The Mitochondrial Mechanism In The Autophagy Model Induced By XOD In Human Hepatic Cell Line L-02

?Background?Autophagy is a kind of highly conservative self digestion process, and it can help digest and degrade organelles or macromolecule proteins into nutritional ingredients, which can be reused, achieving the goal of clearing damaged organelles and effectively making use of self nutritional ingredients. Autophagy is a common biological phenomenon, playing a vital role in many kinds of physical and pathological conditions. Under normal conditions, autophagy is at a relatively low level, but it can be stimulated significantly under various stress conditions. Oxidative stress occurs when ROS are over produced and can not be scavenged. Although the relevance between ROS and autophgy has been reported by many literatures, the detail mechanism still remains unknown. Moreover, in previous studies, autophagy is often accompanied with apoptosis or necrosis, which is a pathological and damaged autophagy. Little is known about physiological autophagy induced by oxidative stress.Xanthine oxidaese(XOD) is a kind of oxidizing enzyme, which can catalyze and decompose xanthine into uric acid with ROS production. In the present study, we found XOD could effectively induce autophagy in L-02 cells. However, what kind of advantages it has, and what role of ROS in the process remain unknown. Systematic study on the above questions will provide data to help understanding the role and molecular mechanism of oxidative stress in autophagy. ?Aims?1.To establish a new kind of cell autophagy model induced by XOD;2.To study mitochondrial oxidative stress and molecular mechanism of autophagy induced by XOD. ?Methods?1. L-02 cells were cultured and treated with several kinds of oxidants including H2O2?GOX?t-BHP?PQ and XOD, autophagy inhibitor(3-MA), antioxidants(NAC and MitoQ);2. LC3?( the marker protein of autophagy and its distribution in cells) was detected through IF, and autophagysomes were detected through MDC;3. Protein level of autophagy related protein such as LC3?, p62, and the activation of signal pathways including ERK?AMPK and p38 were detected through Western Blot;4. Morphology and quantity of autophagosomes and mitophagy were observed with TEM;5. DCFH-DA and Mito-SOX Red were used to detect total ROS and mitochondrial ROS respectively;6. After dyeing with JC-1, mitochondrial membrane potential was observed through laser scanning confocal microscopy;7. SOD2 and CAT assay kits were used to detect the enzymes activity of cells after transfected with virus;8. Mitochondrial DNA copy number was detected through fluorescence quantitive PCR.9. mRFP-GFP-LC3 adenovirus was used to transfect cells for observing the change of autophagy flux;10. Atg5 siRNA was used to inhibit the protein expression of Atg5;11. L-02 were transfected SOD2, CAT and M-CAT lentivirus respaectively, or co-transfected with SOD2&CAT, SOD2&M-CAT to overexpress antioxidant proteins.12. The apoptosis and necrosis were deteced by Annexin V-FITC/PI kit with flow cytometery. ?Results?1. Cell viability decreased as the concentrations of oxidants increased, LC3?protein level has been increased obviously at a certain dose of oxidants, such as, H2O2, t-BHP, PQ and XOD.2. IF, Western Blot and MDC results showed that high concentration of XOD(?10mU/ml) obviously increased the accumulation of LC3 fluorescence puncta and blue fluorescence puncta of autolysosome, with LC3?protein level increased. TEM results showed that there were more autophagysomes in cells after treated with XOD than the untreated ones.After trasfected with mRFP-GFP-LC3 adenovirus, the results showed that autophagy flux had been increased by XOD.3. After treated with XOD, rapamycin and EBSS, autophagic indicators, including MDC, IF-LC3, LC3?protein level and autophagosome number observation through TEM, all showed that XOD could induce autophagy more effectively than the other two normal inducers.4. Although t-BHP, PQ and XOD could induce autophagy obviously at certain dosage, t-BHP and PQ could induce apoptosis at the same time.5. After treated with XOD, intracellular and mitochondrial ROS level both increased obviously, and after co-treated with antioxidants, LC3? protein level decreased. Besides, ROS level decreased though over expressing antioxidant genes, and LC3?protein level was reversed into a relative low level accordingly.6. Phosphorylation level of proteins related with ROS signaling pathways, including AKT, p38/MAPK, ERK and AMPK were increased, and after overexpressing antioxidant proteins, AKT and AMPK phosphorylation levels were both effectively antagonized.7. ROS level and cell death rate were both enforced after autophagy were blocked.8. Mitophagy was observed after treated with XOD and the number of mitochondria was evidently decreased. ?Conclusions?We successfully established an autophagy model induced by XOD in L-02 cells. XOD could induce autophagy more effectively without apoptosis or necrosis. Mitochondrial ROS play a key role in this process. By activation of AMPK and AKT pathway, mitochondrial ROS stimulate autopahgy, including mitophagy. The ROS-induced mitophagy results to decreased mitochondria number leading to decreased ROS level. This feed back ROS regulation benefit for the cell survival...