Effects Of Astaxanthin On H₂O₂-induced Oxidative Stress Response In Primary Mouse Hepatocyte

The level of endogenous reactive oxygen species?ROS?is higher than the ability of endogenous antioxidant defense system removing ROS When the body suffers from various harmful stimuli;then pathological reactions will appear in cells and tissues,which is referred as oxidative stress.Application of animal nutrition regulation methods to prevent animal diseases is focus of research recently,such as adjusting nutitions and dietary supplements.Therefore,it is of great practical significance to prevent oxidative stress diseases in animals by searching for natural compounds with antioxidant effects and studying their protective mechanisms.Astaxanthin is a natural strong antioxidant in animal cell biofilms and lipid-rich tissues.Keap1-Nrf2-ARE is an important endogenous antioxidant pathway activated in the body's cells when exposed to H₂O₂.In this study,H₂O₂-induced mouse primary hepatocyte was used as an oxidative stress model to investigate the effects of astaxanthin on its antioxidant capacity and its relationship with Keap1-Nrf2-ARE signaling pathway.In this study,the method of mouse primary hepatocyte which was established by myself was used to isolate and culture mouse primary hepatocyte.The cells were induced with H₂O₂ to construct an oxidative stress model.Flow cytometry was used to detect the changes of apoptosis rate,viable cell rate and ROS in primary hepatocyte.Biochemical and fluorescence quantitative PCR were used to determine the changes in activity and content of MDA,CAT,SOD,GSH-Px and?-GCS in cells.Western-blot was used to detect the change of relative expression of Nrf2 protein in nucleus.The changes of Keap1 and Nrf2 protein expression and mRNA relative expression were detected by ELISA and fluorescence quantitative PCR.The effects of astaxanthin on antioxidant capacity were explored.The results are presented:?1?This experiment established a highly efficient and stable method for successfully isolating and culturing mouse primary hepatocyte.?2?Using MDA as an index,to determine the best time and concentration of H₂O₂ stimulation was 10?mol/L H₂O₂ for 3h.At this time,the cell apoptosis rate,ROS and the content of MDA were significantly increased?P<0.05?,and an oxidative stress model was successfully constructed.The cells were pre-protected with different concentrations of astaxanthin,and then stimulated with H₂O₂ 10?mol/L for 3h.Similarly,the best treated time and concentration of astaxanthin's pre-protected was 5?g/mL for 3h,and the cell apoptosis rate,MDA and the content of ROS was significantly decreased?P<0.05?.Therefore,this experiment preliminarily indicated that astaxanthin had protective effects on H2O2-induced oxidative stress in mouse primary hepatocytes.?3?Astaxanthin significantly increased the content of non-enzymatic antioxidant GSH in cells of the protective group?P<0.05?,the activity and content of SOD,CAT,GSH-Px and?-GCS in the antioxidant enzyme system?P<0.05?,and improved the anti-oxidation ability of mouse primary hepatocytes,when researching the antioxidant capacity of astaxanthin on cells.?4?Compared with H₂O₂ group,astaxanthin can significantly inhibit nuclear transfer of Nrf2 in the protection group cells?P<0.05?,and Keap1 and Nrf2decoupling.In addition,the relative expression levels of Keap1 and Nrf2 proteins and genes in the cells were significantly reduced?P<0.05?.In conclusion,this study found that astaxanthin has protective effects on H2O2-induced oxidative stress in mouse primary liver cells.This protection is achieved by inhibiting the uncoupling of Keap1 protein and Nrf2 protein and the nuclear transfer of Nrf2 protein,so that the activation of Keap1-Nrf2-ARE signaling pathway was inhibited,and the activity and content of antioxidant enzymes such as SOD and GSH-Px were increased in the cells to increase the antioxidant capacity of mouse primary hepatocytes.