Effects Of NIX On The Mitophagy Mediated By CCCP

ObjectiveRecent studies have discovered a new protein which belongs to Bcl-2 homology domain 3-only proapoptotic protein, was named NIX, it was also called Bcl-2/E1B(BLIP3L).it’s encoding product has TM domain and BH3 domain of Bcl-2.NIX domain located in the outer membrane of mitochondria, through the BH3 domain interact with the proteins of E1B19 K, Bcl-X1, Bcl-2 caused the release of cytochrome c, lead apoptosis. A large number of studies have confirmed that NIX protein played an important role in promoting cell survival, and more and more evidence showed that the occurrence of many human diseases are closely related with NIX promote cell death and survival. In this paper we mainly discusses the effects of carbonyl cyanide m-chlorophenylhydrazone(CCCP) on mitophagy; the influence of CCCP on the expression of NIX in PC12cells; and the regulation of NIX on mitophagy which treated with CCCP.this study will contribute to understand the mechanisms of NIX on mitophagy mediated by CCCP,and provide the perfect solution on prevention and treatment of human disease.MethodsTo construct the lentivirus models which the gene of NIX overexpression and NIX sh RNA.We build the control cell lines, NIX overexpression cell line, NIX sh RNA cell line model through Virus infection PC12 cells, three kinds of cells are treated with CCCP at(0, 10, 20, 30)μmol/l and for different time(0, 2, 4, 6)h, then comparing the content of reactive oxygen species( ROS) in the cells,and to discuss the effects of NIX on mitophagy which mediated by CCCP.Results1. The lentivirus models which the gene of NIX overexpression and NIX sh RNA were constructed successfully. They could be used in the experiment.2. The control cell lines(NC), NIX overexpression cell lines(NIX) and NIX sh RNA interference cell lines(NIX sh RNA)were constructed successfully. Compared with the control cells, the protein expression of NIX in NIX overexpression cells increased significantly. The difference was statistically significant(P<0.05). Comparedwith the control cells, the protein expression of NIX in NIX shRNA interference cells decreased significantly. The difference was statistically significant(P<0.05)3. Compared with the blank control, the ROS increased significantly in the control cells, NIX overexpression cells and NIX sh RNA interference cells after CCCP’s dispose.The difference was statistically significant(P<0.05).4. Compared with the control cells, the content of reactive oxygen species increased in NIX overexpression cells. The difference was statistically significant(P<0.05).5. Compared with the control cells, the content of reactive oxygen species decreased in NIX sh RNA interference cells. The difference was statistically significant(P<0.05).6. Compared with the blank control,the protein expression of NIX all decreased significantly in the control cells and NIX overexpression cells after CCCP’s dispose,The difference was statistically significant(P<0.05).7. Compared with the blank control, after CCCP’s dispose, the autophagy increased obviously in NC. The mitophagy decreased significantly. The difference was statistically significant(P<0.05). Compared with the blank control, the autophagy and mitophagy in NIX overexpression cell increased obviously. The difference was statistically significant(P<0.05).8. Compared with the control cells, when the concentration of CCCP was 30μmol/l,the autophagy and mitophagy in NIX overexpression cells increased obviously. The difference was statistically significant(P<0.05)..9. Compared with the blank control, after CCCP’s dispose, the autophagy in NIX sh RNA interference cells increased obviously. The mitophagy decreased significantly.The difference was statistically significant(P<0.05)10. Compared with the control cells, after CCCP’s dispose, the autophagy and mitophagy in NIX sh RNA interference cell had a significant decrease. The difference was statistically significant(P<0.05).Conclusions1. CCCP could promote the production of ROS.2. The production of ROS is decrease in NIX overexpresstion or NIX sh RNA interference cells.3. The degradation of NIX is decrease in PC12 cell after treated with CCCP.4. NIX can promote the mitophagy mediate by CCCP.