The Effects Of Hypoxia On Cell Apoptosis And The Activity Of Antioxidant Enzymes In Megalobrama Amblycephala

Megalobrama amblycephala, also called Wuchang fish, is a kind of improved economic freshwater fish. In recent years, with the deterioration of natural environment and human activity intervention, its living environment often appeared low oxygen, restricting the development of M. amblycephala breeding. In our study, the structure changes of different tissues of M. amblycephala were observed after hypoxia treatment, and the activities of antioxidant enzymes were analyzed in the liver in hypoxia and hypoxia-recovery conditions. The influence of low oxygen on myocardial cell apoptosis and bcl-2 expression were also studied by TUNEL and fluorescence quantitative PCR. Additionally, the changes of MDA content and SOD activity were analyzed in heart mitochondria extracted using sucrose gradient centrifugation method. The main results were as follows: 1. Effects of hypoxia on structure of different tissues of M. amblycephalaDifferent tissues(heart, liver, kidney, muscle, spleen) were sampled and observed based on HE staining after severe hypoxic treatment(DO:0.5 mg/L), and the results revealed that greater influence of hypoxia on the heart and kidney, which muscle fibers in heart showed swelling and disorder in arrangement in severe hypoxia conditions, broken capillaries were observed in spleen. However, hypoxia effects on structure of muscle, liver spleen were relatively lesser.The results showed based on electron microscope that under moderate hypoxia(DO:2.5±0.5 mg/L for 2 h,4 h), myocardial mitochondrial structure were broken, mitochondria was swelling and disorder, but in server hypoxia(DO:0.5 mg/L), some mitochondria were fractured. 2. Cardiac cell apoptosis caused by hypoxiaIn order to further study hypoxia effects on heart tissue of M. amblycephala, cardiac cell apoptosis and bcl-2 m RNA levels were analyzed by TUNEL(terminal dexynucleotidyl transferase Td T mediated d UTP Nick end labeling) and fluorescence quantitative PCR after hypoxia treatment. The results showed that hypoxia induced cardiac cell apoptosis, and with the increase of hypoxia time and the decrease of hypoxia concentration, apoptosis index gradually increased. In addition, the bcl-2 m RNA levels in heart gradually decreased with the increase of hypoxia treatment in the moderate hypoxia group(DO:2.5±0.5 mg/L), but significantly increased in the severe hypoxia group(DO:0.5 mg/L). 3. The effect of hypoxia on SOD and MDA of heart mitochondriaBased on sucrose gradient centrifugation, heart mitochondria were separated after hypoxia treatment, and the SOD and MDA activity of heart mitochondria were detected after protein concentration was measured by BCA kit. The results showed that both of MDA and SOD reduced with the increase of hypoxia treatment in the moderate hypoxia group(DO:2.5±0.5 mg/L), but increased again in severe hypoxia group(DO:0.5 mg/L). 4. Effect of hypoxia on parameters of antioxidant enzymes in liverThe results about various parameters of antioxidant enzymes detected in M. amblycephala liver after hypoxia treatment revealed that hypoxia decreased both SOD and CAT enzymes activities and hydrogen peroxide(H2O2) content in liver. On the other hand, the nitric oxide(NO) level in liver gradually increased with the increase of hypoxia time and the decrease of hypoxia concentration. The total ROS level fluctuated which gradually decreased with the increase of hypoxia time, but increased after reoxygenation and server hypoxia treatement.