S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with unclear etiology and limited treatment options. The median survival time for IPF patients is approximately 2-3 years and there is no effective intervention to treat IPF other than lung transplantation. As important components of lung tissue, endothelial cells (ECs) are associated with pulmonary diseases. However, the role of endothelial dysfunction in pulmonary fibrosis (PF) is incompletely understood. Sphingosine-1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor highly expressed in lung ECs. Its expression is markedly reduced in patients with IPF. Herein, we generated an endothelial-conditional S1pr1 knockout mouse model which exhibited inflammation and fibrosis with or without bleomycin (BLM) challenge. Selective activation of S1PR1 with an S1PR1 agonist, IMMH002, exerted a potent therapeutic effect in mice with bleomycin-induced fibrosis by protecting the integrity of the endothelial barrier. These results suggest that S1PR1 might be a promising drug target for IPF therapy.

Effect of FTY720 on transfusion-related acute lung injury: a preliminary investigation / 中国输血杂志

【Objective】 To investigate the effects and mechanisms of different doses of fingolimod (FTY720) on non-antibody-mediated transfusion-related acute lung injury (TRALI). 【Methods】 A TRALI mouse model was constructed using lipopolysaccharide (LPS) pre-stimulation and platelets (Plt) of different storage days for second strike. The success of the modeling was determined by protein concentration in lung tissue homogenates, myeloperoxidase (MPo) activity, lung wet/dry weight ratio (W/D ratio), lung tissue damage score and pathological sections. Ceramide and sphingosine-1-phosphate (S1P) contents in platelets of different storage days were detected. FTY720 was administered 1 h after LPS injection to investigate the role of FTY720 in TRALI. The expression levels of vascular endothelial cadherin (VE-cadherin) and zonula occludens-1 (ZO-1) were analyzed by WB. 【Results】 Mice infused with stored 5-day Plt (d5Plt group) exhibited typical signs of TRALI, and the differences in lung tissue homogenate protein concentration (6 546.38±409.50) μg/mL, MPO activity (49.38±4.43) U/L, W/D ratio 4.79±0.21, and lung tissue damage score 7.24±0.38 from the rest of the groups were statistically significant (P<0.05). With the increase of platelet storage time, the ceramide content gradually increased and S1P content gradually decreased, and the ratio of the two was imbalanced. d5Plt showed statistically significant differences (P<0.01) in ceramide content (58.37±5.69) μmol/L and S1P content (149.81±4.86) nmol/L from the rest of the groups. After preventive administration of FTY720, 1 mg/kg FTY720 had no significant effect on TRALI mice, whose lung tissue homogenate protein concentration (6 170.26±545.50) μg/mL, MPO activity (45.97±4.79) U/L, W/D ratio 4.88±0.25, and lung tissue damage score 7.92±0.65 were significantly higher than those of the normal and LPS control groups (P<0.01). The low-dose (0.5, 0.2, and 0.1 mg/kg) FTY720 group alleviated lung injury, and its protein concentration, MPO activity, W/D ratio, and lung tissue injury score were significantly lower than those of the d5Plt group (P<0.05). Pathological sections also showed similar results. In terms of endothelial intercellular junction protein expression, the VE-cadherin expression levels in the 1 mg/kg FTY720 group were significantly lower than those in the normal and LPS control groups (P<0.05), and the VE-cadherin and ZO-1 expression levels in the low-dose (0.5, 0.2, and 0.1 mg/kg) FTY720 group were significantly higher than those in the d5Plt group (P<0.05), which tended to be normalized. 【Conclusion】 In this study, a TRALI mouse model was successfully established by one strike of LPS and two strikes of d5Plt. Low doses of FTY720 (0.5, 0.2, 0.1 mg/kg) were protective against TRALI, while high doses of FTY720 (1 mg/kg) may aggravate the symptoms of TRALI. This protective effect may be somewhat dependent on the expression of VE-cadherin and ZO-1.

Effects of fty-720 on pulmonary fibrosis in mice via tgf-pl/p38 mapk/nf-kb signaling pathway / 中国药理学通报

Aim To investigate the effect of FTY-720 on bleomycin-induced pulmonary fibrosis in mice and its mechanism. Methods Forty specific pathogen-free healthy male BALB/c mice were randomly divided into control group, BLM group, NS + FTY-720 control group, and BLM + FTY-720 group, with 5 mice in each group, and the mice were sacrificed on 7th day and 14th day for a total of 8 groups. Lung tissue inflammation and pulmonary fibrosis were observed by HE staining and Masson staining; Bronchoalveolar lavage fluid (BALF) cells were counted by Swiss-Giemsa staining; BALF protein content was determined by BCA assay; the expression levels of inflammatory cytokines IL-IP and TNF-a in BALF supernatant were measured by ELISA; the expression levels of COL1A1 in lung tissues were detected by immunohistochemis-try; and the expression levels of TGF-|31, p-p38 MAPK and NF-kB were detected by Western blot. Results FTY-720 significantly reduced BLM-induced increase of extracellular collagen deposition in lung tissues of mice, reduced IL-1(3 and TNF-a content in BALF of mice, and reduced the protein content and cell number in BALF of mice; FTY-720 inhibited TGF-(31 activity and p38 MAPK phosphorylation, and then inhibited the expression and activation of NF-kB. Conclusions FTY-720 inhibits bleomycin-induced lung fibrosis in mice by inhibiting the TGF-(31/p38 MAPK/NF-kB signaling pathway.

Evaluation of Neutrophil Activation Status According to the Phenotypes of Adult Asthma

PURPOSE: Neutrophils are considered key effector cells in the pathogenic mechanisms of airway inflammation in asthma. This study assessed the activation status of neutrophils in adult asthmatics, and the therapeutic potential of FTY720, a synthetic sphingosine-1-phosphate analog, on activated neutrophils using an in vitro stimulation model. METHODS: We isolated peripheral blood neutrophils (PBNs) from 59 asthmatic patients (including 20 aspirin-exacerbated respiratory disease [AERD] and 39 aspirin-tolerant asthma [ATA] groups). PBNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharide (LPS) and their activation status was determined based on reactive oxygen species (ROS) production, cell surface expression of CD11b, interleukin (IL)-8 and matrix metallopeptidase (MMP)-9 release. PBNs were primed with FTY720 to evaluate its anti-inflammatory action. RESULTS: In vitro PBN stimulation with fMLP or LPS induced a significant increase in ROS/CD11b/IL-8/MMP-9 levels (P < 0.05 for all). In asthmatics, fMLP-induced ROS level was significantly correlated with values of forced expiratory volume in 1 second/forced vital capacity (r = −0.278; P = 0.036), maximal mid-expiratory flow (r = −0.309; P = 0.019) and PC20 methacholine (r = −0.302; P = 0.029). In addition, ROS levels were significantly higher in patients with AERD and in those with severe asthma than in those with ATA or non-severe asthma (P < 0.05 for all). FTY720 treatment could suppress ROS/CD11b levels, and LPS-induced IL-8 and MMP-9 levels (P < 0.05 for all). Responders to FTY720 treatment had significantly higher neutrophil counts in sputum (P = 0.004). CONCLUSIONS: Our findings suggest a useful in vitro PBN stimulation model for evaluating the neutrophil functional status and the therapeutic potentials of neutrophil-targeting candidates in asthmatics.

Effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts / 中国修复重建外科杂志

Objective: To investigate the effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts. Methods: Murine RAW264.7 macrophages were induced into osteoclasts by dexamethasone and 1α, 25-dihydroxyvitamin D 3, and identified by tartrate resistant acid phosphatase (TRAP) staining. Then, the osteoclasts were divided into 2 groups. The osteoclasts were treated with 400 ng/mL FTY720-P in experimental group and without FTY720-P in control group, respectively. After 48 hours of culture, the cells in 2 groups were detected by real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining. The expressions of EphA2, EphrinA2, RhoA, and the bone reconstruction associated proteins［bone morphogenetic protein 2 (BMP-2) and transform growth factor β 1 (TGF-β 1)］were analyzed and compared. Results: RAW264.7 cells were successfully induced into osteoclasts identified by TRAP staining. Compared with control group, the relative expressions of EphA2 and EphrinA2 mRNAs and proteins in experimental group significantly decreased after 48 hours ( P<0.05), and the relative expression of RhoA protein also significantly decreased ( P<0.05). The relative expressions of BMP-2 and TGF-β 1 mRNAs were significantly increased ( P<0.05), and those protein expressions were enhanced. Conclusion: FTY720-P can down-regulate the expression of RhoA and promote the expressions of TGF- β 1 and BMP-2 by affecting the transduction of EphA2-EphrinA2 bidirectional signaling in osteoclasts.

Effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells / 中国修复重建外科杂志

Methods: The MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.

Effect of fingolimod on proliferation and migration of neural stem cells and astrocytes and its protective mechanism against spinal cord injury in mice / 吉林大学学报(医学版)

Objective; To investigate the effects of fingolimod (FTY720) on the migration and proliferation of neural stem cells (NSCs) and astrocytes of the mice, and to elucidate the protective effect of FTY720 on spinal cord injury (SCI) of the mice and its mechanism. Methods; The NSCs and astrocytes were cultured in vitro and divided into control group (treated with PBS) and experiment group (treated with 0. 1 μmol · L-1 FTY720). The SCI mice were divided into control group (given PBS by oral, n=10) and expriment group (given 1 mg · kg-1 · d-1 FTY720 by oral, n=10). The number of migration cells of NSCs and astrocytes in two groups was detected by Transwell experiment, and the number of migration cells and the number of proliferative neurospheres of NSCs were detected by immunofluorescence staining. The SCI void areas of the mice in two groups were detected by HE staining, and the dynamic recovery of motor function of the mice was assessed by Basso Mouse Scale (BMS) behavioral score. Results; The Transwell detection results showed that compared with control group, the number of migration cells of NSCs in expriment group was increased (P<0. 05), and the number of migration cells of astrocytes in expriment group was decreased (P<0. 05). The immunofluorescence detection results showed that compared with control group, the number of proliferative neurospheres of NSCs in expriment group was increased (P<0. 05), and the number of astrocytes in damaged SCI in expriment group was decreased (P<0. 05). The HE staining results showed that compared with control group, the SCI void area was decreased (P<0. 05). The BMS score results showed that the BMS score of the mice in expriment group was higher than that in control group (P<0. 05). Conclusion; FTY720 can promote the proliferation and migration of NSCs, inhibit the migration of astrocytes, decrease the formation of glial scar, and promote the motor function recovery of the mice after SCI.

Sphingosine 1-Phosphate Receptor Modulators and Drug Discovery

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.

Experimental research of pregnancy immune tolerance induced by a novel immunosuppressive agent FTY720 via blocking the S1P signaling pathway / 上海交通大学学报（医学版）

Objective · To investigate the effects and mechanisms of pregnancy immune tolerance induced by a novel immunosuppressive agent FTY720 and to provide experimental basis for the clinical treatment of unexplained recurrent spontaneous abortion. Methods · The mice of spontaneous abortion model were used as subjects. The effects of intraperitoneal injection of FTY720 on the embryo loss rate in mice of spontaneous abortion model and on the expression of sphingosine-1-phosphate (S1P) in the decidual tissue were observed. S1P-siRNA lentiviral vectors and S1P-overexpression gene lentiviral vectors were constructed and transfected into dendritic cells (DCs) from mouse bone marrow. The effects of FTY720 on the embryo loss rate in mice of spontaneous abortion model after adoptive transferring of these two types of lentiviral vectors were observed. Results · FTY720 had no significant effect on the embryo loss rate in normal pregnant mice. Intraperitoneal injection of FTY720 significantly reduced the embryo loss rate in mice of spontaneous abortion model. The expression of S1P in the decidual tissue in mice of spontaneous abortion model was low. After adoptive transferring of S1P-siRNA lentiviral vector transfected DCs, FTY720 could slightly reduce the embryo loss rate in mice of abortion mouse model, but the effect was far less than that of before adoptive transferring of S1P-siRNA lentivirus transfected DCs. After adoptive transferring of the S1P-overexpression gene lentiviral vector transfected DCs, FTY720 could significantly reduce the rate of embryo loss in mice of spontaneous abortion model and the effect was more significant than that of before adoptive transfecting of S1P-overexpression lentiviral vector transfected DCs. Conclusion · FTY720 is safe. The induction of pregnancy immune tolerance may be related to the blockage of S1P signaling pathway.

Experimental research of pregnancy immune tolerance induced by a novel immunosuppressive agent FTY720 via blocking the S1P signaling pathway / 上海交通大学学报（医学版）

Objective · To investigate the effects and mechanisms of pregnancy immune tolerance induced by a novel immunosuppressive agent FTY720 and to provide experimental basis for the clinical treatment of unexplained recurrent spontaneous abortion. Methods · The mice of spontaneous abortion model were used as subjects. The effects of intraperitoneal injection of FTY720 on the embryo loss rate in mice of spontaneous abortion model and on the expression of sphingosine-1-phosphate (S1P) in the decidual tissue were observed. S1P-siRNA lentiviral vectors and S1P-overexpression gene lentiviral vectors were constructed and transfected into dendritic cells (DCs) from mouse bone marrow. The effects of FTY720 on the embryo loss rate in mice of spontaneous abortion model after adoptive transferring of these two types of lentiviral vectors were observed. Results · FTY720 had no significant effect on the embryo loss rate in normal pregnant mice. Intraperitoneal injection of FTY720 significantly reduced the embryo loss rate in mice of spontaneous abortion model. The expression of S1P in the decidual tissue in mice of spontaneous abortion model was low. After adoptive transferring of S1P-siRNA lentiviral vector transfected DCs, FTY720 could slightly reduce the embryo loss rate in mice of abortion mouse model, but the effect was far less than that of before adoptive transferring of S1P-siRNA lentivirus transfected DCs. After adoptive transferring of the S1P-overexpression gene lentiviral vector transfected DCs, FTY720 could significantly reduce the rate of embryo loss in mice of spontaneous abortion model and the effect was more significant than that of before adoptive transfecting of S1P-overexpression lentiviral vector transfected DCs. Conclusion · FTY720 is safe. The induction of pregnancy immune tolerance may be related to the blockage of S1P signaling pathway.

Study on effect of FTY720 combined with gecitabine on proliferation and apoptosis of non-small cell lung cancer tumor-related cell-lines / 重庆医学

Objective To investigate the effect of FTY720 and gemcitabine on the proliferation and apoptosis of H520 and A549 cells in non-small cell lung cancer(NSCLC) cell line.Methods The interventional influence on the in vitro cultured NSCLC A549 and H520 cells was performed by selecting 0,2,4,6,8,10 μmol/L concentrations of FTY720,then the absorbance value was detected at 24,48,72 h after culture and the proliferation inhibiting effects of FTY720 on A549 and H520 were observed under the condition of different concentration of FTY720;adding single 7 μmol/L of FTY720,single 0.2 μmol/L gemcitabine and 37 μmol / L FTY720 combined with 0.2 mol/L gemcitabine into A549 and H520 cells lines,then the differences of inhibition and apoptosis after 48 h in the cells of each group were observed.Results The inhibitory effect of different concentrations of FTY270 on NSCLC A549 and H520 cell lines was statistically significant difference (P＜0.05).The proliferation inhibiting effect of FTY720 on NSCLC H520 and A549 cell lines had the correlation with the concentration and time.The apoptosis rate of FTY720 combined with gemcitabine on A549 and H520 cells was significantly higher than that of single use in these two drugs (P＜0.05).Conclusion FTY720 combined with gemcitabine can significantly inhibit the proliferation of A549 and H520 in human NSCLC,and can effectively promote the apoptosis of cancer cells,and has the higher clinical value.

Effects of FTY720 pretreatment on lung injury induced by limb ischemia-reperfusion in rats / 中华麻醉学杂志

Objective To evaluate the effects of FTY720 pretreatment on lung injury induced by limb ischemia-reperfusion (I/R) in rats.Methods Thirty healthy male Sprague-Dawley rats,weighing 230-260 g,were divided into 3 groups (n=10 each) using a random number table:sham operation group (group S),I/R group and FTY720 group (group F).Bilateral hind limb I/R was induced by applying rubber band tourniquet high around each thigh for 3 h followed by 3 h of reperfusion.FTY720 was given by intragastric gavage at 3 mg · kg-1 · d-1 for 7 consecutive days before I/R in group F,while the equal volume of normal saline was given instead in group I/R.At the end of reperfusion,blood samples were collected from the carotid artery for blood gas analysis,and oxygenation index (OI) was calculated.The rats were then sacrificed and lungs removed for examination of pathological changes (with light microscope) and for determination of apoptosis rate (using flow cytometry) and expression of CCAAT/enhancer-binding protein homologous protein (CHOP),caspase-12,c-Jun N-terminal kinase (JNK),Bcl-2 and Bax protein in lung tissues (using Western blot).Bax/Bcl-2 ratio was calculated.Results Compared with group S,OI was significantly decreased in group I/R,and apoptosis rate was increased,the expression of CHOP,caspase-12,JNK and Bax protein was up-regulated,and the expression of Bcl-2 was down-regulated,and Bax/Bcl-2 ratio was increased in I/R and F groups (P＜0.05).Compared with group I/R,OI was significantly increased,apoptosis rate was decreased,the expression of caspase-12,CHOP,Bax and JNK protein was down-regulated,and the expression of Bcl-2 was up-regulated,and Bax/Bcl-2 ratio was decreased in group F (P＜0.05).The pathological changes of lungs were significantly attenuated in group F as compared with group I/R.Conclusion FTY720 pretreatment can attenuate lung injury induced by limb I/R in rats,and the mechanism may be related to inhibition of endoplasmic reticulum stress-related cell apoptosis.

Sphingosine 1-phosphate signaling pathway:a novel target for regulation of epithelial and endothelial barrier function / 中国药科大学学报

@#Sphingosine 1-phosphate(S1P)is a pleiotropic sphingolipid metabolite that has been shown to regulate important physiological function by activation of its G-protein-coupled receptors S1PRs. S1P has been identified as an important signaling molecule in maintaining epithelial and endothelial barrier function. S1P signaling pathway is involved in epithelial and endothelial barrier function by regulation of adherens junction and tight junction assembly, cytoskeletal reorganization, and focal adhesion formation. Thus, S1P signaling pathway may become a novel therapeutic target for cell barrier dysfunction during some illnesses such as acute lung injury, inflammatory bowel disease and sepsis. In this review, the research progress of S1P signaling pathway in regulating epithelial and endothelial barrier function and the application of S1P in barrier dysfunction-related diseases were summarized, so as to provide references for future research.

Mechanism of FTY720 on cardiovascular complications of diabetes mellitus / 实用医学杂志

Objective To study the mechanism of FTY720 on type Ι diabetic rats. Methods The typeⅠdiabetes rat model was established by feeding male SD rats with high energy urea and injecting into the abdominal cavity with low dose cephalosporins (STZ, 30 mg/kg). In the following, the treated rats were divided into two groups: model group and FTY720 group. Another group of untreated rats was assigned as normal control group. FTY720 group was given 1 mg/kg FTY720, the normal control group and model group was given the equal amount of water. Results The cardiac function of FTY720 group was recovered markeyly. FTY720 activated the expression of vascular endothelial cells S1P1 , diabetes and reduced the expessions of S1P3 and PKCβ Ⅱand it restored the migration ability of diabetes cardiovascular endothelial cell , as well as the abnormally elevated cardiovascular endothelial cells induced by high sugar permeability. Conclusion The S1P1 and S1P3 cut is an important reaction pathway to the complications of diabetes and cardiovascular dysfunction. FTY720 may reduce the damage to core blood vessels caused by diabetes , and pathological angiogenesis which functionally depends on the PKCβⅡ signaling pathways. Therefore, FTY720 may provide a potential therapy for cardiovascular function impaired by diabetes.

Protective effect of FTY720 on hepatic injury in experimental hepatic fibrosis mice / 吉林大学学报(医学版)

Objective To observe the protective effects of FTY720 on the Con A-induced mouse hepatic fibrosis injury,and to find the possible mechanisms of protective effects.Methods The pathologic models of hepatic fibrosis injury in the mice caused by Con A were set up.Forty mice were randomly divided into control group, model group,high dose of FTY720 (4 mg·kg-1 )group and low dose of FTY720 (1 mg·kg-1 )dose group (n=10).The serum alanine aminotransferase (ALT)and asparate aminotransferase (AST)activities,hepatic index and pathological changes of hepatic tissue were detected .Results Compared with model group,the serum ALT and AST activities in low and high doses of FTY720 groups were decreased significantly (P < 0.05 or P < 0.01).The optical microscope results showed that there were inflammatory cells and hepatocellular necrosis in model group. The masson staining results showed that there were surrounding fiber bundle and hepatic lobule fusion in model group;compared with model group,the damage degree in low and high doses of FTY720 groups was reduced.The protective effects of FTY720 on hepatic injury showed linear relation to the drug dose.Conclusion FTY720 could decrease the levels of ALT/AST,thus FTY720 alleviate hepatic damage degree and delay the process of hepatic fibrosis.The protective effects of FTY720 on hepatic injury in experimental hepatic fibrosis mice may be related to the mechanisms mentioned above.

Effects of FTY720 on neurological function and blood-spinal cord barrier of rats with acute spinal cord injury / 解放军医学杂志

Objective To investigate the effects of a novel immunomodulator FTY720 (Fingolimod) on nerve function and blood-spinal cord barrier (BSCB) of rats with acute spinal cord injury. Methods One hundred and forty-four adult Sprague- Dawley (SD) rats were randomly divided into four groups with 36 each: normal control group (NG Group): rats without any treatment; sham-operated group (SO Group): rats' spinal cords were exposed by laminectomy without injury; hemisection group (HS Group): rats underwent spinal cord hemisection followed by intraperitoneal injection of normal saline; FTY720 treatment group (FTY720 Group): rats underwent spinal cord hemisection followed by intraperitoneal injection of FTY720 [1mg/(kg.d)] for 7 days. The neurological function was assessed by Basso Beatlie Bresnahan (BBB) scores, grid walking, N1 and P1 delay of motor evoked potential (MEP) and somatosensory evoked potentials (SEP), histological evaluation with light microscope with HE staining, and determination of blood-spinal cord barrier permeability with EB at different time points after injury. Results The nerve function of rats in HS group and FTY720 group was impaired after hemisection injury without signs of recovery up to Day 28 after damage as compared with NG group or SO group. The recovery of motor function in FTY720 treatment group was earlier than in HS group. The BBB scores, the results of grid walking test, and the latent period of SEP-P1 showed statistically significant difference between FTY720 group and HS group from Day 7 to 28 after injury (P<0.05). Comparing the pathological picture at Day 14 and Day 28 after injury, the number of chronic inflammatory cells, the degree of glial cell reaction, and the size of syringomyelia cavities in gray matter in FTY720 group were significantly less than those in HS group. In addition, the leakage of EB from the damaged BSCB increased in HS group and FTY720 group than in NG group and SO group through 7 days after injury (P<0.01), while at each time point the leakage of EB was less in FTY720 group than in HS group (P<0.05), and the most significant difference was observed on Day 3 after injury. Conclusion FTY720 can lower the permeability of blood-spinal cord barrier at acute phase of spinal cord injury, effectively promotes the recovery of nerve function after acute injury phase, and it provides certain potential neuroprotective effects.

Role of sphingosine-1-phosphate receptor 1 in pain modulation / 中国药理学通报

Sphingosine-1-phosphate receptor 1(S1PR1) is a new member of G protein-coupled receptor family. A great body of data suggest S1PR1 is capable of regulating lots of downstream signaling molecules and cellular processes. It is found that S1P/S1PR1 plays an important role in the development and mainte-nance of pain. However, it is controversial whether activation of S1PR1 would enhance or attenuate pain. Here, recent studies ＜br> and current perspectives are discussed in order to better under-stand the biological and pathological roles of S1PR1 in pain mod-ulation.

Apoptosis of K562 cells induced by a new immunosuppressive agent FTY720 and its mechanism / 吉林大学学报(医学版)

Objective To study the apoptosis of K562 cells induced by a new immunosuppressive agent FTY720 and its mechanism,and to provide experimental basis for the treatment of leukemia in clinic.Methods The K562 cells were cultured in vitro and divided into blank control group and FTY720 treatment group.The K562 cells in FTY720 treatment group were treated with 6μmol·L-1 FTY720 for 3,6,and 12 h,or treated with different concentrations of FTY720 (2,4,6,8μmol·L-1)for 24 h.The apoptosis,level of reactive oxygen species (ROS),mitochondrial membrane potential(MMP)and cell cycle were measured by flow cytometry.The inhibitory rate of proliferation of K562 cells after treated with FTY720 was detected by WST-1 reducting assay.Results The results of flow cytometry showed that the percentages of apoptotic cells were increased after treated with 6μmol·L-1 FTY720 for 3,6,and 12 h with the prolongation of time compared with blank control group(P<0.01).The percentages of apoptotic cells were also increased after treated with different concentrations of FTY720 for 24 h compared with blank control group(P<0.01).Compared with blank control group,the ROS levels were increased with the increasing of FTY720 concentration,while the MMP was decreased(P<0.01).Compared with blank control group,the percentage of cells at G0/G1 phase was increased,while those at S and G2/M phases were decreased with the increasing of FTY720 concentration (P<0.05).The WST-1 reduction assay results indicated that the inhibitory rates of proliferation of K562 cells after treated with 2,4,6,and 8μmol·L-1 FTY720 for 72 h were (24.0±4.1)%,(46.4±3.9)%,(67.0±4.8)%,and (88.2±5.6)%,respectively,compared with blank control group.The concentration of FTY720 to result the inhibitory rate of 50% (IC50 )on K562 cells was 5.5μmol·L-1 .Conclusion FTY720 could inhibit the proliferation of K562 cells by blocking cell cycle and inducing apoptosis through provoking ROS.

Anti-rejection study in mice skin transplantation of a novel immunosuppressant SYL934 / 中国药理学通报

Aim To determine the effect of SYL934 , a novel immunosuppressant, on skin allograft rejec-tion. Methods HTRF-IP1 assay was used to evaluate the agonistic activity of SYL934-P, the active form of SYL934 in vivo, on S1P1 and S1P3 in vitro. SD rat peripheral blood lymphocytes ( PBL ) test and heart rate test was used to assess the in vivo immunosuppres-sive effect and heart rate effect of SYL934 . Mice skin graft transplantation experiment was used to observe the effect of SYL934 on skin allograft refection. ResultsSYL934-P selectively activated S1 P1 but not S1 P3 in vitro. Single orally administration of SD rats with SYL934 decreased the PBL significantly and played an obviously immunosuppressant role, but did not affect the heart rate. Daily orally administration of mice with SYL934 significantly increased the survival rate of al-lografts of skin slice in mice. Conclusion SYL934 has great selectivity in vitro and good activity in vivo, which indicated it potential use as an anti-rejection drug in skin transplantation.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished. .