| Objective:The present research was designed to study expression of AQP2,AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris..And iTRAQ proteomics technology was applied to investigate the change of protein expression spectrum with unprocessed and processed Euphorbia lathyris,The proteomic profiles and potential biomarkers was investigated in intestinal tissue of unprocessed and processed Euphorbia lathyris,in order to illustrate the potential mechanism of Euphorbia lathyris processing attenuated.Method:1 KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris,Euphorbia factor L1.The expression of AQP2,4,8 were determined by immunohistochemical staining,measuring the amount of mRNA(previously amplified by RT-PCR)in intestines samples from all groups.2.The proteomic profiles of each group were analysised and identificated by iTRAQ proteomics combined Q-Exactive mass spectrometry.Bioinformatics analysis including GO analysis,pathway analysis,network of interaction analysis analysis were done with the corresponding software.The expression of different protein and its upstream and downstream protein was tested by western blotting to determine the initial results.Results:1 Comparing to the normal control,the immunohistochemical staining results showed that AQP2,AQP 4 and AQP 8 were decreased significantly(P<0.05)encode by unprocessed and processed Euphorbia lathyris(Semen Euphorbiae and Semen Euphorbiae Pulveratun).AQP2,AQP 4 and AQP 8 were not decreased significantly by Euphorbia factor L1.There had no differences on the level of AQP2,AQP 8 and the related mRNA between the high-dose group of semen Euphorbiae and semen Euphorbiae Pulveratun extract and positive control group,while significantly reduced with the normal control.AQP4 and mRNA expressed almost the equal levels;there had no significantly differences on all groups.But Euphorbia factor L1 induced the changes of expression on AQP2,AQP8 and the related mRNA differently compared to the normal control.2 Through analysis with software,it was found that there were 7 differentially expressed proteins in the intestines tissues of rats intersection of differentially expressed proteins of Semen Euphorbiae and semen Euphorbiae Pulveratum,Euphorbia factor L1,of which,5 proteins were down-regulated and 2 proteins up-regulated.There were 295 differentially expressed proteins in the intestines tissues of rats in union of differentially expressed proteins of Semen Euphorbiae and semen Euphorbiae Pulveratun,Euphorbia factor L1,of which,70 proteins were down-regulated and 225 proteins up-regulated.Analysis of GO of intersection and union proteins were found.Through analysis of the pathways of these proteins,15 signaling pathways were found in each group;meanwhile,the protein interaction network graph was drawn.Pathway analysis found that experimental groups with different pathway has the difference,compared with normal group.Semen Euphorbiae extract group,compared with normal group,was found active degree higher pathway in immune response pathway mainly,also relates to the path of development,G protein and other physiological processes.Euphorbia factor L1 group,compared with normal group,was found physiological processes were relatively singly regulated.The main signaling pathway includes Ang/Tie2,NF/kB and so on.Semen Euphorbiae Pulveratun extract group,compared with normal group,was found main signaling pathway were B-Raf pathway,epithelial cells to mesenchymal transition(EMT),cell endocytosis and so on.Semen Euphorbiae Pulveratun extract group regulated interleukin relatived signaling pathways unsignificantly.Euphorbia factor L1 was decreased in the Semen Euphorbiae Pulveratun extract group.Meanwhile,the inflammation that interleukin reduced was not responsed obviously.It provides that attenuation of Euphorbia lathyris.has relationship with the inflammation that interleukin reduced.The same signaling pathways between semen Euphorbiae and semen Euphorbiae Pulveratun extract groups were G proteins,Eph/Ephrin and so on.Attenuation and synergy of Euphorbia lathyris could couse by the processes of cytoskeleton,glycolysis and gluconeogenesis.Mutual interaction network analysis the intersection result only has one down-regulated protein that was ang-4.The union results were analyzed three networks.Firstly,the antigen processing and presentation of peptide or polysaccharide antigen via MHC class Ⅱ has 5 down-regulated proteins and 5 up-regulated proteins.The MHC class Ⅱ was the important role in the network.Secondly,regulation of response to stress has 3 up-regulated proteins.RelA/P65 and ubiquitin has a close connection with other protein in the network.Thirdly,interferon-gamma-mediated signaling pathway has 1 down-regulated proteins and 6 up-regulated proteins.The STAT1 has the important role in the network.Western blot results of Ang-4 and STAT1 showed results consistent with the previous proteomics.Conclusion:After processed,regulation of AQPs by semen Euphorbiae were decreased.So intestinal mucosal inflammation and damage were weakened.The intestinal toxicity and the capacity of was decreased obviously induced by Euphorbia lathyris.It may be one of mechanism attenuated of Euphorbia lathyris.Identified differentially expressed proteins mainly constituted by part of the cell.The expression sites of them located within cells and organelles.Metabolic processes and immune response,transduction molecules and protein binding function etc.molecular biological function were significantly affected.Pathway analysis found that experimental groups with different pathway has the difference,compared with normal group.Interaction network analysis showed that Euphorbia lathyris.could regulate a number of key proteins,then regulated multiple paths of the body.Information of proteins and related biological information obtained through proteomics,provide experimental data and basis for explain the toxic mechanism and target of Euphorbia lathyris.processing. |
