Studies On Transmembrane Activating Effects Of S1PR3 Peptide Agonist In Sepsis

Part 1 Studies on transmembrane activating effects of myristoyl-glycine modified peptide derived from S1PR3Objective:To study the effects of the myristoyl-glycine modified peptide which derived from sphingosine 1-phosphate receptor 3(S1PR3)on activation of S1PR3-MAPK(mitogen-activated protein kinases,MAPK)axis in THP-1 cell.Methods:The myristoyl-glycine modified peptide which derived from the second intracellular loop of sphingosine 1-phosphate receptor 3(S1PR3)was named GPS-725.017.The impact of GPS-725.017 on phosphorylation levels of MAPK and JNK was detected by western blot.Comparison of protein expression among or within groups was analyzed by multivariate analysis of variance.Results:Compared with the solvent-treat group,10 min after 30 μmol/L or 50 μmol/L GPS-725.017 stimulated,phosphorylation of ERK significantly increased in THP-1 cells(30 μmol/L,3.10±0.27 vs.7.98±0.45,p<0.001;50 μmol/L,4.78±0.44 vs.25.98±2.32,p<0.001);after 50 μmol/L GPS-725.017 stimulated THP-1 cells for 5 min,10 min,20 min or 30 min,p-ERK or p-JNK level raised at different time points(p<0.01 vs.solvent group).Conclusions:GPS-725.017,a kind of myristoyl-glycine modified peptide derived from S1PR3,could traverse cytomembrane and activate S1PR3-MAPKs axis for THP-1 cell.Part 2 Analysis of the application of S1PR3 peptide agonist in sepsis treatmentObjective:In the first part of our study,the results show that GPS-725.017 can activate the S1PR3-MAPK signaling in THP-1 cells to promote their function.Therefore,we further to study the effects of GPS-725.017 on E.coli infected mouse macrophages,and determine the capability of bacterial clearance by mouse macrophages.Then in a cecal ligation and puncture(CLP)model,septic mice were treated with GPS-725.017 and the prognosis was evaluated.Methods:1.The E.coli clearance in macrophages was measured by gentamycin protection.The macrophages were obtained from the abdominal cavity of mice.The experimental group was pretreated with 10μM GPS-725.017,whereas control group was treated with PBS.2.The septic mice model were established by cecal ligation and puncture followed by GPS-725.017 injection,and their prognosis were observed.Statistical data was analyzed by SPSS 16.0 software or Prism 6.0.All data were presented in mean±SD.Comparisons among groups were analyzed using multivariate analysis of variance with the Bonferroni post-test.Differences with p<0.05 was considered significant.Results:1.To study the function of bacterial clearance in mouse macrophages after treated with 10μmol/L GPS-725.017.Compared with the control group,experimental group displayed significant bacterial clearance at 3h and 6h post GPS-725.017 treatment(p<0.01).2.The prognosis of septic mice was evaluated after CLP model establishment as well as 10 days post-GPS-725.017 administration.The survival rate was significantly higher in GPS-725.017-treatment group(n=30)than control group(n=27)(p<0.05).Conclusions:1.The peptide agonist GPS-725.017 derived from S1PR3 can enhance the function of bacterial clearance of macrophage.2.GPS-725.017 has the capability of improving the septic mice survival.