The JNK And PI3K-AKT Signaling Pathways Are Involved In Apoptosis Induced By Anagrapha Falcifera Multiple Nuclear Polyhedrosis Virus In Spodoptera Litura SI-1

Apoptosis is programed death process controlled by intracellular gene, which are taken place in multicellular organisms under physiological or pathological condition. apoptotic studies having been carried out for a long time.as the signal transduction pathways had been played a increasingly important role in the cell cycle, proliferation, differentiation and apoptosis as well as a variety of life activities, people show a growing emphasis on the relationship between apoptosis and cell signal transduction pathways. JNK signaling pathway is an important branch of the MAPK pathway, it is also called stress-activated protein kinase, which plays an important role in many physiological and pathological processes of cell cycle, apoptosis and cell stress, etc; PI3K signaling pathway was also knowing by almost people because of its participation in a variety of cellular activities, especially in the regulation of tumor cell proliferation and survival. AKT is a major downstream effector of PI3K which is also known as protein kinase B (PKB), activated AKT could regulate cell function through phosphorylation of varieties of enzymes, kinases or transcription factors. All these studies on the relationship between cellular signal transduction pathways and apoptosis were expanded in mammalian cells. Our previous studies have demonstrated that mitochondria-mediated apoptosis signaling pathway was involved in AfMNPV induced apoptosis SL-1cells, cytochrome C release from intermembrane into the cytoplasm and induce of a series of downstream apoptotic cascade reaction. So we continue to explore if there is a relationship between JNK and PI3K-AKT signaling pathway and AfMNPV induced apoptosis process in SL-1cells.SP600125and Wortmannin are the specific inhibitors in JNK and PI3K-AKT signall ing pathway. The tolerance test in SL-1which was a cell line of Spodoptera litura sugges t-ed it was basic non-toxic to the cell growth when the concentration of inhibitors was lo wer than50μM. To better understand the cell kinetics we counted the SL-1cells and dre w the growth curve, the results revealed that the curve was a simple "S" in normal culture conditions and the growth trend didn’t change when added the optimal concentration of these two inhibitors into the culture medium separately. Then we used DAPI staining th e cells and observed under fluorescence microscope, found that apoptotic bodies in AfM NPV-induced SL-1cells were significantly reduced after treatment with SP600125or Wo rtmannin which was consistent with the measurement of the flow cytometry. Based on th e fact that the apoptosis rate in experimental group was obviously lower than control gro upwe preliminary identified the PI3K-AKT signalling pathway participated in the cell de ath process induced by AfMNPV in SL-1.Next, I measured caspase-3activity and apoptosis-related proteins by Western Blot, trying to explore whether JNK and PI3K-AKT signal pathway were involved in AfMNPV induced apoptosis in SL-1, if so, how did signaling pathway protein changed in apoptosis process.In the result, caspase-3was activated and cut in to17KD fragment started downstream apoptotic events. But when added in JNK and PI3K signaling pathway inhibitors respectively,the apoptosis degree was significantly declined,and the apoptosis effect were not very different if we put these inhibitors together.Immediately, we examined changes upstream apoptotic events of cytochrome C, consistently with our predicte results,the cytosolic cytochrome C decreased significantly After added in two signaling pathway inhibitors separately in apoptotic SL-1, All of the data shows that JNK and PI3K signaling pathway were activated in the initial stage of AfMNPV induced apoptosis in SL-1and involved in the whole process of apoptosis.Jnkl, jnk2and jnk3were JNK’s encoding gene and its coded product JNK1and JNK2were widely exists in various tissues,When JNK pathway was activated, the corresponding protein JNK1and JNK2would be phosphorylated and thus would be functioned. Therefore I detect expression of JNK1/2and PI3K pathway’s key effector AKT with Western blot hybridization, as the result said that JNK and PI3K-AKT signaling pathway were activated in AfMNPV induced apoptosis in SL-1cells by activating of JNK1/2and AKT,and cell cycle of SL-1was also be impacted, on the other hand, Either cleaved caspase-3or activated JNK1/2and AKT were extended in phosphorylation with apoptosis prolonged. Therefore, we conclude that JNK and PI3K-AKT signaling pathway were involved in AfMNPV induced apoptosis in SL-1, coordinated adjust to apoptotic response and affect the cell cycle.