Curcumin Attenuates Oxidative Stress In RAW264.7 Cell Via Activating Nrf2-Keap1-ARE Signal Pathway

Large-scale livestock and poultry breeding environment often leads to oxidative stress.Macrophages play an important role in the immune system,which are vulnerable to reactive oxygen species and result in their death.Curcumin is the main active composition of the root of turmeric,which is a natural phenolic yellow pigment with antioxidant effects.In our study,we chose Nrf2-Keapl-ARE signal pathway as research model,to study the protective effects of curcumin on macrophages cell under oxidative stress in vitro.Here,we used RAW264.7 cell as research model and oxidative damage was induced by H2O2.The safe concentration of curcumin and protective effect of curcumin on the cell under oxidative stress were measured by MTT assay.At the mean time,we measured the indexes of anti-oxidant and apoptosis.In addition,we observed the effect of curcumin on genes and proteins level of Nrf2-Keapl-ARE signailing pathway by using RT-PCR and Western-blot.We also measured the effect of curcumin on apoptosis genes transcriptional level.Furthermore,the translocation of Nrf2 protein was also investigated by analysis of total and nuclear proteins with Western-blot.(1)The effects of different concentration of H2O2 on the viability of RAW264.7 cell were evaluated with MTT assay.The IC50 value was determined to be 467.46μM,so we chose 500μM of H2O2 as the optimum treatment dose for RAW264.7.(2)Our data showed that the safe concentration of curcumin for RAW264.7 was 0～20μM,so we chose 5μM,10μM,20μM as low,medium and high dose,respectively.(3)The effects of different concentration of curcumin and treatment time on the viability of RAW264.7 cell under oxidative stress were evaluated with MTT assay.After treated with H2O2 for 4h and 8h,low dose and medium dose pre-treated group(16h)can promote cell viability while high dose pre-treated group suppressed.But when cell suffer from 24h of H2O2 treatment,The protective effect was vanished.No difference in cell viability was observed in co-treated experimental group compared with positive control.(4)We also detect the cellular ROS level in RAW264.7 treated with different concentration of curcumin(5μM,10μM,20μM,treated for 24h)under different H2O2 treatment time(4h,8h and 24h).We found that the groups treated with curcumin could lower the ROS level.Curcumin could elevate the activity of CAT,SOD and GSH-PX to improve the capacity of cell to eliminate ROS,and at the mean time,lower the MDA level.(5)The activity of caspase-3 was also detected,in 4h H2O2 treated group,low dose and medium dose pre-treated group could increase the activity of caspase-3 in 4h of H2O2 treated while medium dose and high dose pre-treated groups could increase the activity of caspase-3 in 8h of H2O2 treatment.Furthermore,our study showed that curcumin could prevent cell from apoptosis to increase cell viability,especially in preventing transformation of early to late stage of apoptosis.(6)Here,we found that,Curcumin could up-regulate Nrf2 expression after H2O2 treated for 4 hours.Meanwhile,curcumin could promote the translocation of Nrf2 to the nucleus to up-regulate the expression of HO-1 and Nqol in low and medium dose group.HO-1,GLCM and GCLC are all regulate by ARE element,curcumin could enhance HO-1 expression while inhibit GCLC.Taken together,it can be inferred that it required some time from the acumulation to nucleus of Nrf2 to the activation of anti-oxidant genes.Curcumin could induced the expression of Nrf2 in protein level after H2O2 treated for 8 hours.And there is a negative correlativity between PI3K and Nrf2 protein expression.Our findings revealed that curcumin could resist oxidative stress by activating Nrf2 protein,leading Nrf2 to accumulate in nuclear,which involved in transcriptional activation of specific Nrf2 dependent genes.On the other hand,curcumin could scavenge ROS by increasing the activity of the antioxidant enzyme.In summary,curcumin could resist oxidant by increasing the transcriptional activity of antioxidant genes and the activity of antioxidant enzyme that potentially inhibit apoptosis process.