Study On The Proteomics Of Melamine With Or Without Cyanuric Acid On Hepatotoxicity

Currently on the toxicity of melamine and cyanuric acid research mainly around the kidney,urinary,nerve,reproductive,immune,etc,no scholars have studied the toxicity of melamine in terms of overall differential proteins.In this experiment,iTRAQ quantitative proteomics technique is used to investigate the toxicity of melamine with or without cyanuric acid in mice.The differences of all the proteins in the liver of mice are analyzed to determine the main differential protein of melamine substances which has toxic effects on the liver,find and make sure the target protein and then verify it.And then analysis the protein which belongs to the cell signaling pathway,thus reveal the mechanism of toxic action of this kind of substance in the liver of mice,so as to improve the toxicological data of melamine.In this study,thirty SPF male mice of KM with 22-24g weight are randomly divided into blank control group,melamine group(30 mg·kg-1)and melamine with cyanuric acid group(15 + 15 mg·kg-1).The experiment groups are fed for 28 days and once irrigation stomach every other day,the 29 day asepsis to remove the liver tissue made of sample protein.SDS-PAGE is used to analysis.The differential protein is obtained by iTRAQ technique which mass spectrometry authenticates and protein retrievals.The differential proteins are analyzed by bioinformatics such as GO analysis and KEGG pathway analysis,to find out the significant biological function of the differential protein,the metabolic pathway and the signal transduction pathway.The differential proteins PDIA6,HSP70 and HSPA4L were screened and verified by Western-blot.The result shows:1.A total of 20284 unique peptides and Identified 3695 differentially expressed proteins are obtained by proteomics iTRAQ technique from the liver of mice.Compared with melamine group and blank control group,there are 166 differential proteins,including 130 up-regulated proteins and 36 down regulated proteins.Compared with melamine and cyanuric acid group and blank control group,there are 242 differential proteins,including 161 up-regulated proteins and 81 down regulated proteins(up-regulation>1.2,down-regulation<0.83,p<0.05).2.The difference of the proteins involved in the melamine group is 69,the molecular function is 23,the cell composition is 23,the combination of 51 different proteins are involved in the biological process,46 are involved in the molecular function,Cell fraction.3.There are 10 KEGG enrichment pathway in the melamine group,and there are about 13 KEGG enrichment pathway in the combined group.4.In this study,Western-blot method is used to detect the expression levels of HSP70,PDIA6 and HSPA4L.Compared with the blank control group,the protein expression level of HSP70 and HSPA4L in melamine and cyanuric acid group is significantly lower than that in the control group,and the protein expression level of PDIA6 is significantly higher than that in blank group(p<0.05).Compared with he blank control group,the protein expression level of HSPA4L is lower than that in the control group(p<0.05),but the other protein expression level is not statistically significant.The results can be seen that western-blot determination and iTRAQ determination of differential protein expression trend is consistent.It also shows that melamine and cyanuric acid combined with HSP70 and HSPA4L expression down-regulated,PDIA6 expression up-regulated,endoplasmic reticulum stress pathway to promote cell apoptosis play a positive role.This study also show that advanced differential proteomics techniques(iTRAQ tag method)that is used to study the toxic effects of melamine with or without cyanuric acid on liver is reliable,and to provide some microscopic material basis for melamine research.