Effects Of Hypoxia Stress On Mitochondrial Autophagy And Energy Metabolism In Rgcs Of Goldfish (Carassius Auratus)

Freshwater bodies often endanger the survival of fish because of uneven and insufficient dissolved oxygen.Mitochondria are the main consumers of oxygen in cells.Regulating the function and stability of mitochondria is one of the important strategies for fish to cope with hypoxia.Radial glial cells exist in the brain of adult goldfish,and their morphology and function are different from other glial cells.As the main source of cell energy,mitochondria is one of the main indicators to judge the state of cells.Under the condition of hypoxia,the permeability of cell membrane increases,and the entry of macromolecules into the mitochondrial matrix increases the swelling area of mitochondria,which leads to the release of apoptotic factors such as Cytc,resulting in apoptosis.In this study,goldfish radial glial cells were taken as the research object to explore the changes of mitochondrial function,dynamic balance and energy metabolism of radial glial cells exposed to different oxygen concentrations.1.Effects of different oxygen concentrations on the viability of RGC cells:the results of cell viability test showed that the cell viability of normoxic group and 15%O₂group increased with the decrease of oxygen concentration within 48 hours;the cell viability of 10%O₂group was the highest at 24 hours and then decreased at 48 hours;and the cell viability of3%O₂group decreased with the decrease of oxygen concentration.Therefore,the 24h time point was selected as the hypoxia cultivation time in the follow-up experiment.The results showed that the proliferation rate of RGC cells under moderate hypoxia was higher than that of normoxic culture at a certain time.2.Mitochondrial ROS production and effect on membrane potential of RGC cells under different oxygen concentrations:the results of ROS production showed that there was no significant change in ROS production in 15%O₂group compared with normoxic group at 24h,while ROS production in 10%O₂group increased significantly,while ROS production decreased significantly in 3%O₂group.The results of mitochondrial membrane potential showed that the mitochondrial membrane potential increased with the decrease of oxygen concentration,and there was no significant difference between 10%O₂group and normoxic group,but significantly decreased in 3%O₂group.The results showed that RGC cells did not reduce the production of ROS because of cell proliferation,and the continuous decrease of membrane potential indicated the instability of cell state.3.Effects of different oxygen concentrations on mitochondrial autophagy in RGC cells:the results of mitochondrial lysosome co-localization showed that the fluorescence intensity of mitochondria and nucleus was the most in 10%O₂group and significantly decreased in 3%O₂group,and the lysosome fluorescence increased with the increase of oxygen concentration and reached the highest in 3%O₂.The results of fluorescence quantitative analysis showed that the expressions of major genes in pink1,parkin,bnip3,nix,fundc1 and lc3 were significantly higher than those in normoxia.The results showed that mitochondrial autophagy was activated under hypoxia.4.Effects of different oxygen concentrations on the oxidative phosphorylation function of RGC mitochondria:the results of cell ATP content showed that ATP content decreased with the decrease of oxygen concentration,and there was no significant relationship between ATP content and cell proliferation,and the results of cell citrate synthase（CS）activity showed that the cell increased but had no significant difference in10%O₂group,and then decreased significantly in 3%O₂group.The results of lactate dehydrogenase（LDH）activity showed that LDH activity decreased with the decrease of oxygen concentration,and there was a significant difference between 10%O₂group and 3%O₂group.The results showed that RGC cells mainly carried out aerobic respiration under normoxic condition,aerobic respiration and anaerobic respiration under10%O₂,and anaerobic respiration under 3%O₂.Fluorescence quantitative analysis showed that the expression of mitochondrial electron transport chain related genes nd4,coⅠ,atp6 and related nuclear genes ndufa1,uqcr1,surf1 and atp5b decreased significantly.To sum up,hypoxia stress increased ROS production and decreased mitochondrial membrane potential in RGC cells,and the cell homeostasis was broken.Moderate hypoxia activates mitochondrial autophagy,which accelerates the growth of cells at a certain time point,which is characterized by an increase in the activity of mitochondria and nuclei.With the decrease of oxygen concentration,the main respiratory mode of RGC cells is aerobic respiration-aerobic respiration/anaerobic respiration-anaerobic respiration.The respiratory mode of RGC cells directly affects the production of ATP and reduces the main source of cell energy.In this process,the production and accumulation of ROS harms the function of mitochondria,which in turn affects the process of RGC cells participating in neuroendocrine.