S1P/S1PR2的心血管调节作用及其信号通路

<正>1-磷酸鞘氨醇(sphingosine 1-phosphate,S1P)是近年来发现的在心血管研究中具有重要生理功能的一种膜磷脂类代谢产物。S1P通过与细胞表面的受体即1-磷酸鞘氨醇受体(sphingosine-1-phosphate receptos,S1PRs)作用而发挥其广泛的生物学效应。S1PR是G蛋白耦联受体家族成员之一,包括S1PR1、S1PR2、S1PR3、S1PR4、S1PR5,这些受体主要与G蛋白α亚基耦联而发挥不同的生物学功能[1]。S1P对心     

抑制鞘氨醇激酶-2活性加重血管紧张素II诱导的心肌细胞肥大

目的 本实验旨在明确鞘氨醇激酶（sphingosine kinase,SphK）2在血管紧张素（angiotensin,Ang）II诱导的心肌细胞肥大中的作用。方法 分离并体外培养SD乳鼠心肌细胞。给予AngII（10 μmol/L）处理24h诱导心肌细胞肥大。AngII刺激时分别给予溶媒或SphK2特异性抑制剂ABC294640（1 μmol/L）共处理。采用蛋白免疫印迹法检测心肌细胞SphK2蛋白表达。采用酶联免疫吸附试验（enzyme-linked immunosorbant assay,ELISA）检测心肌细胞细胞核1-磷酸鞘氨醇（sphingosine 1-phosphate,S1P）水平。采用结晶紫染色观察AngII诱导的心肌细胞肥大程度。采用实时定量聚合酶链式反应（real time-polymerase chain reaction,RT-PCR）检测心肌细胞肥大标志基因心房钠尿肽（atrial natriuretic peptide,ANP）、脑钠尿肽（brain natriuretic peptide,BNP）和β-肌球蛋白重链（β-myosin heavy chain,β-MHC）mRNA表达水平。结果 与对照组比较,AngII处理上调心肌细胞SphK2蛋白表达和S1P浓度（均P<0.05）,并增加心肌细胞横截面积与ANP、BNP和β-MHC mRNA表达水平（均P<0.05）。与溶媒组比较,ABC294640处理显著降低了心肌细胞核S1P浓度,并进一步增加了AngII处理后的心肌细胞横截面积和肥大标志基因mRNA表达水平（均P<0.05）。结论 抑制SphK2活性加重AngII诱导的心肌细胞肥大。SphK2有可能成为治疗病理性心肌肥大的新靶点。     

S1PR2通过抑制AKT/GSK-3β减少高糖诱导的内皮增殖

目的通过下调1-磷酸鞘氨醇2受体(S1PR2),观察其对体外高糖培养的脐静脉内皮细胞增殖的影响并探讨其通路。方法体外培养脐静脉内皮细胞,加入30 mmol/L的D-葡萄糖干预72 h后,检测S1PR2受体变化,内皮增殖情况,通过下调S1PR2后,查看S1PR2影响内皮增殖的可能通路。结果与正常组细胞相比,高糖组内皮细胞存活率降低,S1PR2表达增加,p-AKT/AKT、p-GSK-3β/GSK-3β、β-catenin、cyclin D1表达降低;通过在高糖干预的内皮细胞中加入S1PR2特异性拮抗剂(JTE-013)后,与高糖组相比,内皮细胞存活率增加,p-AKT/AKT、p-GSK-3β/GSK-3β、β-catenin、cyclin D1表达增加,差异均有统计学意义(P0.05)。结论 S1PR2参与高糖诱导的内皮细胞增殖,其通路可能与AKT/GSK-3β/β-catenin/cyclin D1相关。     

阿芬太尼调节SphK1/S1P信号通路保护心肌缺血再灌注损伤大鼠

[目的]探究阿芬太尼对心肌缺血再灌注损伤(MIRI)大鼠的作用及在该过程中对鞘氨醇激酶1(SphK1)/鞘氨醇-1-磷酸(S1P)信号通路的调节机制。[方法]将SPF级SD雄性大鼠随机分为假手术组、模型组、阳性药物组(复方丹参组)和阿芬太尼低剂量组、阿芬太尼高剂量组、阿芬太尼高剂量+SphK1激动剂组(阿芬太尼+PMA组),每组20只。除假手术组,其余组均利用结扎左前降支冠状动脉后再灌注复制MIRI模型。全自动生物化学分析仪检测血清乳酸脱氢酶(LDH)、肌酸激酶(CK)和谷草转氨酶(AST)的活性;TTC检测大鼠心肌梗死面积;HE染色观察大鼠心肌组织形态学特征;TUNEL染色检测大鼠心肌细胞凋亡;ELISA检测血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)及S1P的水平;试剂盒检测心肌组织中丙二醛(MDA)含量和超氧化物歧化酶(SOD)的活性;Western blot检测心肌组织SphK1蛋白表达。[结果]相较于假手术组,模型组大鼠心肌组织病理损伤严重,血清中心肌损伤标志物LDH、CK和AST的活性,心肌梗死面积和心肌细胞凋亡率,TNF-α、I...     

鞘氨醇激酶/1-磷酸鞘氨醇信号通路在糖尿病及其并发症中作用的研究进展

全球糖尿病及其并发症发病率不断增加, 对公共卫生构成重大威胁, 开发对糖尿病及其并发症有效的治疗方法十分必要。鞘氨醇激酶(SphK)/1-磷酸鞘氨醇(S1P)信号轴是鞘脂代谢途径中重要的信号通路。越来越多的证据表明, SphK/S1P信号通路在糖尿病和相关并发症中起着关键作用。该文就SphK/S1P信号通路在糖尿病及其并发症中作用的研究进展作一综述, 以期为其治疗靶点提供思路。     

1-磷酸鞘氨醇及其受体与动脉粥样硬化研究进展

<正>动脉粥样硬化(AS)的发病机制尚未完全清楚,研究表明,AS是一种慢性免疫-炎症性疾病,是血管壁对各种损伤的一种异常反应~(〔1〕)。大量流行病学数据表明高密度脂蛋白(HDL)具有抗AS作用,近些年发现与HDL结合的1-磷酸鞘氨醇(S1P)在抗AS中发挥着重要作用~(〔1〕)。S1P影响着不同细胞的增殖、迁移、黏附、炎症等,在心血管、神经、免疫、呼吸等多个系统有着重要的作用,S1PR激动剂FTY720已经应用于临床治疗多发     

模拟失重大鼠胸主动脉和腹主动脉溶酶体相关分子表达改变

目的 研究四周尾部悬吊模拟失重大鼠胸主动脉(thoracic aorta,TA)和腹主动脉(abdominal aorta,AA)溶酶体相关分子的基因和蛋白表达改变.方法 采用4周尾部悬吊大鼠模拟失重对心血管系统的影响,通过Western blot技术、免疫组织化学染色和实时定量聚合酶链反应(quantitative ...     

1-磷酸鞘氨醇对人胎儿肺成纤维细胞中α-平滑肌肌动蛋白表达的作用

目的探讨1-磷酸鞘氨醇(S1P)通过1-磷酸鞘氨醇受体2(S1P2)及RhoA/Rho激酶通路对人胎儿肺成纤维细胞(HFL-1)中α-平滑肌肌动蛋白(α-SMA)表达的作用。方法本研究为实验研究。培养14～19代HFL-1。应用蛋白质印迹法分别检测不同浓度(10-7 mol/L、10-6 mol/L、10-5 mol/L)S1P刺激下HFL-1中α-SMA的表达, 10-6 mol/L S1P及10-6 mol/L S1P2拮抗剂JTE-013刺激下HFL-1中α-SMA、RhoA的表达, 10 μg/L RhoA抑制剂C3胞外酶、10-5 mol/L Rho激酶抑制剂Y27632、10-6 mol/L S1P刺激下HIF-1中α-SMA的表达。结果 S1P 10-6 mol/L组、S1P 10-5 mol/L组α-SMA的相对表达量0.641(0.591, 0.747)、0.662(0.633, 0.810)较空白对照组0.116(0.089, 0.456)升高, 差异均有统计学意义(均P<0.05)。S1P组α-SMA的相对表达量0.907(0.466, 1.629)较空白对...     

鞘氨醇-1-磷酸对心脏肥大的作用及对心脏微血管内皮细胞的影响

目的探讨鞘氨醇-1-磷酸(S1P)对心脏肥大(CH)的作用与对心脏微血管内皮细胞(CMECs)功能及血管新生的调控,及对磷脂酰肌醇3-激酶/蛋白激酶B/血管内皮生长因子(PI3K/AKT/VEGF)通路的影响。方法收集51例CH患者和65例正常志愿者外周血,及其死亡捐赠的左心室心肌组织,采用免疫组织化学法(IHC)检测两组心肌组织S1P含量,酶联免疫吸附测定法(ELISA)检测外周血S1P、人二氢-S1P(DH-S1P)和血管内皮生长因子(VEGF)的含量。采用免疫荧光法(IF)分别对两组心肌组织进行S1P和分化簇31(CD31)、S1P和Alpha-平滑肌肌动蛋白(α-SMA)共定位染色。采用免疫印迹法(WB)检测心肌组织S1P、S1P受体1-3(S1PR1-3)、α-SMA、CD31及PI3K/AKT通路的变化。分离培养原代CMECs,将第3代CMECs接种于12孔板中,建立肥大CMECs模型,分为两组:去氧肾上腺素(PE)组和PE+S1P(OE)组,每组6孔。OE组转染1μg的PDEST42-S1P-V5质粒24 h,IF对细胞进行染色。结果CH患者心肌组织S1P和血清S1P、DH-S1P和VEGF含量显著低于正常志愿者(t=6.994、7.822、5.982、9.811,P<0.05),且血清VEGF和S1P含量呈显著正相关性(r=0.427,P>0.01)。相比于正常志愿者,CH患者心肌组织CD31+S1P+双阳性共定位细胞占CD31阳性细胞比例显著降低(t=18.214,P<0.05);α-SMA+S1P+双阳性共定位细胞占α-SMA阳性细胞比例也显著降低(t=12.451,P<0.05)。与正常志愿者相比,CH患者心肌组织S1P、S1PR3、CD31和α-SMA蛋白含量明显降低(t=4.254、4.492、15.803、9.941,P<0.05),S1PR2含量明显增高(t=6.828,P<0.05),S1PR1含量无明显变化,差异无统计学意义(P>0.05)。CH患者心脏组织p-PI3K、p-AKT和p-eNOS蛋白表达量明显低于正常志愿者(t=12.340、15.597、8.624,P<0.05)。相比于PE组,OE组中S1P和α-SMA双阳性共定位细胞占α-SMA阳性细胞比例显著增加,细胞培养上清中S1P和VEGF蛋白水平显著增加(t=6.894、5.213,P<0.05)。结论低水平的S1P可能通过抑制CMECs血管新生和间充质转换,及下调PI3K/AKT/eNOS通路在CH的发生发展中发挥重要作用。     

血清1-磷酸鞘氨醇水平对老年急性脑梗死患者颈动脉粥样硬化斑块形成有一定预测价值

摘要 目的：探讨血清1-磷酸鞘氨醇（S1P）水平对老年急性脑梗死（ACI）患者颈动脉粥样硬化（CAS）斑块形成的预测价值。方法： 采用横断面法选取入院24h内接受颈部血管超声检查的老年ACI患者223例,根据有无CAS斑块分为内膜增厚组（72例）和斑块组（151例）,另选取同期健康体检者70例作为对照组。检测3组受试者血清S1P水平,采用单因素和多因素Logistic回归分析S1P与老年ACI患者CAS斑块形成的相关性,采用受试者工作特征（ROC）曲线分析S1P对颈动脉不稳定斑块的预测价值。结果：单因素分析显示,患者年龄、胆固醇（TC）,甘油三酯（TG）、血清S1P水平与CAS斑块的形成有关（P均＜0.05）。斑块组S1P水平显著高于内膜增厚组 [(712.63±132.34) ng/mL vs. (656.86±130.58)ng/mL,P＜0.05]。多因素Logistic回归显示,S1P是影响CAS斑块形成的独立危险因素（OR>1,P<0.05）。ROC曲线显示,当S1P截断值为685ng/mL时,曲线下面积为0.97（95%CI：0.970~0.980,P=0.001）,敏感度为95.8%,特异性为89.4%。结论： 血清S1P水平是老年ACI患者CAS斑块形成的独立危险因素。     

间断人工重力在模拟失重所致胸主动脉炎症反应中的对抗作用

目的 观察模拟失重大鼠胸主动脉炎症反应变化以及间断人工重力对抗模拟失重所致变化的作用.方法 采用尾部悬吊方法建立模拟失重大鼠模型,将45只雄性Sprague-Dawley大鼠随机分为3组,每组15只(n=15).即对照(CON)组、4周尾部悬吊(HU)组和1 h/d间断人工重力(IAG)组.建模成功后,分离大鼠胸主动脉...     

Melatonin prevents deregulation of the sphingosine kinase/sphingosine 1‐phosphate signaling pathway in a mouse model of diethylnitrosamine‐induced hepatocellular carcinoma

The sphingosine kinase (SphK)/sphingosine 1‐phosphate (S1P) pathway is involved in multiple biological processes, including carcinogenesis. Melatonin shows beneficial effects in cell and animal models of hepatocellular carcinoma, but it is unknown if they are associated with the modulation of the SphK/S1P system, along with different downstream signaling pathways modified in cancer. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight i.p) once a week for 8 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p. beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Melatonin alleviated the distortion of normal hepatic architecture, lowered the incidence of preneoplastic/neoplastic lesions, and inhibited the expression of proliferative/cell cycle regulatory proteins (Ki67, PCNA, cyclin D1, cyclin E, CDK4, and CDK6). S1P levels and expression of SphK1, SphK2, and S1P receptors (S1PR1/S1PR3) were significantly elevated in DEN‐treated mice. However, there was a decreased expression of S1P lyase. These effects were significantly abrogated in a time‐ and dose‐dependent manner by melatonin, which also increased S1PR2 expression. Following DEN treatment, mice exhibited increased phosphorylation of PI3K, AKT, mTOR, STAT3, ERK, and p38, and a higher expression of NF‐κB p50 and p65 subunits. Melatonin administration significantly inhibited those changes. Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the protective effects of melatonin in hepatocarcinogenesis.     

Expression of SphK1 impairs degranulation and motility of RBL-2H3 mast cells by desensitizing S1P receptors

Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting a variety of biologically active substances, including sphingosine-1 phosphate (S1P). Sphingosine kinase 1 (SphK1) and formation of S1P, which leads to transactivation of S1P receptors and their downstream signaling pathways, regulates mast-cell functions initiated by cross-linking of the high-affinity immunoglobulin E (IgE) receptor FcepsilonRI. Surprisingly, overexpression of SphK1 in rat basophilic leukemia (RBL)-2H3 mast cells impaired degranulation as well as migration toward antigen. These effects were reversed by serum withdrawal, yet the increased formation and secretion of S1P were the same as in the presence of serum. Nonetheless, serum increased localization of SphK1 at the plasma membrane. This restricted formation of S1P induced internalization and desensitization of S1P receptors on the surface of mast cells as determined by confocal immunofluorescence microscopy, aberrant S1P receptor signaling, and lack of S1P receptor coupling to G proteins. Serum starvation, which significantly reduced membrane-associated SphK1 activity, restored S1P receptor functions. Our results have important implications for mast-cell migration and degranulation as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream.     

Melatonin inhibits the sphingosine kinase 1/sphingosine‐1‐phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin

The sphingosine kinase (SphK)1/sphingosine‐1‐phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV‐infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll‐like receptor (TLR)4, tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6, nuclear factor‐kappa B (NF‐κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.     

Deletion of the sphingosine kinase-1 gene influences cell fate during hypoxia and glucose deprivation in adult mouse cardiomyocytes

OBJECTIVES: Activation of sphingosine kinase (SphK), which has two known isoforms, is responsible for the synthesis of sphingosine 1-phosphate (S1P), a cell survival factor. We tested the following hypotheses: 1] cardiac myocytes null for the SphK1 gene are more vulnerable to the stress of hypoxia+glucose deprivation; 2] the monoganglioside GM-1, which activates SphK via protein kinase C epsilon, is ineffective in SphK1-null myocytes; 3] S1P generated by SphK activation requires cellular export to be cardioprotective. METHODS: We cultured adult mouse cardiac myocytes from wildtype and SphK1-null mice (deletion of exons 3-6) and measured cell viability by trypan blue exclusion. RESULTS: In wildtype adult mouse cardiomyocytes subjected to 4 h of hypoxic stress+glucose deprivation, cell viability was significantly higher than in SphK1-null cardiomyocytes. SphK1-null cells also displayed more mitochondrial cytochrome C release. Cell death induced by hypoxia+glucose deprivation was substantially prevented by pretreatment with exogenous S1P in both wildtype and SphK1-null myocytes, but S1P was effective at a lower concentration in wildtype cells. Hence, the absence of the Sphk1 gene did not affect receptor coupling or downstream signal transduction. Pretreatment for 1 h with 1 microM of the monoganglioside GM-1 increased survival in wildtype cells, but not in SphK1-null myocytes. Thus, activation of SphK1 by GM-1 leads to cell survival. In wildtype cells, enhanced survival produced by GM-1 was abrogated by pretreatment either with 300 nM of the S1P(1) receptor-selective antagonist VPC23019 or with 100 ng/ml of pertussis toxin for 16 h before exposure to hypoxia+glucose deprivation. CONCLUSION: As the effect of GM-1 is blocked both at the receptor and the G-protein (Gi) levels, we conclude that S1P generated by GM-1 treatment must be exported from the cell and acts in a paracrine or autocrine manner to couple with its cognate receptor.     

Curcumin ameliorates diabetic nephropathy by inhibiting the activation of the SphK1-S1P signaling pathway

Curcumin, a major polyphenol from the golden spice Curcuma longa commonly known as turmeric, has been recently discovered to have renoprotective effects on diabetic nephropathy (DN). However, the mechanisms underlying these effects remain unclear. We previously demonstrated that the sphingosine kinase 1-sphingosine 1-phosphate (SphK1-S1P) signaling pathway plays a pivotal role in the pathogenesis of DN. This study aims to investigate whether the renoprotective effects of curcumin on DN are associated with its inhibitory effects on the SphK1-S1P signaling pathway. Our results demonstrated that the expression and activity of SphK1 and the production of S1P were significantly down-regulated by curcumin in diabetic rat kidneys and glomerular mesangial cells (GMCs) exposed to high glucose (HG). Simultaneously, SphK1-S1P-mediated fibronectin (FN) and transforming growth factor-beta 1 (TGF-β1) overproduction were inhibited. In addition, curcumin dose dependently reduced SphK1 expression and activity in GMCs transfected with SphK^WT and significantly suppressed the increase in SphK1-mediated FN levels. Furthermore, curcumin inhibited the DNA-binding activity of activator protein 1 (AP-1), and c-Jun small interference RNA (c-Jun-siRNA) reversed the HG-induced up-regulation of SphK1. These findings suggested that down-regulation of the SphK1-S1P pathway is probably a novel mechanism by which curcumin improves the progression of DN. Inhibiting AP-1 activation is one of the therapeutic targets of curcumin to modulate the SphK1-S1P signaling pathway, thereby preventing diabetic renal fibrosis.     

Distinct roles of sphingosine kinases 1 and 2 in human mast-cell functions

Sphingosine-1-phosphate (S1P) is now emerging as a potent lipid mediator produced by mast cells that contributes to inflammatory and allergic responses. In contrast to its weak effect on degranulation of murine mast cells, S1P potently induced degranulation of the human LAD2 mast-cell line and cord blood-derived human mast cells (hMCs). S1P also stimulated production and secretion of cytokines, TNF-alpha and IL-6, and markedly enhanced secretion of a chemokine, CCL2/MCP-1, important modulators of inflammation. S1P is produced in mast cells by the 2 sphingosine kinases, SphK1 and SphK2. SphK1 but not SphK2 plays a critical role in IgE/Ag-induced degranulation, migration toward antigen, and CCL2 secretion from hMCs, as determined by specifically down-regulating their expression. However, both isoenzymes were required for efficient TNF-alpha secretion. Taken together, our data suggest that differential formation of S1P by SphK1 and SphK2 has distinct and important actions in hMCs.