Amodio Rosalba, De Ruvo Caterina, Sacchetti Andrea, Di Santo Angelomaria, Martelli Nicola, Di Matteo Vincenzo, Lorenzet Roberto, Poggi Andreina, Rotilio Domenico, Cacchio Marisa, Esposito Ennio
Laboratory of Neurophysiology, Istituto di Ricerche Farmacologiche "Mario Negri", Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti 66030, Italy.
Int J Dev Neurosci. 2003 Nov;21(7):379-89. doi: 10.1016/s0736-5748(03)00090-x.
Primary cultures of cerebellar granule neurons (CGNs) were prepared from 8-day-old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. To induce apoptosis, culture medium was replaced with serum-free medium (containing 5mM KCl) 8 days after plating. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling (TUNEL) method, and by flow cytometry. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by low K(+) (5mM) concentrations, the potential anti-apoptotic effect of caffeic acid phenethyl ester (CAPE), a potent flavonoid antioxidant, was tested in this experimental model. It was found that CAPE (10 microg/ml) promoted cell survival and was capable of blocking the apoptotic process as assayed by both TUNEL and flow cytometric methods. The same concentration of CAPE prevented the formation of ROS induced by low K(+). Since there is evidence that low K(+)-induced apoptosis in CGNs is associated with a drop in intracellular Ca(2+) concentration (Ca(2+)), activation of the cell death effector proteases caspase-3 and caspase-9, and of the transcription factor nuclear factor kappa B (NF-kappaB), the interference of CAPE with these purported mediators of apoptosis was also evaluated. It was found that CAPE did not interfere with the marked decrease in Ca(2+) induced by low K(+), whereas it completely blocked caspase-3, caspase-9, and NF-kappaB activation. It is concluded that CAPE could exert its anti-apoptotic effect in CGNs by blocking ROS formation and by inhibiting caspase activity.
小脑颗粒神经元（CGNs）的原代培养物取自8日龄的Wistar大鼠，并在含有高浓度（25 mM）氯化钾的合适培养基中培养。为诱导细胞凋亡，接种8天后将培养基换成无血清培养基（含5 mM氯化钾）。通过末端脱氧核苷酸转移酶介导的dUTP-荧光素缺口末端标记（TUNEL）法和流式细胞术检测细胞凋亡。由于有证据表明活性氧（ROS）生成增加参与了低钾（5 mM）浓度诱导的细胞凋亡，因此在该实验模型中测试了一种强效类黄酮抗氧化剂咖啡酸苯乙酯（CAPE）的潜在抗凋亡作用。结果发现，CAPE（10微克/毫升）可促进细胞存活，并能够通过TUNEL法和流式细胞术检测阻断凋亡过程。相同浓度的CAPE可防止低钾诱导的ROS形成。由于有证据表明CGNs中低钾诱导的细胞凋亡与细胞内钙浓度（[Ca2+]i）下降、细胞死亡效应蛋白酶caspase-3和caspase-9以及转录因子核因子κB（NF-κB）的激活有关，因此还评估了CAPE对这些所谓凋亡介质的干扰作用。结果发现，CAPE不干扰低钾诱导的[Ca2+]i显著下降，而它完全阻断了caspase-3、caspase-9和NF-κB的激活。结论是，CAPE可通过阻断ROS形成和抑制caspase活性在CGNs中发挥抗凋亡作用。
