Effects of Rotenone Exposure on Apoptosis in rAAV-NDI1-infected Neural Stem Cell Line of Minipig

Background: The overall objective of this study is to confirm that normal expression of yeast NADH- dehydrogenase (NDI1) can occur in neural stem cell lines of minipig with resistance to rotenone exposure, an environmental factor responsible for dysfunction of mitochondrial enzyme complex I. In modern society, there are many diseases that cannot be treated. Diseases including LHON, Parkinson disease and dystonia, have been associated with defects in mitochondrial complexes. This experiment was performed to demonstrate that it was not sensitive. The overall objective of this study is to confirm that normal expression of yeast NADH-dehydrogenase (NDI1) can occur in neural stem cell lines of minipig with resistance to rotenone exposure, an environmental factor responsible for dysfunction of mitochondrial enzyme complex I. Methods: A Mini Pig Neural stem cell line (MPV) was used for transfection of the NDI gene, DMEM/F-12 culture medium and MPV was inoculated in a six-well plate (Corning, USA) at a concentration of 1´105 cells/3ml/well, with inoculation of MPV at 37°C for 24 hours, 5% CO2, followed by incubation in an incubator with 95% humidity and attached. The plate containing MPV cells was treated with recombinant adeno-associated virus ndi1 (rAAV-ndi1) for transfection, with periodic replacement with a cell selection culture solution containing 10% FBS, 1% P/S and 0.2 mM rotenone. The experiment was performed for restoration of mitochondrial activity of thawed cells. RNA was extracted from MPV cells and transfection of MPV cells with the NDI1 gene was performed using Trizol (Invitrogen, USA). Reverse transcription PCR (RT-PCR) and Western blot were performed to confirm normal expression of the NDI1 gene in MPV cells transfected with the NDI1 gene. Immunofluorescence was performed to determine the presence of the NDI1 protein in the cell and the cell count was used for LUNA and the rate of cell death for rotenone was determined using the MTS [3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. Result: rAAV-NDI1 was successfully introduced into MPV cells and the proliferation rates for the cells were compared with those of the transformed cells; after three days, the non-infected cells were killed and the infected cells proliferated. The results after differentiating the cell lines were similar to those of previously reported studies. Toxicity analysis on rotenone was also performed using the MTS assay and the rates of cell death over three days were compared; the results showed significantly lower levels of NDI1-transformed minipig neural stem cells compared with those of minipig. The present work will be a complementary contribution to the comprehensive study of the scorpion sting syndrome. These results were similar to those of previously reported studies. Previous studies have reported that oxidoreductive stress can be a cause of apoptosis. Therefore, conduct of additional studies for measurement of ROS and oxygen utilization to determine oxidative stress in cells will be necessary.