Effects of azadirachtin on proliferation and apoptosis of Plutella xyllostella embryonic cells
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摘要:目的
探讨印楝素对小菜蛾Plutella xyllostella胚胎细胞增殖和凋亡的影响及可能的机制。
方法将体外培养的小菜蛾胚胎细胞分为印楝素处理组和未处理组，采用CCK-8试剂盒检测细胞增殖的抑制率，激光共聚焦镜检PI染色后的细胞死亡和DAPI染色后的凋亡小体，通过Western-blotting检测各组细胞Caspase-3的表达情况以及通路蛋白Akt的磷酸化水平。
结果印楝素对小菜蛾胚胎细胞有明显的增殖抑制作用，且呈现浓度依赖性，24 h的IC50为4.4 μg·mL-1。印楝素处理后的细胞经PI染色后能明显观察到死亡细胞，DAPI染色后可见凋亡小体；Caspase-3蛋白发生剪切，并且抑制Akt的磷酸化水平。
结论印楝素对小菜蛾胚胎细胞的增殖有明显的抑制作用，并通过抑制Akt信号通路的活化，诱导细胞产生依赖于Caspase-3的Ⅰ型凋亡。
Abstract:ObjectiveTo investigate the effect and mechanism of azadirachtin on proliferation and apoptosis in Plutella xyllostella embryonic cells.
MethodP. xyllostella embryonic cells were divided into azadirachtin treated and untreated groups, the inhibition rate of cells proliferation was detected using CCK-8 kit. The cell death and apoptotic body were observed using laser confocal microscope after stained by PI and DAPI respectively. The expression of Caspase-3 and the phosphorylation of pathway protein Akt were detected using Western-blotting.
ResultAzadirachtin had significant inhibitory effect on the proliferation of P. xyllostella embryonic cells, and the effect was dose-dependent. IC50 was 4.4 μg·mL-1 in 24 h. Dead cells could be observed in azadirachtin treated group after stained by PI, and apoptotic body could be found in the same group after stained by DAPI. Caspase-3 protein was cleaved and Akt phosphorylation was inhibited.
ConclusionAzadirachtin has significant inhibitory effect on the proliferation of P. xyllostella embryonic cells. By inhibiting the activation of Akt signaling pathway, azadirachtin can induce cells to produce Caspase-3-dependent type I apoptosis.
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Keywords:
- azadirachtin /
- Plutella xyllostella /
- embryonic cell /
- apoptosis /
- proliferation
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图 1 印楝素处理小菜蛾胚胎细胞24 h的细胞抑制率
Figure 1. The inhibitory rate of azadirachtin on Plutella xyllostella embryonic cells in 24 h after treatment
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图 2 印楝素处理小菜蛾胚胎细胞后的PI染色镜检结果
A:未经处理的正常细胞(CK)，B、C、D分别为:1.25、2.5、5 μg·mL-1印楝素处理24 h；箭头所指为死亡细胞。
Figure 2. Microscopic examination of Plutella xyllostella embryonic cells stained with PI after treatment of azadirachtin
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图 3 不同浓度印楝素处理PI染色的细胞数与细胞总数百分比(PI)
Figure 3. The percentage of PI staining cells in total cells after treated with different concentrations of azadirachtin
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图 4 印楝素诱导小菜蛾胚胎细胞的凋亡小体
A:未经处理的正常细胞(CK)，B、C、D、E、F分别为: 0.625、1.25、2.5、5、10 μg·mL-1印楝素处理24 h；箭头表示细胞中被DAPI染色的凋亡小体。
Figure 4. Apototic bodies of Plutella xyllostella embryonic cells induced by azadirachtin
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图 5 凋亡小体发生率(统计样本中出现凋亡小体的点数与细胞总数之比，DAPI)
Figure 5. Incidence rate of apoptotic bodies (percentage of apoptotic bodies in total cells, DAPI)
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图 6 Western-blotting检测印楝素诱导小菜蛾胚胎细胞中裂解Caspase-3表达
CK:未经处理的正常细胞，A、B、C、D分别为:1.25、2.5、5、10 μg·mL-1印楝素处理24 h。
Figure 6. Western-blotting results of cleaved Caspase-3 levels in Plutella xyllostella embryonic cells induced by azadirachtin
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