| 【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. |
