RhoA蛋白参与凝血酶对胎鼠大脑皮层神经元的损伤

RhoA is involved in thrombin-induced neuron injury in the cortex of fetal rats

目的探讨凝血酶诱导的胎鼠大脑皮层神经元损伤与RhoA蛋白活化表达的关系。方法体外培养胎鼠大脑皮层神经元8d后，采用Westernblotting检测不同浓度凝血酶（0、1、10、30和100U／mL）作用3h后及30U／mL凝血酶作用不同时间（0、0．5、1、3和6h）对皮层神经元I让oA蛋白活化表达的影响：RhoA蛋白活化抑制剂ExoenzymeC3预处理0．5h及30U／mL凝血酶作用皮层神经元3h后，行Westernblotting检测RhoA蛋白活化表达的情况，并采用Hoechst33258核染色及CCK-8法观察ExoenzyrneC3预处理和未处理时凝血酶对皮层神经元的损伤情况。结果在浓度实验中，30U／mL凝血酶作用皮层神经元3h后能明显增加RhoA膜蛋白表达，与0U／mL组相比差异有统计学意义（P〈0．05）；而各浓度凝血酶对RhoA总蛋白表达无明显影响。在时程实验中，与其他时间点相比，30U／mL凝血酶在3h时能明显增加RhoA膜蛋白表达，差异有统计学意义（P〈0．05），但对RhoA总蛋白表达也无明显影响。在ExoenzymeC3干预实验中，ExoenzymeC3预处理组与凝血酶组相比，RhoA膜蛋白表达明显降低，差异有统计学意义（P〈0．05）；Hoechst33258核染色显示．与凝血酶组相比，ExoenzymeC3预处理组中核浓染细胞数量明显降低，差异有统计学意义（P〈0．05）；CCK-8法检测细胞活性显示，凝血酶组细胞存活率明显低于ExoenzymeC3预处理组，差异有统计学意义（P〈0．05）。结论凝血酶诱导的胎鼠大脑皮层神经元损伤与RhoA膜蛋白的高表达即RhoA蛋白活化表达有关。

Objective To investigate the relationship between the activation of RhoA and thrombin-induced neuron injury in the cortex of fetal rats. Methods The neurons from the fetal rat cortex were cultured in vitro for 8 d; and then, they were treated by thrombin at different concentrations （0, 1, 10, 30 and 100 U/mL） for 3 h, and by 30 U/mL thrombin at different times （0, 0.5, 1, 3 and 6 h）; Western blotting was used to examine the effects of these different treatments on the activation of RhoA. The neurons were pretreated with Exoenzyme C3, the RhoA inhibitor, for 0.5 h, and incubated with 30 U/ml thrombin for 3 h; and then, Western blotting was employed to examine the activation of RhoA; besides that, the injuries of these neurons with the presence and absence of Exoenzyme C3 were observed by Hoechst33258 staining and CCK-8 assay. Results The activation of RhoA expressing in membrane with the treatment of 30 U/mL thrombin for 3 h was significantly increased as compared with that under the treatment of 0 U/mL （P〈0.05）; and the total RhoA showed no significant changes with the treatment of all concentrations. The 3 h site with thrombin （30 U/mL） could more significantly induce RhoA expression as compared with other time sites （P〈0.05）, and the total RhoA showed no significant changes under the treatment of all time sites. Pretreatment of neurons with Exoenzyme C3 could significantly down-regulate RhoA level as compared with those without pretreatment （P〈0.05）; meanwhile, Hoechst33258 staining indicated that the number of brightly stained neurons in the Exoenzyme C3 presence group was dramatically decreased as compared with that in the Exoenzyme C3 absence group （P〈0.05）, and CCK-8 assay showed that the cell survival rate in Exoenzyme C3 absence group significantly decreased as compared with that in the Exoenzyme C3 presence group （P〈0.05）. Conclusion RhoA in membrane is related to the thrombin-induced neuron injury in the cortex of fetal rats.