印楝素A与印楝素B对粉纹夜蛾BTI-Tn-5B1-4的细胞毒性

为了明确印楝素A和B活性差异的机理，本研究比较了印楝素A和印楝素B对粉纹夜蛾Trichoplusia ni离体培养胚胎细胞系BTI-Tn-5B1-4的毒性。结果表明：印楝素A与印楝素B对BTI-Tn-5B1-4细胞具有良好的增殖抑制活性，处理后3 d，其IC₅₀值分别为2.9 μg/mL和9.85 μg/mL，印楝素A的细胞毒力显著高于印楝素B。倒置显微镜观察发现，印楝素A和印楝素B处理可导致细胞变形，贴壁能力下降，并出现明显空泡，印楝素A的影响明显高于印楝素B。流式细胞仪检测结果表明，印楝素可导致BTI-Tn-5B1-4细胞体积显著膨大，印楝素A处理细胞体积增大程度显著高于印楝素B；印楝素可以明显影响BTI-Tn-5B1-4细胞膜电位，1.25 μg/mL印楝素A和印楝素B处理后3 d，细胞DiBAC4（3）荧光强度分别增加88.12%和55.37%，印楝素A的影响显著高于印楝素B。荧光显微镜观察发现，印楝素对BTI-Tn-5B1-4细胞核具有明显影响，印楝素B的影响明显高于印楝素A，印楝素B处理后，细胞核受损细胞数更多，受损程度更严重。结果显示印楝素A和印楝素B的细胞作用机理存在差异，本研究从细胞学水平解释了印楝素的生长发育抑制作用机理。

Toxicity of azadirachtin A and azadirachtin B on Trichoplusia ni (Lepidoptera:Noctuidae)BTI-Tn-5B1-4 cells

To clear the mechanism of activity differences of azadirachtin A and azadirachtin B, the toxicities of azadirachtin A and azadirachtin B on Trichoplusia ni BTI-Tn-5B1-4 cell line were compared. The results showed that both azadirachtin A and azadirachtin B had excellent inhibitory effect on BTI-Tn-5B1-4 cell proliferation. After T. ni BTI-Tn-5B1-4 cells were treated with azadirachtin A and azadirachtin B for 3 d, their IC50 values were 2.09 μg/mL and 9.85 μg/mL, respectively, suggesting that the toxicity of azadirachtin A was greater than that of azadirachtin B. Under inverted microscope, it was found that both azadirachtin A and azadirachtin B could induce cell deformation, adhesion capacity reduction and vacuole appearance, and azadirachtin A showed obviously greater effect than azadirachtin B. Azadirachtin treated-cells were commonly bigger than the control, and the cells treated with azadirachtin A were bigger than those with azadirachtin B through flow cytometry analysis. Azadirachtin can also change membrane potential of BTI-Tn-5B1-4 cells After BTI-Tn-5B1-4 cells were treated for 3 d, DiBAC₄ (3) fluorescence intensity in azadirachtin A and azadirachtin B-treated cells was 88.12% and 55.37%, respectively, showing that azadirachtin A had greater effect than azadirachtin B. Under fluorescence microscope, it was found that azadirachtin could influence cell nucleus, and azadirachtin B affected more remarkably than azadirachtin A, based on the results that the amount of cells with damaged-nucleus treated with azadirachtin B was larger, and damage in cell nucleus was more severe. The results suggest that there exists mechanism difference between azadirachtin A and azadirachtin B, and provide the clues to explain the mechanism of azadirachtin that inhibits insect growth at the cell level.