltransferase; Sphingosine; Tandem Mass Spectrometry; Up-Regulation

Sphingolipids are present in membranes of all eukaryotic cells. Bioactive sphingolipids also function as signaling molecules that regulate cellular processes such as proliferation, migration, and apoptosis. Human cytomegalovirus (HCMV) exploits a variety of cellular signaling pathways to promote its own replication. However, whether HCMV modulates lipid signaling pathways is an essentially unexplored area of research in virus-host cell interactions. In this study, we examined the accumulation of the bioactive sphingolipids and the enzymes responsible for the biosynthesis and degradation of these lipids. HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK), the enzyme that generates sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). We also utilized a mass spectrometry approach to generate a sphingolipidomic profile of HCMV-infected cells. We show that HCMV infection results in increased levels of dhS1P and ceramide at 24 h, suggesting an enhancement of de novo sphingolipid synthesis. Subsequently dihydrosphingosine and dhS1P decrease at 48 h consistent with attenuation of de novo sphingolipid synthesis. Finally, we present evidence that de novo sphingolipid synthesis and sphingosine kinase activity directly impact virus gene expression and virus growth. Together, these findings demonstrate that host cell sphingolipids are dynamically regulated upon infection with a herpes virus in a manner that impacts virus replication.

Topics: Apoptosis; Cell Line, Tumor; Cytomegalovirus; Gene Expression Regulation, Viral; Genes, Immediate-Early; Humans; Lysophospholipids; Mass Spectrometry; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); RNA Interference; Sphingolipids; Sphingosine; Time Factors; Virus Replication

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.

Topics: Animals; Behavior, Animal; Cyclic AMP; Fluorescence Resonance Energy Transfer; Lysophospholipids; Mice; Mice, Knockout; Neurons; Pain; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Spine

Fumonisins are causative agents of diseases in mice and rats, including liver and renal toxicities, as well as cancer, and are specific inhibitors of ceramide synthase in the metabolism of sphingolipid. The purpose of this study was to determine whether an elevated level of sphingoid base 1-phosphate was related to the expressions of metabolism enzymes in the liver of fumonisin B1 (FB1)-treated mice and acted as a contributing factor to hepatotoxicity. In our previous study, FB1 was confirmed to be toxic to both liver and kidneys, coupled with simultaneous elevation of sphinganine 1-phosphate. ICR mice were treated intraperitoneally with 10 mg/kg/day FB1 for 5 days, with the concentrations of sphingolipid metabolites in the serum and liver measured using HPLC following Bligh-Dyer extraction. The levels of sphingoid bases and their 1-phosphates in the serum and liver were markedly elevated in response to treatment with FB1. In the liver, FB1 increased the expression of sphingosine kinase and inhibited the expression of sphingosine 1-phosphate lyase. The cleaved form of caspase-3 was detected in the liver of FB1-treated mice, indicating the occurrence of apoptosis in the liver following exposure to FB1. The expressions of proapoptotic signaling molecules, such as phosphorylated forms of c-Jun N-terminus kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), were increased in the liver of FB1-treated mice. In conclusion, these results suggest the elevation of sphingoid base 1-phosphate, as a result of the activation of sphingosine kinase and the inhibition of sphingosine 1-phosphate lyase, may be a major target for FB1-induced hepatotoxicity via the activation of an apoptotic signaling pathway.

Topics: Aldehyde-Lyases; Animals; Caspase 3; Chemical and Drug Induced Liver Injury; Fumonisins; Liver; Lysophospholipids; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; Mycotoxins; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes formation of important regulators of inter- and intracellular signaling, sphingosine-1 phosphate (S1P), and dihydrosphingosine 1-phosphate (dhS1P). In this study, we investigated the role of SphK1 in the regulation of expression of matrix metalloproteinase 1 (MMP1) in dermal fibroblasts, a key event in regulation of extra cellular matrix. We show that overexpression of SphK1 up-regulated MMP1 protein, MMP1 mRNA, and MMP1 promoter activity, and this action of SphK1 required activation of the ERK1/2-Ets1 and NF-kappaB pathways. Furthermore, experiments using SphK1 specific siRNA demonstrated that SphK1 is required for the TNF-alpha stimulation of MMP1. Additional data revealed a specific role of dhS1P, and not S1P, as a mediator of SphK1-dependent activation of ERK1/2 and up-regulation of MMP1. The stimulatory effect of dhS1P was sensitive to pertussis toxin, suggesting a possible involvement of a G-protein-coupled receptor. In contrast, S1P, but not dhS1P, stimulated the induction of COX-2, which demonstrated selective actions of these two closely related bioactive lipids. In conclusion, this study describes a novel mode of SphK1 signaling through generation of dhS1P with a key role in mediating transcriptional responses to TNF-alpha. This is the first report of selective function of dhS1P as compared with the better studied S1P.

Topics: Ceramides; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Lysophospholipids; Matrix Metalloproteinase 1; NF-kappa B; Pertussis Toxin; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Protein c-ets-1; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Up-Regulation

Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.

Topics: Avian Sarcoma Viruses; Calcium; Cell Movement; Cells, Cultured; Endothelial Cells; Gene Expression; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Serine C-Palmitoyltransferase; Signal Transduction; Sphingosine; Tumor Necrosis Factor-alpha; Up-Regulation

We had found previously that neurotrophin-3 (NT-3) is a potent stimulator of cAMP-response element binding protein (CREB) phosphorylation in cultured oligodendrocyte progenitors. Here, we show that CREB phosphorylation in these cells is also highly stimulated by sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is known to be a potent mediator of numerous biological processes. Moreover, CREB phosphorylation in response to NT-3 involves sphingosine kinase 1 (SphK1), the enzyme that synthesizes S1P. Immunocytochemistry and confocal microscopy indicated that NT-3 induces translocation of SphK1 from the cytoplasm to the plasma membrane of oligodendrocytes, a process accompanied by increased SphK1 activity in the membrane fraction where its substrate sphingosine resides. To examine the involvement of SphK1 in NT-3 function, SphK1 expression was down-regulated by treatment with SphK1 sequence-specific small interfering RNA. Remarkably, the capacity of NT-3 to protect oligodendrocyte progenitors from apoptotic cell death induced by growth factor deprivation was abolished by down-regulating the expression of SphK1, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Altogether, these results suggest that SphK1 plays a crucial role in the stimulation of oligodendrocyte progenitor survival by NT-3, and demonstrate a functional link between NT-3 and S1P signaling, adding to the complexity of mechanisms that modulate neurotrophin function and oligodendrocyte development.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Membrane; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Drug Interactions; Gene Expression Regulation; In Situ Nick-End Labeling; Lysophospholipids; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Neurotrophin 3; Oligodendroglia; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Sphingosine; Stem Cells; Thiazoles; Thiazolidinediones; Thiones; Time Factors