创伤性颅脑损伤后S1PR1通过ERK调控海马区神经干细胞增殖的实验研究

Role of ERK in S1PRl-mediated proliferation of hippocampal stem cells following traumatic brain injury ： An experimental study

目的探讨创伤性颅脑损伤（TBI）后细胞外信号调节蛋白激酶（ERK）在1-磷酸鞘氨醇受体1（S1PR1）介导的海马区神经干细胞增殖中的作用。方法采用控制性皮质损伤法制备大鼠TBI模型。将112只SD大鼠按照随机数字表法均分为假损伤组、TBI组、TBI并S1PR1抑制剂VPC23019干预组（TBI＋VPC组）、TBI并ERK抑制剂U0126干预组（TBI＋U0126组），每组各28只。采用Western blot方法检测各组海马区S1PR1、磷酸化ERK（pERK）以及总ERK（tERK）的表达水平。通过免疫荧光双标染色检测各组海马齿状回颗粒细胞层下区神经干细胞的增殖能力。结果在TBI后7、14以及21 d，与假损伤组相比，TBI组S1PR1和pERK的表达水平明显增加（均P〈0.05），海马区神经干细胞的增殖能力增强（均P〈0.05）；与TBI组相比，TBI＋VPC组S1PR1和pERK的表达均下调（均P〈0.05），海马区神经干细胞的增殖能力下降（均P〈0.05）；与TBI组相比，TBI＋U0126组S1PR1的表达水平无明显变化（均P〉0.05），但pERK的表达水平显著下降（均P〈0.05），神经干细胞的增殖能力明显受到抑制（均P〈0.05）。各时间点四组间tERK表达量的差异无统计学意义（均P〉0.05）。结论TBI后，海马区S1PR1表达的增加可引起pERK表达上调和神经干细胞增殖能力增强，抑制pERK可阻碍TBI后S1PR1介导的海马区神经干细胞增殖，提示TBI后S1PR1通过ERK调节海马区内源性神经再生与修复。

Objective To explore the role of extracellular signal regulated protein kinase （ERK） in sphingosine-l-phosphate receptor 1 （S1PR1）-mediated neural stem cell （NSC） proliferation in hippocampus following traumatic brain injury （TBI）. Methods Rat model of TBI was induced by a controlled cortical impact device. A total of 112 Sprague Dawley rats were randomly and evenly divided into sham, TBI, TBI ＋ VPC and TBI ＋ U0126 groups according to random number table. Rats in TB1 ＋ VPC group received 7 intraperitoneal injections of VPC23019 （S1PR1 antagonist） at scheduled time points following TBI. Rats in TBI ＋ U0126 group were intravenously administrated with U0126 （ ERK inhibitor） via tail vein at 20 rain before TBI. The levels of S1PR1, phosphate ERK （pERK） and total ERK （tERK） in hippocampus were determined by Western blot. NSC proliferation in subgranular zone of hippocampus was assessed by double- labeled immunofluorescence. Results Compared with sham group, the expressions of hippocampal S1PR1 and pERK in TBI group were significantly increased, and the proliferation of NSCs in subgranular zone was activated at 7, 14 and 21 days after TBI （all P 〈0.05）. However, compared with TBI group, both proteins were down-regulated and NSC proliferation was evidently inhibited in TBI ＋ VPC group （ all P 〈 0.05 ）. In particular, the pERK expression and NSC proliferation in TBI ＋ U0126 group were significantly decreased （ all P 〈0.05） , whereas the level of S1PR1 maintained at the same level compared with TBI group （ all P 〉 0. 05 ）. In addition, the difference in tERK expression between the 4 groups was not statistically significant （all P 〉 0.05 ）. Conclusions Increased S1PR1 could trigger up-regulation of pERK expression and enhance NSC proliferation in hippocampus after TBI. Inhibition of ERK cascade could prevent S1PR1- mediated NSC proliferation in hippocampus. It is suggested that S1PR1 might regulate the proliferation of endogenous NSCs via ERK signaling pathway post trauma.