Molecular Mechanism Of Mitophagy In Hep G2 Cells Induced By Recombinant Buckwheat Trypsin Inhibitor

Mitochondria are morphologically dynamic organelles that shift between tubular reticular networks and fragmented structures through mitochondrial fusion and fission.Changes in mitochondrial dynamics regulate the morphology and function.Mitophagy,a kind of selective autophagy,is a process that the damaged mitochondria are packaged into autophagosomes and then degraded by fusion with lysosomes,thus maintaining stability of the cell environment.Studies have found that abnormal mitochondrial autophagy is closely related to many diseases such as neurodegenerative disease,cardiovascular disease,and cancer.Our previous studies have demonstrated that recombination buckwheat trypsin inhibitor(rBTI),a member of the potato type I proteinase inhibitor family and derived from tartary buckwheat extracts,could suppress tumor cells proliferation by inducing cells autophagy.But the mechanism of rBTI-induced autophagy is unclear.In this study,the autophagy level of Hep G2 cell,which treated with rBTI,was observed with transmission electron microscopy,MDC staining,Western Blot,the plasmid pEGFP-LC3 transfection and the confocal laser scanning microscope.Western Blot was used to examine the mitochondrial protein and marker proteins involved in autophagy.The morphology of mitochondria was observed by the plasmid pDsRed2-mito transfection and the confocal laser scanning microscope.The effect of rBTI on mitochondrial fusion and fission was detected by Western Blot and qRT-PCR.The colocalization of mitochondrion and autophagy marker proteins LC3 was analyzed with the plasmid pEGFP-LC3 and pDsRed2-mito cotransfection.We detected the mitochondrial membrane potential of cells with confocal laser scanning microscope and flow cytometer,detected the level of ROS with flow cytometer,and detected the activity of SOD and CAT,and the content of GSH with eliasa.The relationship between ROS and the autophagy rBTI-induced was analyzed with flow cytometer,the plasmid pEGFP-LC3 transfection and Western Blot.Our results indicated that the rBTI induced autophagy in Hep G2 cell.This was supported by the increase of the autophagolysosome structures packaged with mitochondria,the level of autophagy,GFP-LC3 punctate structures,and the level of the marker proteins involved in autophagy.At the same time,we found rBTI induced mitochondrial fragmentation.After treated with rBTI,the Red-Mito punctate structures increased,the level of mitochondrial protein Cox IV and Tom20 reduced significantly,and the mitochondia fission protein Fisl increased while the mitochondia fusion protein Mfnl decreased in Hep G2 cell.It was also noted that rBTI highly increased colocalization of mitochondria and autophagy marker proteins LC3 in treated cells compared to non-treated cells.These appearances indicated the type of autophagy rBTI-induced is mitophagy.We also found that rBTI caused mitochondrial dysfunction in Hep G2 cell and this may be the immediate cause of rBTI-induced mitophagy.This occurs through enhancing depolarization of the mitochondrial membrane potential;decreasing the ATP level;increasing reactive oxygen species(ROS).At the same time,rBTI also resulted in the rise of the superoxide dismutase and catalase activity and glutathione peroxidase(GSH)content,and changes in the GSH/oxidized glutathione ratio.These results indicated that rBTI increased the antioxidant capacity of Hep G2 cell,and this may be caused by the increase of ROS level rBTI-induced.Furthermore,ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 cell mitophagy to remove dysfunctional mitochondria.The work provides an important basis and experimental evidence for further research on mitophagy mechanism induced by rBTI in tumor cell.