Juglone induces apoptosis in Sf9 cells through reactive oxygen species-mediated caspase-dependent mitochondrial pathway

Juglone is a naphthoquinone compound that has been shown to have insecticidal activity, but its mechanism of action remains to be fully elucidated. The results of this study indicate that juglone inhibits Sf9 cell proliferation in a time- and concentration-dependent manner. Typical apoptotic features, such as cell membrane blebs and apoptotic bodies were observed by microscopy and DAPI staining. Additionally, a series of DNA fragments appearing as a ladder-like pattern of bands at intervals of about 200 bp, a hallmark of apoptosis, were detected by the DNA fragmentation assay. Flow cytometry analysis revealed that juglone induced G2/M phase arrest of Sf9 cells, as indicated by significant increase in the proportion of G2/M phase cells from 28.8 to 55.74%, and the proportion of apoptotic cells from 5.05 to 33.12% compared to control cells, implying that juglone can induce apoptosis in Sf9 cells. In addition, intracellular biochemical assays confirmed that in Sf9 cells juglone also significantly induced the loss of mitochondrial membrane potential, the production of reactive oxygen species (ROS) and the mRNA expression levels of key apoptotic genes (Apaf-1, Caspase-3, Caspase-9, Cytochrome C) in the mitochondrial apoptosis pathway. Thus, it was confirmed that, in Sf9 cells, juglone induces cell apoptosis by activating the caspase-dependent mitochondrial apoptosis pathway through excessive production of ROS. The findings of this study provide a new perspective for studying the toxicology and mode of action of juglone at the cellular level.