Study On The Mechanism Of ARE Pathway Of Zhibaidihuang Decoction Based On Anti Oxidation

Objective: Traditional Chinese medicine believes that "fire and heat" is the main cause of various diseases and should be treated with drugs cold and cool in nature.Nourish yin and purge fire is often used in clinical practice,Zhibaidihuang decoction is one of the representative prescription.Previously,the effective treatment of Zhibaidihuang decoction has a lot of research reports however the mechanism of this activity remains to be elucidated.This study intends to make changes by comparing Zhibaidihuang decoction treatment group and normal rats the signs index changes,energy metabolism and oxidative stress,from oxidative stress signaling pathway mediated by Nrf2/ARE,through the analysis of differences in metabolite and metabolic clear Zhibaidihuang decoction in target protein regulated by /ARE signaling pathway after Nrf2 the comprehensive analysis and discussion,the role of Zhibaidihuang Decoction on regulation of oxidative stress,the mechanism of nourish yin and purge fire,provide a theoretical basis for the treatment of "Shang Huo".Methods:(1)The dual luciferase reporter assay for screening pathway Zhibaidihuang Decoction sensitive detection of RT-PCR,ARE signaling pathway related genes(Nrf2,Keap1)expression determination by phosphomolybdic acid colorimetric method for its ATP content,cell oxygen consumption rate of active oxygen and ROS,and the detection of SOD by xanthine oxidase concentration level.To determine the downstream target proteins regulated by the Nrf2/ARE signaling pathway.(2)The SD rats were intragastric administration by Zhibaidihuang Decoction,determination of changes to drug group based index and inflammatory factors,and the establishment of rat serum urine metabolism group based on LC-MS analysis method,combined with multivariate data analysis method for serum and urine metabolites between the rats,analysis mechanism of Zhibaidihuang Decoction on oxidative stress pathway and the change of different metabolites and oxidative metabolites association.Results:(1)The body weight,spleen pancreas index and blood routine of the rats in the administration group and the control group were measured.The dual luciferase reporter gene we screened ARE this reaction the original,and then determine the Nrf2/ARE signaling pathway through the cell to detect the content of ATP,HepG2 cells and the detection rate of oxygen consumption determination,SOD method of xanthine oxidase concentration level,group content of ATP elevated significantly compared with control group,cell oxygen consumption rate increased,SOD level decreased.(2)The body weight and spleen pancreas index of rats in the administration group and the control group were measured.The results showed that there was no significant difference in weight and spleen and pancreas index,and there were many changes in the blood routine.The results show that the use of LC-MS to determine the metabolism of Zhibaidihuang Decoction group compared with the control group there are many obvious differences in relative metabolites.In serum samples,the three metabolic pathways of fatty acid biosynthesis,glycerol phospholipid metabolism and linoleic acid metabolism pathway have been significantly changed.Which group compared with the control group decreased Zhidan palmitic acid,linoleic acid,oleic acid,and lysophosphatidylcholine(LysoPC 16:1(9Z)),LysoPC(14:0),LysoPC(20:0),LysoPC(16:0),LysoPC(18:2(9Z,12Z),LysoPC(20:4)(5Z,8Z,11 Z,14Z)rose).Valine urine metabolism group,leucine and isoleucine biosynthesis,glycerol phospholipid metabolism and phenylalanine,tyrosine and tryptophan biosynthesis have significant changes in the group,Zhibai phenylpyruvic acid,leucine and valine choline phosphate content relative to the control group on the rise.Conclusion:(1)Oxidative stress protective mechanism of Zhibaidihuang decoction may prompt the increased activity of ROS to HepG2 cells to hypoxia stress in advance,so as to stimulate Nrf2 and Keap1 dissociation and activation,thus further start of gene transcription and expression of downstream protection ARE,play a key role in protection against oxidative damage.(2)Through analyzing the differences in serum and urine of rats can be further confirmed that the metabolite zbdhd can provide energy for the oxidation of the body,improve the antioxidant capacity,as the body of the oxidative stress plays a protective role,also can promote protein synthesis,regulate immune function,alleviate inflammatory reaction.