Department of General and Vascular Surgery, Medical University of Silesia, Ziołowa 45/47, PL 40-635 Katowice, Poland.
Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Poland.
Brain Res. 2014 Mar 17;1553:1-11. doi: 10.1016/j.brainres.2014.01.017. Epub 2014 Jan 22.
The protective potential of rapamycin has been reported in a few experimental models of brain ischemia, both in vivo and in vitro. Although the precise cellular processes underlying the neuroprotective effects of rapamycin in experimental models of stroke remain unknown, the current experimental data suggest that the mechanism of action of the drug may result from the mTOR-mediated autophagy induction. However, it is unclear whether the activation of autophagy acts as a pro-death or pro-survival factor in vascular endothelial cells in ischemic brain damage. It seems to be very important, since stroke affects not only neurons and astrocytes but also microvessels. In the present study, we used human umbilical vein endothelial cells (HUVEC) subjected to ischemia-simulating conditions (combined oxygen and glucose deprivation, OGD) for 6h to determine potential effect of rapamycin-induced autophagy on HUVEC damage. The drug at concentrations of 100 and 1000nM increased the expression of Beclin 1 and LC3-II together with a significant increase in the p62 degradation in ischemic HUVEC. Treatment with rapamycin in OGD significantly increased the cell viability, indicating that the drug exerts cytoprotective effect. The inhibition of Beclin 1 by siRNAs significantly attenuated the expression of autophagy-related proteins and reduced HUVEC viability following OGD and rapamycin treatment. Our findings demonstrated that toxicity of simulated ischemia conditions were enhanced in HUVEC when autophagy was blocked, and that rapamycin effectively prevented OGD-evoked damage by induction of protective autophagy via inhibition of the mTOR pathway.
雷帕霉素的保护潜力已在一些脑缺血的体内和体外实验模型中得到报道。尽管雷帕霉素在中风实验模型中神经保护作用的确切细胞过程尚不清楚，但目前的实验数据表明，该药物的作用机制可能源于 mTOR 介导的自噬诱导。然而，尚不清楚自噬的激活在缺血性脑损伤中的血管内皮细胞中是作为促死亡还是促存活因子起作用。这似乎非常重要，因为中风不仅影响神经元和星形胶质细胞，还影响微血管。在本研究中，我们使用经历缺血模拟条件（氧和葡萄糖剥夺，OGD）6 小时的人脐静脉内皮细胞（HUVEC），以确定雷帕霉素诱导的自噬对 HUVEC 损伤的潜在影响。该药物在 100 和 1000nM 的浓度下增加了 Beclin 1 和 LC3-II 的表达，同时 p62 降解也显著增加。OGD 中的雷帕霉素处理显著增加了细胞活力，表明该药物发挥了细胞保护作用。siRNAs 对 Beclin 1 的抑制显著减弱了自噬相关蛋白的表达，并减少了 OGD 和雷帕霉素处理后的 HUVEC 活力。我们的研究结果表明，当自噬被阻断时，模拟缺血条件的 HUVEC 毒性增强，而雷帕霉素通过抑制 mTOR 通路有效预防 OGD 引起的损伤。
