The Effects Of Temperature And Nitrite On Antioxidative System In Kidney Cell, Erythrocyte, And Hepatocyte In Vitro Of Grass Carp Ctenopharyngodon Idellus

In order to investigate that effects of temperature and nitrite on antioxidative function in cells of grass carp (ctenopharyngodon idellus) and to understand the cellular mechanism underlying, we conducted this study though three aspects described as follows:(1) the study of temperature effect on the cellular oxidant and antioxidant indicators as well as the relative expression level of antioxidant enzyme gene in the CIK cells;(2) the study of nitrite effect on oxidant and antioxidant indicators, Hb, metHb, and the relative expression level of the relevant antioxidant enzyme gene in the blood cells;(3) the study of nitrite effect on the antioxidant performance indicators in the hepatocyte.The CIK cell in the study (1) is a kind of passage cell and it is comes from the fisheries college of Huazhong agricultural university. The fish weight in the study (2) and (3)is about65.8±5.5g, the blood cell and hepatocyte were both obtained in the sterile conditions, we set three experiment groups (1.0mg/L,10.0mg/L,100.0mg/L), one negative control (0mg/L) and three repetition in each group in these experiments. The exposure time in the blood and the hepatocyte experiment are0h,3h,6h,12h,24h and0h,6h,12h,24h,48h,96h respectively. The result were described as follows:1. The effect of temperature on the oxidative stress level in CIK cellsUnder the condition of acute temperature exposure, the increased4℃and reduced4℃were both cause the oxidative stress situation changed. We found that the ROS level, the mitochondrial membrane potential (△ψm), the MDA content, and protein carbonyl content were all significantly influenced by the cool and heat stress treatment, the activity of TAC is up to the highest level at20℃, both the cool and heat treatment were cause the GSH activities higher than the control. The activity of SOD, CAT and GPx are higher than the control at the cool and heat treatment, GR activity showed increased with the temperature rising gradually. Through the fluorescent quantitation investigated of sod, cat, gpx gene, no matter the heat or cool treatment, the transcript level of these three antioxidant enzyme gene were all higher than their control group. 2. The effect of nitrite exposure on the oxidative and antioxidative indicators and anti-oxidative relative gene’s transcript level of blood cells in grass carpThe nitrite exposure showed significant influence on the the cellular ROS level, the content of Hb and MDA, the mitochondrial membrane potential (△ψm) and the key antioxidant enzyme’s gene expression level in blood cells of grass carp. These result showed that nitrite effect on the blood cells was dose-effective and time-effective. The ROS level, mitochondrial membrane potential (△ψm) and the MDA content were all showed increased first and then reduced with the concentration of nitrite increasing, the CAT, SOD, GPx, GST, y-GCS, LDH activity appeared increased first and then declined when the nitrite concentration improved at each time point, the GSH and TAC activity were also showed increased first and then declined when the nitrite concentration improved. The transcript level of sod, cat, gpx were also showed increased first and then declined. The ROS level improved was the sign of free radical metabolism ability variation and the oxidative stress status exacerbation, and the lower concentration of nitrite with longer exposure time would definitely damage the cell’s antioxidative repair ability.3. The effect of nitrite on the nonspecific immunity and antioxidative response performance in isolated hepatic cells of grass carpThe results showed that nitrite effect the nonspecific immunity and antioxidant ability significantly, the activity of SOD, CAT, LDH, GST, y-GCS, GSH and TAC were all showed increased first and then declined when the nitrite concentration improved or the exposure time prolonged, the lower concentration of nitrite with longer exposure time cause the enzyme activity improved significantly which suggested that the nonspecific immunity and the radical remove ability were both improved, on the other side, it also suggested the oxidative stress status was serious improved than the control.