Studies On The Antioxidant Of ESW Polypeptide And Its Effects In Nrf2-ARE Signal Pathway

The Eupolyphaga sinensis Walker(ESW)is an insect Eupolyphaga Families.The main components of proteins,polysaccharides,polypeptides were extracted from the ESW by modern extraction technology.Studies have shown that ESW is an important traditional Chinese medicine,the active ingredients of which are capable of regulating blood lipids,anti-tumor,immune regulation,anti-oxidation.Currently there was little depth studies on the mechanism of the active ingredients extracted from ESW.This investigation was to prepare ESW polypeptide by enzymatic hydrolysis,to understand free radical scavenging and antioxidant aging of ESW polypeptide in vitro and in vivo experiments.The role of ESW polypeptide was analyzed in Nrf2-ARE signaling pathway by RT-PCR.The results would to elucidate clearly the antioxidant mechanism of ESW polypeptide,which would provide experimental evidence for the future development of ESW polypeptide as antioxidants.① Experiment One The purification and identification of ESW polypeptide extractionThe extract of ESW polypeptide was prepared by enzymatic hydrolysis.The concentration and molecular weight of ESW polypeptide were determined by the biuret and Tricine-SDS-PAGE.Results showed that the concentration of ESW polypeptide was 45mg/mL and the molecular weight of that about 8～9kDa small peptide.② Experiment Two Free radical scavening of ESW polypeptideThe methods of Fe2+-Errosin and pyrogallic acid and ultraviolet spectrophotometry were used to determinate the effect of ESW polypeptide to free radical scavenging,hemolysis of red blood cell,mitochondrial swelling and MDA generation in tissues.Spectrophotometry was used to analyze the determination of iron reduction ability and inhibition of the β-carotene-linoleic acid oxidation and inhibition of linoleic acid oxidation of ESW polypeptide.Results showed that there was an increase with the rising of ESW concentration in the scavening rate of free radical,hemolysis rate of red blood cells,the mitochondrial swelling and the content of MDA in tissues for ESW polypeptide compared with the control group.The results revealed that some kinds of free radical could be were removed by ESW polypeptide.③ Experiment Three The investigation to the antioxidant of ESW polypeptideThe mouse was gavaged at different doses of ESW polypeptide for 20 consecutive days in vivo.The SOD,CAT and GSH-PX activity in blood,serum and tissues,and MDA content in tissues were detected by the method of Xanthine oxidase,colorimetry and TBA.The organ index of mice was calculated in terms of mice tissue and body weight.The results show that there wwas an increase in the SOD,CAT and GSH-PX activity in tissues and blood,and a decrease in the MDA content in tissues compared with the aging group.The results revealed that ESW polypeptide could improve the SOD and CAT and GSH-PX activity and reduce the MDA content,which indicate the significant antioxidant of ESW peptide.④ Experiment Four Studies on the antioxidant aging of ESW polypeptideAging mice model was established by injection of D-galactose,while the mice was gavaged at different doses of ESW polypeptide for 20 consecutive days.The mice were observed generally,and the organ index of mice was calculated in terms of mice tissue and body weight.It was measured for the mice movement and stress resistance.Xanthine oxidase,colorimetric,TBA and DNTB methods were applied to determined the SOD,CAT and GSH-PX activity,the contents of MDA and GSH.Results showed that in the aging model group there was a decrease in food intake,an decline in the mice movement capacity and stress resistance ability,a decrease in SOD,CAT and GSH-PX activity in tissues,blood and serum from aging mice,an increase in MDA contents,while as a reduce in GSH contents in aging mice tissues.However,in ESW polypeptide groups there was an enhance in mice movement and stress resistance ability,and an improving in SOD,CAT and GSH-PX activity,and an increase GSH contents,a reduce in MDA contents.The results revealed that the ESW polypeptide could improve the activity of antioxidant enzyme,delay oxidant aging.⑤ Experiment Five The study of ESW polypeptide in Nrf2-ARE signal pathwayCaco-2 cells were treated with ESW polypeptide,H2O2 and tBHQ.The contents of GSH and ROS,and HO-1 activity in Caco-2 were determined by using the DNTB,fluorescence probe and spectrophotometry method.The expression of Nrf2 in the nucleus was detected by immunofluorescrnce and immunohistochemistery.The expression of Nrf2 and HO-1 mRNA and protein in Caco-2 were evaluated by RT-PCR and Western Blot.Results showed that there was an increase in the GSH contents,a decrease in the ROS contents,an increase in the activity of HO-1 and expression of Nrf2 and HO-1 mRNA and protein in ESW polypeptide groups.The results revealed that ESW polypeptide could initiate antioxidant components in Nrf2-ARE signal pathway,which showed the mechanism of ESW polypeptide antioxidant.In summary,ESW polypeptide was the small polypeptide with 8～9kDa,ESW polypeptide had scavening free radical in vivo,and could improve the activity of antioxidant enzyme,as well as reduce lipid peroxide production,which showed a good antioxidant function and delayed the oxidative aging.ESW polypeptide could increase intracellular Nrf2 and HO-1 protein and mRNA expression,improve the intracellular GSH contents and HO-1 activity by starting antioxidant components in Nrf2-ARE signaling pathway,which presented antioxidant effects,thus revealed the antioxidant mechanisms of ESW polypeptide.