OBJECTIVES: To investigate the role of sphingosine-1-phosphate receptor 5 (S1PR5) in modulating barrier function of mouse brain microvascular endothelial cells with oxygen-glucose deprivation and reoxygenation (OGD/R). METHODS: Mouse brain microvascular endothelial cells (bEnd.3) were exposed to OGD/R to induce barrier dysfunction following treatment with S1PR5-specific agonist A971432 or lentivirus-mediated transfection with a S1PR5-specific siRNA, a S1PR5-overexpressing plasmid, or their respective negative control sequences. The changes in viability and endothelial barrier permeability of the treated cells were evaluated with CCK-8 assay and FITC-dextran permeability assay; the levels of intracellular reactive oxygen species (ROS) and localization and expression levels of the proteins related with barrier function and oxidative stress were detected using immunofluorescence staining, DCFH-DA probe and Western blotting. RESULTS: S1PR5 activation obviously enhanced viability of bEnd.3 cells exposed to OGD/R (P<0.0001). Both activation and overexpression of S1PR5 reduced FITC-dextran leakage, while S1PR5 knockdown significantly increased FITC-dextran leakage in the exposed bEnd.3 cells. Activation and overexpression of S1PR5 both increased the cellular expressions of the barrier proteins ZO-1 and occludin, while S1PR5 knockdown produced the opposite effect. In cells exposed to OGD/R, ROS production was significantly reduced by S1PR5 activation and overexpression but increased following S1PR5 knockdown. Overexpression of S1PR5 obviously increased the expressions of the antioxidant proteins Nrf2, HO-1 and SOD2 in the exposed cells. CONCLUSIONS S1PR5 activation and overexpression significantly improve cell viability and reduce permeability of a mouse brain microvascular endothelial cell model of OGD/R, the mechanism of which may involve the reduction in ROS production and upregulation of the antioxidant proteins.
Animals ; Mice ; Oxidative Stress ; Endothelial Cells/cytology* ; Brain/blood supply* ; Reactive Oxygen Species/metabolism* ; Receptors, Lysosphingolipid/metabolism* ; Sphingosine-1-Phosphate Receptors ; Blood-Brain Barrier/metabolism* ; Glucose ; Cell Line ; Oxygen/metabolism* ; NF-E2-Related Factor 2/metabolism*

Transmembrane protein, also known as integral membrane protein, can be distributed in the lipid bilayer or across the entire membrane, and it plays an important role in cell signal transduction. It has been discovered that multiple transmembrane proteins are involved in the regulation of tumor signals. Recent studies have revealed that the abnormal expression of some transmembrane protein is closely related to the occurrence, development and prognosis of non-Hodgkin's lymphoma (NHL), including programmed cell death protein 1 (PD-1), TMEM30A, NOTCH1, TOLL-like receptor (TLR), sphingosine-1-phosphate receptor, TRAIL, etc. The study on these transmembrane proteins and related genes has important clinical significance for the treatment and prognosis of NHL, and it may become a new therapeutic target. At present, there have been some research results in this field at home and abroad. This article reviewed the research progress of transmembrane protein that has inhibitory effects on NHL in recent years.
Humans ; Lipid Bilayers ; Lymphoma, Non-Hodgkin/therapy* ; Prognosis ; Programmed Cell Death 1 Receptor ; Sphingosine-1-Phosphate Receptors

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Chlorpromazine ; Humans ; Lymphoma ; Sphingosine-1-Phosphate Receptors

BACKGROUND: Endothelial cells play a key role in the cytokine storm caused by influenza A virus. MicroRNA-155 (miR-155) is an important regulator in inflammation. Its role in the inflammatory response to influenza A infection, however, has yet to be elucidated. In this study, we explored the role as well as the underlying mechanism of miR-155 in the cytokine production in influenza A-infected endothelial cells. METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were infected with the influenza A virus strain H1N1. The efficiency of H1N1 infection was confirmed by immunofluorescence. The expression levels of proinflammatory cytokines and miR-155 were determined using real-time polymerase chain reaction. A dual-luciferase reporter assay characterized the interaction between miR-155 and sphingosine-1-phosphate receptor 1 (S1PR1). Changes in the target protein levels were determined using Western blot analysis. RESULTS: MiR-155 was elevated in response to the H1N1 infection in HPMECs (24 h post-infection vs. 0 h post-infection, 3.875 ± 0.062 vs. 1.043 ± 0.013, P = 0.001). Over-expression of miR-155 enhanced inflammatory cytokine production (miR-155 mimic vs. negative control, all P < 0.05 in regard of cytokine levels) and activation of nuclear factor kappa B in infected HPMECs (miR-155 mimic vs. negative control, P = 0.004), and down-regulation of miR-155 had the opposite effect. In addition, S1PR1 was a direct target of miR-155 in the HPMECs. Inhibition of miR-155 enhanced the expression of the S1PR1 protein. Down-regulation of S1PR1 decreased the inhibitory effect of the miR-155 blockade on H1N1-induced cytokine production and nuclear factor kappa B activation in HPMECs. CONCLUSION MiR-155 maybe modulate influenza A-induced inflammatory response by targeting S1PR1.
Down-Regulation ; Endothelial Cells ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza A virus ; Influenza, Human/genetics* ; MicroRNAs/genetics* ; Sphingosine-1-Phosphate Receptors

OBJECTIVE: To investigate the effect of sphingosine-1-phosphate receptor 2 (S1PR2) specific antagonist JTE-013 on the proliferation of human chronic myeloid leukemia (CML) cell line K562. METHODS: K562 cells were treated with JTE-013 (0, 0.5, 1, 5, 10, 20 μmol/L) for 24 and 48 hours respectively, CCK8 assay was used to detect the cell viability. K562 cells were treated with JTE-013 (0, 5, 10, 20 μmol/L) for 24 hours, propidium iodide (PI) DNA staining was used to analyze the cell cycle, Western blot was used to determine the levels of P21 and Cyclin D1 protein expression. RESULTS: JTE-013 inhibited the proliferation of CML cell line K562 in a dose dependent manner (r=-0.971). The proliferation rate of CML cells showed that the activity of CML cells decreased gradually with the increase of JTE-013 concentration (r=-0.971). The detection demonstrated that JTE-013 suppressed tumor cell proliferation through cell cycle arrest in G/G phase. Further detection of the protein expressions of G phase regulators showed that level of P21 increased, and expression of Cyclin D1 decreased. CONCLUSION JTE-013, a S1PR2 antagonist, can inhibit the proliferation of human CML K562 cells, which may be achieved by arresting the cells in G/G phase.
Apoptosis ; Cell Proliferation ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Pyrazoles ; Pyridines ; Receptors, Lysosphingolipid ; Sphingosine-1-Phosphate Receptors

OBJECTIVE: To observe the expressions of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 2 (S1PR2) in hippocampus of epileptic rats and to investigate the pathogenesis of SphK1 and S1PR2 in epilepsy. METHODS: One hundred and eight male Sprague-Dawley (SD) rats were randomly divided into control group (n=48) and pilocarpine (PILO) group (n=60). A robust convulsive status epilepticus (SE) was induced in PILO group rats by the application of pilocarpine. Control group rats were injected with respective of physiological saline. Pilocarpine group was randomly divided into 6 subgroups (n=8): acute group (E6 h, E1 d, E3 d), latent group (E7 d) and chronic group (E30 d, E56 d). Each subgroup has 8 control rats and 8 epileptic rats. Hippocampal tissue and brain slices were obtained from control rats and rats subjected to the Li-PILO model of epilepsy at 6 h, 1 d, 3 d,7 d,30 d and 56 d after status epilepticus (SE). Western blot technique was used to determine the expressions of SphK1 and S1PR2 in hippocampus at different point of time after pilocarpine treatment. Immunofluorescence was applied to detect the activation and proliferation of hippocampal astrocytes and the localization of SphK1 and S1PR2 in rat hippocampal astrocytes. RESULTS: Compared with control group, the levels of SphK1 in acute phase (E3 d), latent phase (E7 d) and chronic phase (E30 d, E56 d) were significantly increased while the expressions of S1PR2 were decreased in acute phase (E3 d), latent phase (E7 d) and chronic phase (E30 d, E56 d)(P＜0.05 or P＜0.01). Immunofluorescence results showed astrocyte activation and proliferation in hippocampus of epileptic (E7 d) rats (P＜0.05). Confocal microscopy confirmed the preferential expressions of SphK1 and S1PR2 in epileptic rat(E7 d)hippocampal astrocytes. CONCLUSION The results indicate that SphK1 and S1PR2 may play an important role in the pathogenesis of epilepsy by regulating the activation and proliferation of hippocampal astrocytes and altering neuronal excitability.
Animals ; Astrocytes ; enzymology ; Epilepsy ; enzymology ; physiopathology ; Hippocampus ; cytology ; enzymology ; Male ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Pilocarpine ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Lysosphingolipid ; metabolism

Sphingosine 1-phosphate (S1P) levels are often found to be elevated in serum, bronchoalveolar lavage, and lung tissue of idiopathic pulmonary fibrosis patients and experimental mouse models. Although the roles of sphingosine kinase 1 and S1P receptors have been implicated in fibrosis, the underlying mechanism of fibrosis via Sphingosine 1-phosphate receptor 2 (S1P₂) has not been fully investigated. Therefore, in this study, the roles of S1P₂ in lung inflammation and fibrosis was investigated by means of a bleomycin-induced lung fibrosis model and lung epithelial cells. Bleomycin was found to induce lung inflammation on day 7 and fibrosis on day 28 of treatment. On the 7(th) day after bleomycin administration, S1P₂ deficient mice exhibited significantly less pulmonary inflammation, including cell infiltration and pro-inflammatory cytokine induction, than the wild type mice. On the 28(th) day after bleomycin treatment, severe inflammation and fibrosis were observed in lung tissues from wild type mice, while lung tissues from S1P₂ deficient mice showed less inflammation and fibrosis. Increase in TGF-β1-induced extracellular matrix accumulation and epithelial-mesenchymal transition were inhibited by JTE-013, a S1P₂ antagonist, in A549 lung epithelial cells. Taken together, pro-inflammatory and pro-fibrotic functions of S1P₂ were elucidated using a bleomycin-induced fibrosis model. Notably, S1P₂ was found to mediate epithelial-mesenchymal transition in fibrotic responses. Therefore, the results of this study indicate that S1P₂ could be a promising therapeutic target for the treatment of pulmonary fibrosis.
Animals ; Bleomycin ; Bronchoalveolar Lavage ; Epithelial Cells ; Epithelial-Mesenchymal Transition ; Extracellular Matrix ; Fibrosis ; Humans ; Idiopathic Pulmonary Fibrosis ; Inflammation ; Lung ; Mice ; Phosphotransferases ; Pneumonia ; Pulmonary Fibrosis ; Receptors, Lysosphingolipid ; Sphingosine

Sphingosine-1-phosphate (S1P) plays an important role in trafficking leukocytes and developing immune disorders including autoimmunity. In the synovium of rheumatoid arthritis (RA) patients, increased expression of S1P was reported, and the interaction between S1P and S1P receptor 1 (S1P1) has been suggested to regulate the expression of inflammatory genes and the proliferation of synovial cells. In this study, we investigated the level of S1P1 mRNA expression in the blood leukocytes of RA patients. In contrast to the previous reports, the expression level of this gene was not correlated to their clinical scores, disease durations and ages. However, S1P1 was transcribed at a significantly lower level in the circulating leukocytes of RA patients when compared to age-, and sex-matched healthy controls. Since these data may suggest the participation of S1P1, further studies are needed to determine the role of this receptor in the pathogenesis of RA.
Arthritis, Rheumatoid* ; Autoimmunity ; Humans ; Immune System Diseases ; Leukocytes* ; Receptors, Lysosphingolipid* ; RNA, Messenger ; Synovial Membrane

Initial discovery on sphingosine 1-phosphate (S1P) as an intracellular second messenger was faced unexpectedly with roles of S1P as a first messenger, which subsequently resulted in cloning of its G protein-coupled receptors, S1P₁₋₅. The molecular identification of S1P receptors opened up a new avenue for pathophysiological research on this lipid mediator. Cellular and molecular in vitro studies and in vivo studies on gene deficient mice have elucidated cellular signaling pathways and the pathophysiological meanings of S1P receptors. Another unexpected finding that fingolimod (FTY720) modulates S1P receptors accelerated drug discovery in this field. Fingolimod was approved as a first-in-class, orally active drug for relapsing multiple sclerosis in 2010, and its applications in other disease conditions are currently under clinical trials. In addition, more selective S1P receptor modulators with better pharmacokinetic profiles and fewer side effects are under development. Some of them are being clinically tested in the contexts of multiple sclerosis and other autoimmune and inflammatory disorders, such as, psoriasis, Crohn’s disease, ulcerative colitis, polymyositis, dermatomyositis, liver failure, renal failure, acute stroke, and transplant rejection. In this review, the authors discuss the state of the art regarding the status of drug discovery efforts targeting S1P receptors and place emphasis on potential clinical applications.
Acute Kidney Injury ; Animals ; Clone Cells ; Cloning, Organism ; Colitis, Ulcerative ; Dermatomyositis ; Drug Discovery* ; Fingolimod Hydrochloride ; Graft Rejection ; In Vitro Techniques ; Liver Failure ; Mice ; Multiple Sclerosis ; Polymyositis ; Psoriasis ; Receptors, Lysosphingolipid* ; Second Messenger Systems ; Sphingosine* ; Stroke

Objective: To screen lentiviral vectors carrying siRNA which can specifically down-regulate the gene expression of the sphingosine-1-phosphate receptor 3 (S1PR3) in the corpus cavernosum smooth muscle (CCSM) cells of rats with spontaneous hypertension (SHT) and investigate the influence of the vectors on the signaling pathways of ROCK1, ROCK2 and eNOS in the CCSM cells of SHT rats. METHODS: Using the S1PR3 mRNA sequence of the rat as an interfering target, we designed and synthesized three pairs of siRNA sequences (siRNA1, 2 and 3) targeting S1PR3 and one pair of negative control, and then constructed and packaged them into lentiviral vectors. We cultured the CCSM cells of SHT and Wistar-Kyoto (WKY) rats in vitro and randomly divided them into groups A (SHT untransfected control), B (SHT transfected and carrying negative control virus), C (SHT transfected and carrying siRNA1 targeting S1PR3), D (SHT transfected and carrying siRNA2 targeting S1PR3), E (SHT transfected and carrying siRNA3 targeting S1PR3), and F (WKY untransfected control). With the multiplicity of infection (MOI) = 60, we transfected the CCSM cells of the SHT rats with the lentiviral vector and then determined the expression of the green fluorescent protein (GFP) as well as the mRNA and protein expressions of S1PR3, ROCK1, ROCK2 and eNOS in the CCSM cells of the SHT and WKY rats by RT-PCR and Western blot. RESULTS: Gene sequencing proved the successful construction of the lentiviral vector. The transfection efficiency of the CCSM cells of the rats was >80% in groups B, C, D and E. Compared with group A, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 exhibited no significant difference in group B but were remarkably decreased in groups C, D, E and F (P< 0.05), most significantly in group E, with the inhibition rates of the mRNA and protein expressions of S1PR3 of (34.2±2.9) and (77.7±4.7)%, those of ROCK1 of (33.3±1.4) and (51.1±7.3)%, and those of ROCK2 of (30.8±3.6) and (58.32±5.5)%, respectively. The mRNA and protein expressions of eNOS in group A showed no significant difference from those in groups B, C, D and E (P>0.05) but remarkably lower than those in group F (P< 0.05). Compared with group F, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 were not significantly different from those in group E (P>0.05) but markedly increased in groups A, B, C and D (P< 0.05), while those of eNOS remarkably decreased in groups A, B, C, D and E (P< 0.05). CONCLUSIONS The three constructed lentiviral vectors carrying siRNA targeting different loci of the S1PR3 gene could significantly inhibit the expression of S1P3 as well as RhoA/Rho kinase signaling pathways in the CCSM cells of SHT rats, and the vector carrying siRNA3 exhibited the highest inhibitory effect.
Animals ; Down-Regulation ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Lentivirus ; genetics ; Male ; Myocytes, Smooth Muscle ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Penis ; metabolism ; RNA, Messenger ; RNA, Small Interfering ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Inbred WKY ; Receptors, Lysosphingolipid ; genetics ; metabolism ; Signal Transduction ; Sphingosine-1-Phosphate Receptors ; Transfection ; rho-Associated Kinases ; metabolism