Myristoyl—glycine修饰的S1PR3特异性激动肽跨膜激活效应研究

Study of the activating effects of myristoyl-glycine modified peptide which derived from S1PR3

目的拟通过合成1-磷酸鞘氨醇受体3（sphingosine 1-phosphate Receptor 3, S1PP,3）特异性激动肽并经肉豆蔻酰甘氨酸（myristoyl—glycine）修饰，研究其激活丝裂原活化蛋白激酶（mitogen-activated protein kinases, MAPKs）通路的效应。方法设计合成源自七次跨膜受体S1PR3胞内第2个环的短肽，并经Myristoyl—glycine修饰，命名为GPS-725．017。Westernblot检测GPS-725．017对单核巨噬细胞（THP一1）MAPKs通路关键分子细胞外调节蛋白激酶 （ extracellularregulated protein kinase, ERK）及应激活化蛋白激酶（stress-activated protein kinase, JNK）磷酸化水平的影响。组内或组问不同浓度和不同时间点蛋白灰度值比较采用多因素方差分析。结果与溶剂对照组相比，30Ixmol／L或50μmol／L的GPS-725．017处理THP-1细胞10min，P-ERK水平显著升高[30μmol／L组：（3．10±0．27）vs．（7．98±0．45），P〈0．01；50μmol／L组：（4．78±0．44）vs．（25．98±2．32），P〈0．01]；50μmo]／L的GPS-725．017处理THP-1细胞5min、10min、20min和30min，与溶剂组相比，各时间点p-ERK或p-JNK水平均显著上升（均为P〈0．01）。结论源于S1PR3胞内第2个环并经Myristoyl—glycine修饰的短肽GPS-725．017，可跨膜显著活化MAPKs通路，本研究为临床靶向调控S1PR3，治疗炎症及脓毒症提供理论依据。

Objective To study the effects of the myristoyl-glycine modified peptide which derived from the second intracellular loop of sphingosine 1-phosphate receptor 3 （S1PR3） on activation of mitogen- activated protein kinases （MAPKs） pathway. Methods The phosphorylation levels of JNK and ERK in THP-1 cells were detected by western blot after GPS-725. 017 stimulation. Statistical data analysis was conducted by multivariate analysis of variance. Results Western blot showed that 10 rain after 30μmol/L or 50 μmol/L GPS-725. 017 stimulated, phosphorylation of ERK significantly increased in comparison with the solvent-treated group [30 μmol/L group： （3. 10 ±0. 27） vs. （7. 98±0. 45）, P 〈0. 01 ; 50 μmol/L group： （4. 78 ±0. 44） vs. （25.98 ±2. 32）, P 〈0. 01] ; after 50 μmol/L GPS-725. 017 stimulated THP- 1 cells for 5 min, 10 min, 20 min or 30 rain, p-ERK or p-JNK level raised at different time points （P 〈 0.01vs. solvent group） . Conclusions GPS-725. 017, a kind of myristoyl-glycine modified peptide derived from S1PR3, could traverse cytomembrane and activate MAPKs pathway. This study provides an implication of targeting S1PR3 for clinical therapy on inflammatory diseases or sepsis.