Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
J Ethnopharmacol. 2011 Sep 2;137(2):971-8. doi: 10.1016/j.jep.2011.07.013. Epub 2011 Jul 12.
Barakol, an anxiolytic agent isolated from Senna siamea leaves which has been traditionally used for producing natural sleep, has been described as toxic to patients.
The aim of current study was to investigate the molecular mechanism of barakol-induced toxicity in mouse embryonal carcinoma P19 cell model.
XTT assay was used to determine cell viability in P19 cells treated with barakol. Apoptotic cells were detected by Hoechst 33342 staining. Intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry using a fluorescent dye, DCFH-DA. Detection of apoptotic protein expression in P19 cells was performed by Western blot analysis. Caspase-9 activity was measured using a fluorescent immunosorbent enzyme assay kit.
Treatment with barakol decreased cell viability in a concentration- and time-dependent manner with an IC(50) value of 1.5mM in 24-h treated cells. A Hoechst 33342 assay revealed that barakol cytotoxicity was due to a significant increase in the number of apoptotic cells. Different scavengers to characterize ROS were utilized and revealed that hydroxyl radicals played a major role in ROS-induced apoptosis in barakol-treated cells. Western blot analysis demonstrated that barakol-induced apoptosis was mediated by the increase in expression ratio of Bax/Bcl-2. Furthermore, increase in caspase-9 activity after exposure to barakol for 24h was also observed. Pretreatment of cells with N-acetyl-l-cysteine (NAC) attenuated intracellular ROS generation, the Bax/Bcl-2 protein expression, and apoptosis.
The mechanism of barakol-mediated toxicity in P19 cells is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-9 activation leading to apoptotic cell death. Pretreatment of cells with NAC could antagonize the toxicity produced by barakol.
从暹罗决明叶子中分离出来的具有镇静作用的巴拉科，传统上被用于产生自然睡眠，已被描述为对患者有毒。
本研究旨在研究巴拉科在小鼠胚胎癌细胞 P19 细胞模型中诱导毒性的分子机制。
用 XTT 法测定巴拉科处理的 P19 细胞的细胞活力。用 Hoechst 33342 染色法检测凋亡细胞。用荧光染料 DCFH-DA 通过流式细胞术分析细胞内活性氧（ROS）的产生。通过 Western blot 分析检测 P19 细胞中凋亡蛋白的表达。用荧光免疫酶测定试剂盒测定 caspase-9 活性。
巴拉科处理以浓度和时间依赖的方式降低细胞活力，在 24 小时处理的细胞中 IC50 值为 1.5mM。Hoechst 33342 测定表明，巴拉科细胞毒性是由于凋亡细胞数量的显著增加。利用不同的清除剂来表征 ROS，结果表明羟基自由基在巴拉科处理细胞的 ROS 诱导凋亡中起主要作用。Western blot 分析表明，巴拉科诱导的凋亡是通过 Bax/Bcl-2 表达比值的增加介导的。此外，暴露于巴拉科 24 小时后还观察到 caspase-9 活性增加。用 N-乙酰-L-半胱氨酸（NAC）预处理细胞可减轻细胞内 ROS 的产生、Bax/Bcl-2 蛋白表达和细胞凋亡。
巴拉科在 P19 细胞中的介导毒性的机制主要与 ROS 的产生有关，随后是 Bax/Bcl-2 比值的失衡，以及 caspase-9 的激活导致细胞凋亡死亡。用 NAC 预处理细胞可以拮抗巴拉科产生的毒性。
