ITRAQ-based Analysis Of Serum Markers And Intervention Of Alpha-1-acid Glycoprotein In Rat Model Of Crush Syndrome

ObjectiveCrush syndrome is the second leading cause of death in earthquake,but there are limited methods for early diagnosis and treatment.Serum differentially expressed proteins in crush syndrome were detected by proteomics method,and Enzyme-linked immunosorbent assay(ELISA)was used to validate the differentially expressed proteins.It provides a basis for the discovery of biomarkers for crush syndrome.The effect of differential expression of α1 acid glycoprotein(AGP)in crush syndrome was studied.MethodPart I: 56 healthy male SD rats Randomly divide into seven groups: Control group(n = 8),CS3kg-0h group(n=8),CS3kg-12 h group(n=8),CS5kg-0h group(n=8),CS5kg-12 h group(n=8),CS5kg-72 h group(n=8).Using a dose of 2% pentobarbital sodium(3ml / kg)for intraperitoneal anesthesia,then place in a prone position,a new type of digital extrusion platform was used to establish a rat crush syndrome model.The lower extremity compression parameter pressure was 3/5 kg and the time was 16 h.Blood was taken through the abdominal aorta at three time points: 0h,12 h,and 72 h after decompression.Screening of differentially expressed proteins in serum by i TRAQ detection and bioinformatics analysis.The related differential proteins were verified by ELISA.Part 2: Randomly divide the rats into Control group,CS group and AGP group according to the principle that each group survives 6 rats after crush.Blood was taken at two time points,12 h and 72 hours after decompression,biochemical index detection and ELISA were used to detect serum levels of TNF-α,IL-6,NGAL,and KIM-1,and the muscle and kidney tissue of each group was taken at 4% The solution was fixed in Marin solution and stained with TUNEL and immunohistochemistry.Results1.The result of i TRAQ: A total of 206 differential proteins were screened in the CS3kg-0h group VS control group;A total of 267 differential proteins were screened in the CS3kg-12 h VS control group;A total of 280 differential proteins were screened in CS3kg-72 h group VS control group;A total of 344 differential proteins were screened in CS5kg-0h group VS control group,A total of 365 differential proteins were screened in CS5kg-12 h group VS control group,A total of 313 differential proteins were screened in CS5kg-72 h group VS control group.Among the 20 up-regulated proteins,neutrophil gelatinase-associated lipocalin,haptoglobin,and alpha 1 acid glycoprotein increased significantly immediately after extrusion,and further increased at 12 h after decompression and decreased at 72 h after decompression.2.Bioinformatics analysis: We performed GO function and KEGG Pathway enrichment analysis of the differentially expressed proteins.In the six comparison groups,the result of GO enrichment analysis has metabolic process in biological processes.Significantly enriched the KEGG pathway involved in differentially expressed proteins was complement and coagulation cascades.3.Verification results: Compared with the normal control group,NGAL immediately increased in the 3kg and 5kg crush groups,and maintained a high level in the subsequent 12 h and 72 h.Compared with the normal control group,AGP increased significantly in the 3kg crush group immediately after decompression,and increased significantly in the 5kg pressure group 12 hours after decompression.Compared with the control group,HP increased significantly at 0h in the 3kg crush group and then decreased,while slightly increased immediately in the 5kg crush group and then increased gradually.4.The role of α1 acidic glycoprotein in crush syndrome: BUN,Cr,K+,and CK in the CS group were significantly higher than those in the control group at 12 hours after decompression.the biochemical indexes of AGP group were not significantly improved compared with CS group.IL-6 and TNF-α in the CS group were significantly higher than those in the Control group 12 hours after decompression,and decreased significantly 72 hours after decompression.IL-6 and TNF-α in AGP group were lower than those in CS group at 12 h and 72 h after decompression.In the CS group,the level of NGAL in serum and urine was significantly higher than in the Control group at 12 h after decompression,and significantly decreased at 72 h after decompression;the blood NGAL in the AGP group was significantly higher than the CS group at 72 h after decompression.Urinary NGAL in the AGP group was significantly lower than that in the CS group 12 hours after decompression.In the CS group,the serum KIM-1 increased significantly at 12 h,72h after decompression.the blood KIM-1 in the AGP group was significantly lower than that in the CS group at 12 h and 72 h after decompression.urine KIM-1 was increased at 12 h after decompression.The pathological results showed that the muscle and kidney damage in the AGP group was alleviate compared with CS group.Renal TUNEL and immunostaining showed that the ratio of apoptotic cells and the expression of Caspase1 and Caspase3 in CS group were significantly higher than those in Control group and that in the AGP group were all lower than those in the CS group.ConclusionIn this study,based on the i TRAQ method,the differentially expressed proteins of crush syndrome were screened out and bioinformatics analysis was performed.It is clear that NGAL and AGP may become new serum biomarkers of crush syndrome and provide new ideas for the early diagnosis of crush syndrome.Bioinformatics analysis found that complement and coagulation cascade pathways may play an important role in the development of crush syndrome.Early administration of exogenous AGP intervention therapy can play an anti-inflammatory role by reducing the expression levels of serum IL-6 and TNF-α.AGP may protect renal injury associated with crush syndrome by changing the expression of NGAL and KIM-1 in kidney and antiapoptotic effect.