| Reactive oxygen species(ROS) is a strong oxidative power of oxygen-containing molecules, ions and free radicals. Oxidative stress is an imbalance between oxidants and antioxidants in cells resulted of excessive accumulation of oxidants. Increased oxidative stress mediated by ROS has been concerned on many diseases in humans, meanwhile, leads to huge of economic loss in aquaculture. Some studies in mammals have demonstrated that L-carnitine(LC) has antioxidant properties as a nutritional additive. However, effects of L-carnitine on antioxidant function of fish are known very little, responsible antioxidant mechanisms of L-carnitine are not fully clear. To study effects of L-carnitine on growth, ROS level, activity and gene expression of antioxidant enzymes of oxidative fish cell, and confirm that regulation of L-carnitine in Keap1-Nrf2-ARE signaling pathways, FHM(fathead minnow muscle cell line) and GCO(Ctenopharyngodon idellus ovary cell line) were chose as research object and cell model of oxidative stress was built by H2O2. Meanwhile, flow cytometry, fluorescence quantitative PCR and immunocytochemistry were used to analysis the levels of ROS and apoptosis, gene expression as well as nuclear translocation of Nrf2 in two cells. This study will provide evidence for elucidating mechanism that L-carnitine protects cells against oxidative damage, which is important for the basic theory of L-carnitine as an antioxidant used in aquaculture and prevention of diseases. This study includes five parts test, the results are as follows:1) To detect effects of H2O2 on FHM and GCO fish cells, screen the concentration and time of H2O2 that lead to inhibition of cell viability and cell survival are around 50%. MTT method and trypan blue staining method were used to determine cell vitality and cell survival rate. To check whether H2O2 could induce FHM and GCO cells oxidative stress in screening condition, using flow cytometry instrument to determine levels of ROS and apoptosis and biochemistry method to calculate levels of MDA and GSH as well as activities change of SOD, CAT, GPx and γ-GCS in cells. The results showed that the impacts of H2O2 on cell viability and cell survival of FHM and GCO cells were time-dose dependent effects. Viability and survival rate of FHM cells treated with 1 m M H2O2 for 1h were decreased to 45% and 55%, respectively. Similarly, viability and survival rate of GCO cells treated with 2.5 m M H2O2 for 1h were decreased to 60% and 45%, respectively. 1 and 2.5 m M H2O2 were significantly increased levels of ROS, apoptosis and MDA(p<0.05), significantly reduced activities of SOD, GPx and γ-GCS in FHM and GCO cells(p<0.05). Under the conditions of this experiment, by checking levels of ROS and apoptosis, and activities of main antioxidant enzymes, the stimulations of 1 m M H2O2 for 1h and 2.5 m M H2O2 for 1h can successfully induce oxidative damage of FHM and GCO cells.2) After treatment of 0.1-1m M L-carnitine for 6 and 12 h in FHM cells, cell viability was significantly higher than that of the control group(p<0.05), treatment of 0.01-1m M L-carnitine for 12 h in GCO cells significantly increased cell viability(p<0.05). Compared with H2O2 group, 0.001-1m M L-carnitine significantly increased viability of FHM cells exposed to 1 m M H2O2(p<0.05), meanwhile, 0.1-5m M L-carnitine significantly increased viability of GCO cells exposed to 2.5 m M H2O2(p<0.05). 0.1-1m M L-carnitine significantly reduced ROS levels of FHM cells(P < 0.05), similarly, 0.05 and 1 m M L-carnitine significantly reduced ROS levels of GCO cells(p<0.05). Compared with the H2O2 group, 0.5 m M Lcarnitine significantly reduced apoptosis and necrosis of oxidative FHM and GCO(p<0.05).3) Compared with H2O2 group, the MDA content of FHM and GCO cells were significantly reduced in 0.1-1m M L-carnitine pre-treatment group(p<0.05). Total GSH content of FHM cells was increased in all L-carnitine pretreatment groups, in addition, total GSH content of 1 m M L-carnitine group reached significant level(p<0.05), pretreatment with 0.1-1 m M L-carnitine significantly increased total GSH content of GCO cells(p<0.05). Compared with the H2O2 group, total SOD activity of FHM cells was significantly increased in 0.1 m M L-carnitine pre-treatment group(p<0.05), 0.5 and 1m M L-carnitine pre-treatment significantly increased total SOD activity of GCO cells(p<0.05). CAT activities of FHM cells in 0.5 and 1m M L-carnitine pre-treatment groups were significantly higher than 1m M H2O2 group(p<0.05), the activity CAT of cells in 0.5m M L-carnitine pretreatment group was significantly higher than that of 2.5 m M H2O2 group(p<0.05). GPx activities of FHM cells in 0.1-1m M L-carnitine pretreatment group were significantly higher than 1m M H2O2 group(p<0.05), and L-carnitine pretreatment had no significant effects on activity of GPx in GCO cells(p>0.05). Compared with H2O2 group, γ-GCS activities of FHM cells were significantly improved in 0.1-1m M Lcarnitine pre-treatment groups(p<0.05), γ-GCS activities of GCO cells were significantly improved in 0.5 and1 m M L-carnitine pre-treatment groups(p<0.05).4) L-carnitine could significantly up-regulate relative expressions of Cu Zn-SOD m RNA in FHM and GCO cells, and significantly up-regulate relative expressions of GPx and GCLC in GCO cells(p<0.05). H2O2 also significantly down-regulate relative expressions of Cu Zn-SOD m RNA, while significantly up-regulate relative expressions of GCLC m RNA in FHM and GCO cells. In oxidative stress condition, L- carnitine(0.1-1m M) pretreatment significantly increased relative expression of GCLC m RNA in FHM(6h) and GCO(12h)(p<0.05), CAT and GPx m RNA relative expressions of GCO cells in 0.5 m M L-carnitine pretreatment group were increased significantly(p<0.05). L-carnitine pretreatment could up-regulate Cu Zn-SOD, CAT and GPx m RNA relative expression of FHM cells.5) Compared with the control group, Nrf2 m RNA relative expression of FHM cells in H2O2 group had no significant difference(p>0.05), however, Nrf2 m RNA relative expression of GCO cells in H2O2 group was significantly decreased(p<0.05). 0.5m M L-carnitine can up-regulate relative expression level of Nrf2 m RNA in normal and oxidative stress conditions(p<0.05). The immunocytochemistry results showed that under the state of oxidative stress, Lcarnitine can obviously promote the nuclear translocation of Nrf2 in FHM and GCO cells.In conclusion, H2O2 can reduce viability, survival rate and antioxidant function of FHM and GCO cells, furtherly induce cells to oxidative stress. Suitable concentration of L-carnitine can improve cell activities of FHM and GCO exposed to H2O2, and inhibit apoptosis and ROS production as well as significantly improve the antioxidant function of cells. L-carnitine can up-regulate relative expression of antioxidant enzymes and Nrf2 in FHM and GCO cells treated by H2O2, meanwhile, L-carnitine has a beneficial role in nuclear translocation of Nrf2. Our finding demonstrated that L-carnitine can promote the growth and antioxidant function of FHM and GCO cells. L-carnitine has a moderating effect of nuclear transcription factor Nrf2 in FHM and GCO, its antioxidant mechanisms involve in Keap1-Nrf2-ARE signal pathway. Under the conditions of this experiment, adding amount of L-carnitine is 0.1-1m M for 6-12 h protected FHM and GCO cells from H2O2-induced oxidative stress. |
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