| Cytochrome c is a class of water-soluble protein,which is synthesized by nuclear genes and attached to the mitochondrial inner membrane surface. As a carrier,cytochrome c plays an important role in the process of electron transfer of the respiratory chain. Once cytochrome c dysfunctional or cytochrome c deletion, the function of respiratory chain will be abnormal, thereby cause a lack of ATP and eventually cell death occur. In addition, cytochrome releases from mitochondria to the cytosol which is the most important events of mitochondria-mediated apoptotic signal that caspase pathway induces apoptosis. Although studies in mammalian cells have confirmed the release of cytochrome c from mitochondria to cytoplasm, thereby affecting apoptosis, but apoptosis in insect cells the same way whether or not there are not fully elucidated. In the study of apoptosis of the insect,role of cytochrome c has some different results. The studies in Drosophila suggest that cytochrome c is not released from mitochondria during apoptosis in that insect, and that cytochrone c plays no role in caspase activation in these cells. In contrast with reports in Drosophila, the release of cytochrome c from mitochondria and consequently activation of caspase-3 were demonstrated during lepidopteran cell apoptosis. The release of cytochrome c from mitochondria in ultraviolet-induced Spedoptera frugiperda Sf-9 cells was observed by western blotting So far, there is universal view that cyt-c can not releases from mitochondria to the cytosol, and caspase activation and apoptosis did not depend on cytochrome c.We had early found that cytochrome c was released from mitochondria to the cytoplasm and can activated apoptosis-related proteins in undergoing apoptotic Spodoptera litura SL-1 cells induced by baculovirus. However, these studies were mainly based on the western blotting.In order to provide further an evidence on the role of cytochrome c on apoptosis induced with baculovirus AfMNPV in lepidopteran cells we used the technique of RNA interference to investigate an effect of interfering cytochrome c. Preliminary studies have demonstrated that scliencing the expression of cytochrome c can inhabit apoptosis induced by baculovirus AfMNPV SL-1 and the mRNA level of cytochrome c in the cells treated with dsRNA decreased and caspase-3 activity decreased. On the basis of previous research, here we continue to use RNAi to deeply research role of cytochrome c in apoptosis of lepidopteran insects.DsRNAs corresponding to the coding regions of cytochrome c were transcribed in Vitro and the transcription productions were identified by electrophoresis. SL-1 cells treated with dsRNA at various lengths of time are infected by AfMNPV for 10h and then visualized by with DAPI staining. Under fluorescence microscope, the number of apoptotic cells induced with AfMNPV and apoptotic bodies decreased significantly and declined with dsRNA treatment time course.DNAs were extracted from cells transfected dsRNA at differenttime periods (24h,48 h and 72h) and analyzed on gel electrophoresis. Level of DNA fragmentation of cells which are transfected with cytochrome c dsRNA is lower than that cells treated without dsRNA.To detect quantitatively apoptotic cells, after cells treated with dsRNA for 48h and infected with AfMNPV cells for 10 h were stained with Annexin V-FITC and PI. Rate of apoptosis in cells treated with different treatments was detected by the flow cytometry FCM analysis showed that the apoptotic ratio in cells infected with alone AFMNPV was 63.2%, in cells treated with eGFP dsRNA plus AfMNPV was 51.88% and in cells the treated with cyto-dsRNA plus virus was 67.49%.The caspase-9 activity assay performed on the cells incubated in the presence of dsRNA and viruses revealed a significantly decrease in caspase-9 activity with respect to the control cells treated with eGFP dsRNA and without cytochrome c dsRNA and other treatments.This is the first report about effect of silencing cytochrome c expression on the activity of caspase-9 in lepidopteran insect, indicating cytochrome c is associated with activity of caspase-9.These results strongly demonstrate that Cyt-c is essential for AfMNPV-induced apoptosis in Spodoptera litura cells and caspase-9 activation is dependent on cytochrome c and cytochrome c play an important role for apoptotic signaling and may be required for Lepidopteran insect apoptosis. We speculate that the role of cytochrome c in Lepidopteran cells and mammalian cells apoptosis is homologous. |
