藁本内酯对糖氧剥夺-再灌注诱导的血管内皮细胞凋亡和ERK磷酸化水平的影响

Effect of ligustilide on cell apoptosis and phosphorylation of ERK in vascular endothelial cells with oxygen-glucose deprivation-reperfusion injury

目的:研究藁本内酯预处理对糖氧剥夺-复糖氧损伤人脐静脉血管内皮细胞(HUVEC)损伤和凋亡自噬蛋白及ERK磷酸化水平的影响。方法:将融合生长的HUVEC给予藁本内酯预处理1h后,用连二亚硫酸钠(Na2S2O4)制备糖氧剥夺-复糖氧损伤(oxygen-glucose deprivation-reperfusion injury,OGD-R)模型,观察细胞一般形态,微板法检测细胞外乳酸脱氢酶(LDH)含量;高内涵分析系统检测Beclin-1、p53的表达与细胞周期,蛋白免疫印迹法测定细胞内ERK磷酸化水平及Bax、Bcl-2蛋白的表达。结果:与溶剂对照组比较,OGD-R损伤的HUVEC细胞形态异常,细胞外LDH含量增加,细胞内Beclin-1、p53、Bax、Bcl-2表达和ERK磷酸化水平明显上升;藁本内酯20、40、60μmol/L可改善OGD-R损伤的HUVEC的形态,降低细胞外LDH含量,降低细胞内Beclin-1、p53、Bax,提升Bcl-2的表达和进一步提高ERK磷酸化水平。结论:藁本内酯预处理对OGD-R损伤的HUVEC有保护作用,可缓解OGDR损伤导致的细胞凋亡与自噬,该作用与ERK相关通路的激活有关。

Obieetive： To observe the damage, apoptosis and phosphorylation of ERK in vascular endothelial cell pretreated with ligustilide （LIG） after oxygen-glucose deprivation-reperfusion （OGD-R） injury. Methods： Human umbilical vein endothelial cells （HUVEC） were involved in the experiment. When fusion growth, cells were grouped into solvent control （ pretreated with 0.1% methyl alcohol）, treatment groups （ pretreated with 20, 40, 60μM of ligustilide, respectively） and model group （pretreated with 0.1% methyl alcohol）. After cells received drug intervention for lh, ischemia-reperfusion injury models were established by sodium dithionite for every group except solvent control. Cellular morphology was abtained by microscope, extracellular lactate dehydrogenase （LDH） content was measured by kit. The expression of p53 and Beclin-1 and the cell cycle were determined by high-content screening （HCS） and analysis system. ERK, p-ERK, Bax and BC1-2 were de- termined by Western blot and analyzed by Quantity One. Results： Cell were ruptured and LDH leaked out from HUVEC after OGD-R injury companied with up-regulation of pro-apoptotic and autophagy proteins such as p53, Bax and Beclin-1, the p-ERK was up-regulated as well, while BC1-2 expression in cells decreased. Ligustilide at the concentration of 20 -60μM protected cell integrity, restrained pro-apoptotic and autophagy proteins expression and promoted anti-apoptotic protein expression. Cells ERK pathway were activated and leaved G0/G1 phase to mitosis after OGD-R injury while ligustilide pretreatment promoted these changes, according to western blot and HCS. Conclusions： Ligustilide protect against OGD-R injury-induced HUVEC damage, apoptotic , autophagy and promote cell division, the ERK pathway may be in-volved.