Research Of The Endogenous Mitochondrial Pathway In ROS-induced Cell Apoptosis In Silkworm, Bombyx Mori

Cell apoptosis is controlled by genes and can death autonomic orderly with a series of morphological and biochemical characteristics,which plays a critical role in diverse biological processes,including cell development,differentiation,proliferation and stress responses.Oxidative stress plays a key role in the regulate and control of cell survival and reactive oxygen species(ROS) contribute to apoptosis by mediating highly oxidative stress.The domesticated silkworm,Bombyx mori(B.mori),is increasing being employed as an insect model in Lepidopteran for research,however,study on the B.mori cell apoptosis induced by oxidative stress is still at primary stage and underlying apoptotic signaling pathway is poorly understood.This study is based on the reported apoptosis-related genes in B.mori and compared to the sf9cells which act as typical representative in Lepidopteran and have strong antioxidant capacity,it determined the mitochondrial apoptosis pathway in B.mori cells was induced by ROS-mediated highly oxidative stress, this will lay a foundation for further analysis of B.mori cell apoptosis mechanism.The main research results are as follows:1.The establishment of apoptotic model of ROS-induced in BmN-SWU1B.mori cellsBmN-SWU1and sf9cells were treated with H2O2at different concentrations (0.1μM,1μM,5μM,10μM) for0-24h,0-12h, respectively.Using the methods of MTT analysis,DAPI staining,flow cytometry analysis and TUNEL staining,we detected cell vitality,morphology,apoptosis rate and DNA breakage,respectively.The results indicate that cell fatality rate were close to50%and DNA fragmentation occurred in BmN-SWU1and Sf9cells with treatment of1μM H2O2for24h and12h,respectively. Cells exhibited a series of apoptotic characteristics in a time-dependent manner, characterized by cell protuberances,cytoplasmic condensation and apoptotic bodies.In conclusion, the median lethal dose was1μM H2O2at24h,12h and were considered the optional treatment conditions in BmN-SWU1and sf9cells,respectively.2.The research of the endogenous mitochondrial pathway in ROS-induced BmN-SWU1cell apoptosis in B.moriWith the treatment of1μM H2O2for different times in the BmN-SWU1and sf9cells,the mitochondrial distribution changed from relatively homogeneous into aggregated,diffused with increasing time,which indicates that H2O2can affect the mitochondrial distribution.Moreover,H2O2induced a loss in mitochondrial transmembrane potential in a time-dependent manner,there was no difference between H2O2treated group at8h and the control group at Oh which were detected by flow cytometry analysis.Meanwhile,the levels of intracellular ROS and Ca2+increased compared to the control group.The results of qPCR and western-blot analysis indicated that there were evident increases in the expression of BmDronc, Bmapaf-1, BmICE, BmEndoG, BmP53, BmDredd, P53and concurrent decreases in the BmBuffy, BmIAP,Bcl-2expression in the BmN-SWU1cells with the treatment of H2O2.Cytochrome c protein expressed in excess and released from mitochondria into the cytoplasm after exposured on H2O2.Our results also showed that Caspase-9and Caspase-3/7activities increased significantly in a time-dependent manner in the BmN-SWU1and sf9cells following treatment with H2O2.In summary,this study has revealed that apoptosis in BmN-SWU1B.mori cells can be induced by H2O2-mediated oxidative stress through a mitochondrial-dependent pathway.