FOXO基因对印楝素诱导sf9细胞凋亡的影响

Effect of FOXO Gene on the Apoptosis of sf9 Cells Induced by Azadirachtin

【目的】探讨印楝素(azadirachtin,AZA)通过转录因子FOXO诱导草地贪夜蛾卵巢细胞sf9凋亡的分子机制。【方法】将体外培养的草地贪夜蛾卵巢细胞分为AZA处理组和未处理组,采用CCK-8法检测AZA对sf9细胞增殖的影响;透射电镜观察sf9细胞的超微结构变化;流式细胞术分选Annexin V-FITC/PI染色的凋亡细胞;Western blot检测p-FOXO及凋亡相关蛋白的表达情况;RNAi FOXO后,检测FOXO基因表达量与Caspase-3的酶活性变化。【结果】CCK-8检测发现AZA可显著抑制sf9细胞增殖,并呈时间和浓度依赖性;通过透射电镜检测到5 mmol/L AZA可破坏sf9细胞的微观结构,导致细胞核收缩,染色质聚集,细胞质空泡状;流式细胞术分选Annexin V-FITC/PI染色的凋亡细胞,发现AZA诱导sf9细胞凋亡与时间呈正相关;进一步通过Western blot检测凋亡相关蛋白,发现AZA可诱导凋亡相关蛋白Bim和Cleaved Caspase-3的表达水平显著增加,而P-FOXO蛋白表达量降低,FOXO总蛋白表达无明显差异;利用qRT-PCR进一步检测发现,FOXO基因表达量与AZA处理时间呈正相关,在此基础上通过RNAi技术沉默FOXO后,经AZA处理发现Caspase-3的酶活性显著降低。【结论】AZA可通过上调FOXO基因抑制草地贪夜蛾卵巢细胞sf9的增殖并诱导其凋亡。

【Objective】The study aimed to investigate the molecular mechanisms of azadirachtin(AZA)inducing the apoptosis of sf9 cells of Spodoptera frugiperda through FOXO transcription factor.【Method】The ovarian cells of culture in vitro S.frugiperda were divided into AZA group and control group.Cell proliferation and microcosmic changes of sf9 cells were analyzed using CCK-8 assay and transmission electron microscopy,respectively.Apoptotic cells stained by Annexin V-FITC/PI were separated with flow cytometry,and the expressions of p-FOXO and apoptosisrelated proteins were determined by Western blot.The expression of FOXO gene and the activity of Caspase-3 were detected by qRT-PCR and RNAi,respectively.【Result】The CCK-8 detection results showed that AZA inhibited the proliferation of sf9 cells significantly with a time and concentration dependent manner.Transmission electron microscopy revealed that 5 mmol/L AZA could obviously trigger changes in the microstructure of sf9 cells such as contraction of the nucleus,chromatin condensation and the development of cytoplasmic vacuoles.The results of flow cytometry revealed that AZA induced apoptosis of sf9 cells which was positively correlated with time.The results of Western blot analysis showed that AZA could significantly increase the expression of apoptosis-related proteins Bim and cleaved Caspase-3.Compared to the control group,the expression of P-FOXO protein was significantly decreased.However,the expression of the total FOXO protein remained apparently unaltered.The qRT-PCR expression analysis showed that the mRNA expression of FOXO transcription factor increased upon prolonged treatment with AZA.Silencing of FOXO in sf9 cells by RNAi interference caused a significant decline in the activity of Caspase-3 protein.【Conclusion】Collectively,the results of the present study show that AZA can inhibit the proliferation of sf9 cells and promote the apoptosis by blocking the phosphorylation of FOXO gene.