Evalution On The LPS–mediated Antioxidant Activity And Mechanisms Of Jateorhizine Using An Optimized Zebrafish Model

Objective:Oxidative stress is caused by oxidative and antioxidant imbalance in vivo.When undergoing a variety of stimuli,excess reactive oxygen species are generated,which result to Oxidative damage,including DNA damage,protein oxidation,then the functional failure of vital organs.Oxidative stress is the leading cause of aging,neurodegenerative diseases,cancer,diabetes,hypertension,Alzheimer’s disease,Parkinson’s disease,neurodegeneration,traumatic distal organ injury,renal tubular epithelial injury etc.Therefore,Searching natural antioxidant compounds has promising clinical value.The establishment of a reliable oxidative stress model is of great significance for revealing the mechanism of oxidative stress and screening for antioxidant drugs and functional foods.The goal of this study is to establish a Simple,sensitive,fast and intuitive detection method for the examination of the antioxidant activities of various compounds.Zebrafish was used as model animal,and the zebrafish embryos were oxidized by three reactive oxygen species inducers including lead acetate,AAPH and lipopolysaccharide,after incubation with selected antioxidant drugs,the amount of ROS can be detected by adding fluorescence probe to the system.Coptis chinensis has antibacterial,antiviral,antiinflammatory,hypoglycemic,hypolipidemic and other effects.jateorhizine（JAT）is one of the main active substances in Coptis chinensis.Previous studies have shown that the Coptis chinensis exerted antioxidant activity.Therefore,Coptis chinensis is considered as a new,natural antioxidant agent due to its high antioxidant activity,little side effects.The aim of present study is to investigate the antioxidant activities of jateorhizine and to explore the underlying mechanismsThe main contents and results of this study are listed as follows:（1）the experiment contains four groups including control group,lipopolysaccharide group and lead acetate group AAPH group.Each group was selected for 6h healthy zebrafish embryo.First,zebrafish embryo was given,lipopolysaccharide（1ppm）,lead acetate(1.6*10^-6ppm),AAPH（12.5ppm）,to detect the effect of different inducers on the survival rate of the embryo.The results showed that the hatching rate of zebrafish embryo was 92%in the control group,87%in the lipopolysaccharide group,83%in the lead acetate group and 69%in the AAPH group.（2）The effect of different reactive oxygen species inducers on the ROS production was investigated by DCFH-DA probe after induction of zebrafish embryos.DCFH-DA probe itself does not fluoresce,but with the biological ROS binding reaction,it will be split into DCF,can emit green fluorescence.By comparing the fluorescence intensity of different groups can know its ROS level.The results showed that the fluorescence intensity of AAPH group was about 1.7 times that of NC group,while that of LPS group and（CH₃COO）₂Pb group was 1.9 and 2.1 times higher than that of normal group.（3）zebrafish embryos were treated by DPPP to detect the degree of lipid peroxidation caused by different reactive oxygen species inducers.The reactive oxygen species produced in body after stress can attack the unsaturated fatty acids in the biofilm,cause lipid peroxidation,and thus form lipid peroxides,which in turn cause cell damage.The results showed that the AAPH group had little difference from the NC group,and the fluorescence intensity of the lipopolysaccharide and AAPH group was 2 times and 2.5 times of that of the NC group.（4）The effect of different reactive oxygen species inducers.on cell death was examined by subjecting the embryo to AO treatment.After oxidative stress,oxidative damage occurs,causing damage to the DNA in the cell.Once combined with DNA,AO probe is emiting green fluorescence,while,and if combined with RNA,lead to the emission of red fluorescence.Compared with NC group,the fluorescence intensity of LPS group and（CH₃COO）₂Pb group was about 2 times and 3times,respectively,and AAPH group was slightly higher than NC group.（5）Zebrafish were divided into control group,the model group and the jateorhizine group.The drug group was fed with 0.02%lipopolysaccharide and 0.02%lipopolysaccharide.After two weeks,take zebrafish serum and liver.Respectively,serum and liver in the detection of superoxide dismutase,malondialdehyde and reduced glutathione activity and content.To investigate the effect of jatrorrhizine on the oxidation target of HepG2 cells induced by lipopolysaccharide,the expression of JNK,ERK1/2,NF-κB,MAPK and AKT,were investigated by Western blot.