Shu Benshui, Wang Wenxiang, Hu Qingbo, Huang Jingfei, Hu Meiying, Zhong Guohua
Laboratory of Insect Toxicology, Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, People's Republic of China.
Arch Insect Biochem Physiol. 2015 Jul;89(3):153-68. doi: 10.1002/arch.21233. Epub 2015 Apr 1.
The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.
印楝素是从印楝树（Azadirachta indica A. Juss）及楝科其他植物中分离得到的一种著名的植物四环三萜类化合物，本研究在草地贪夜蛾培养细胞系（Sf9）中探究了其诱导细胞凋亡的作用。在光学显微镜下，于不同时间观察了用不同浓度印楝素处理的Sf9细胞的形态变化。形态学和生化分析表明，用1.5μg/mL印楝素处理的Sf9细胞呈现出典型的形态变化，这表明细胞发生了凋亡，且出现了清晰的DNA梯形条带。流式细胞术分析显示，在Sf9细胞中，用1.5μg/mL印楝素处理24小时时，细胞凋亡率达到最大值32.66%。Sf9细胞增殖受到抑制，这表明印楝素的作用具有剂量依赖性，在48小时和72小时时的半数有效浓度（EC50）分别为2.727×10⁻⁶和6.348×10⁻⁹μg/mL。用印楝素处理Sf9细胞可显著提高Sf半胱天冬酶 -1的活性，但对拓扑异构酶I的活性没有影响，这表明印楝素在Sf9细胞中诱导的凋亡是通过半胱天冬酶依赖性途径进行的。这些结果不仅为印楝素诱导细胞凋亡提供了一系列形态学、生化和毒理学的综合证据，也为从天然化合物中筛选昆虫细胞凋亡诱导剂提供了参考模型。
