S1PR3特异性激动剂RY-15促进细菌清除的作用研究

S1PR3 agonist RY-15 promotes bacterial clearance

目的通过合成S1PR3特异性激动剂RY-15，研究其促进巨噬细胞清除细菌的作用，为临床转化研究提供依据。方法带FITC的RY-15与THP-1细胞共培养5min、10min、20min、30min，激光共聚焦显微镜下观察RY-15的入胞情况。庆大霉素保护实验观察GY-5和RY-15对巨噬细胞清除细菌能力的影响。Western Blot检测GY-5和RY-15分别对THP-1细胞ERK通路的磷酸化水平。结果激光共聚焦显微镜观察显示，RY-15作用THP-1细胞10min后，开始入胞；30min后，入胞效果显著。庆大霉素保护实验发现，RY-15可以显著降低细菌被吞噬后巨噬细胞内存活的大肠杆菌数量（P〈0．05），而GY-5则对巨噬细胞清除细菌作用没影响（P〉0．05）。Western Blot实验结果显示，GY-5、RY-15均可以使ERK通路磷酸化，而RY-15对其活化的作用更显著。结论S1PR3特异性激动剂RY-15可以进入到细胞内，促进巨噬细胞清除细菌，这一作用可能是通过ERK通路磷酸化来实现的。

Objective RY-15, a specific agonist of Sphingosine 1-phosphate Receptor 3, was synthesized for investigating the function and mechanism of S1PR3 in bacterial clearance. Methods Measure the ability of RY-15 with FITC to enter the THP-1 cell after coculture for 5 min, 10 min, 20 min, 30 min through confocal microscopy. The function of GY-5 and RY-15 in bacterial clearance was observed by gentamicin protection test. The phosphorylation level of ERK and p-ERK in THP-1 cell was detected by Western Blot after GY-5 and RY-15 stimulation for different times. Results According to confocal microscopy, RY-15 started to enter the THP-1 cell after stimulating for 10 min and the effect of entering cell was very obvious after stimulating for 30 min. Compared to GY-5 group, live bacteria in the macrophage were largely decreased in the RY-15 group（P〈0.05）. Conmpared to GY-5 group, the p-ERK level raised largely at different poins. Conclusions RY- 15, a specific agonist of Sphingosine 1-phosphate Receptor 3, can promote bacterial clearance through entering cell and the phosphorylation level of ERK is a possible mechanism.