The Study Of Antioxidant Mechanisms Of The Sonchus Brachyotus Extract Based On The Nrf2-Keap1-ARE Signaling Pathway

Oxidative stress is that when the body is subjected to harmful stimulation(such as chemical poisons,drug metabolism,radiation and light),the production of reactive oxygen species(ROS)in the body is increasing.Therefore,the body’s own antioxidant system is unable to remove too much ROS,resulting in the imbalance between oxidation and antioxidation.Oxidative stress can lead to various forms of tissue damage and lead to many diseases,such as alzheimer’s disease,parkinson’s disease,ulcerative enteritis and so on.Most organisms have antioxidant enzyme defense system,non-enzyme antioxidant defense system and repair system to protect the body from the adverse effects of oxidative stress.In recent years,more and more studies have begun to look for safer and natural antioxidants from biological resources.Plant extract has become a new research and application hotspot because of its high efficiency,non-toxicity and easy to be accepted by consumers.The previous research results of our group have proved that the extract(SBE)has antioxidant effect,but the mechanism of antioxidant effect has not been reported.There are many and complex signal pathways related to antioxidation in the body,and one of the most critical pathways is Nrf2-Keapl-ARE signal pathway.Therefore,on the basis of previous research,this paper takes SBE as the research object and studies the molecular mechanism of antioxidation of SBE based on Nrf2-Keap1-ARE signal pathway.The main results of this paper are as follows:1.Effect of SBE pre-protection on H2O2-induced oxidative damage in Caco-2 cells:In the previous experiment,the H2O2 damage model was successfully constructed.The model was treated with 7.5 mM H2O2 for 24 h.First,the cells were pretreated with different concentrations of SBE(6.25,12.5,25,50,100,200 μg/mL)for 6,12,24 h,and then treated with 7.5 mM H2O2 for 24 h.The cells were collected and the contents of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CAT)were determined.The results showed that SBE pre-protection could significantly reduce the contents of ROS and MDA,and significantly increase the activities of SOD and CAT,indicating that SBE could improve the antioxidant capacity of Caco-2 cells.2.The level of mRNA was used to study the antioxidant mechanism of SBE:based on the antioxidation of SBE,Nrf2-Keapl-ARE pathway was selected to study its antioxidant mechanism,and experiments were carried out from two aspects:first protection and then injury and then repair(the concentration of SBE was 50,100,200 μg/mL,and the treatment time was 12h).The mRNA expression levels of related factors(Nrf2,Keap1,HO-1,NQO1,SOD,CAT,GSH-Px)in Nrf2-Keapl-ARE signal pathway were measured by Real-time fluorescence quantitive PCR(RT-qPCR).The results showed that SBE could significantly increase the expression of Nrf2 gene and the level of downstream cytoprotective gene.The results showed that SBE could regulate the mRNA level of upstream and downstream genes in Nrf2-Keapl-ARE signal pathway,and the effect of SBE pre-protection was better than that of damage before protection.3.Effect of SBE on Nrf2 and Keap1 protein expression:according to the above experimental results,SBE pre-protection injury was selected for protein expression analysis.First,Caco-2 cells were pretreated with different concentrations of SBE(50,100,200 μg/mL)for 12 h,and then treated with 7.5 mM H2O2 for 24 hours.The cells were collected and the protein of Nrf2 and Keap1 were determined by Western blot.The results showed that SBE could significantly increase the protein expression of Nrf2 and decrease the expression of Keap1,indicating that SBE can regulate the protein expression of Nrf2 and Keapl,and play an antioxidant role through Nrf2-Keapl-ARE pathway.