The Regulation Of High Uric Acid In Protein Molecular Network Identified By Proteomics Approach

Objective: High level of uric acid may cause hyperuricemia,which further develops into gout,leading to cardiovascular and chronic kidney disease.Previous studies shown that high uric acid is a pro-oxidant in cells,which induce oxidative stress,mitochondrial dysfunction,and inflammatory response,leading to endothelial dysfunction.However,to date,the molecular mechanism is not very clear.Herein,aiming at human glomerular mesangial cells(HRMC)and gout patients blood samples,we combined the proteomics approach,and biochemical experiments as well as bioinformatics tools to identify abnormal expression of proteins and to characterize their localization,functions,interaction networks and pathways.Accoring to cell behavior,concomitant diseases and clinical biochemical indexes,we explored the effect and mechanism of high uric acid in the protein level.Methods: 1.In this study,we chose human glomerular mesangial cells(HRMC)as the research object,and optimized the appropriate uric acid intervention conditions according to the cell proliferation ability,then analyzed HRMC cells protein expression using mass spectrometry-based proteomic approach,and identified the abnormally expressed proteins.The abnormal proteins were further characterized using bioinformatics tools including GO,string,KEGG and so on.Accroding to HRMC cell behavior,we discussed the effect of high uric acid on HRMC and explored the molecular mechanism on protein level.2.16 patients with gout(8 cases of gout and 8 cases of gout with kidney damage)were selected as the subjects,and 8healthy adults were selected as control group.We used proteomics strategies to identify and quantify the expression of proteins in plasma.Statistical methods were used to screen differentially expressed proteins between gout pateins and healthy adults.The abnormal proteins were further analyzed and discussed according to concomitant diseases concomitant and clinical biochemical indexes.Finally,we explore the potential of the abnormal proteins in plasma of gout patientsas clinical biomarkers.Results: 1.High uric acid(UA)regulates the protein network and cell behavior of human glomerular mesangial cells.(1)The HRMC cells were stimulated with 0.1mM,0.3 mM,0.5 mM and 0.7 mM UA for 6 h,12 h,24 h,48 h and 72 h respectively.Compared with the control group(NC),in 48 hours in 0.5 mM and 0.7 mM group,cell proliferation ability significantly decreased,while in 72 hours and 0.7 mM group,cell proliferation ability also significantly lower and stable.(2)In the HRMC cells treated with 0.7 mM UAfor 48 hours,a total of 1977 proteins were idnetifed,including 314 differential proteins,of which there were 186 up-regulated proteins and128 down-regulated proteins.(3)Bioinformatics analysis showed that high uric acid caused the protein networks and pathways abnormal,particually in endoplasmic reticulum proteins.(4)Cell behavior analysis found that cell apoptosis ratio gradually increase with the increase of uric acid concentration,for examples,early and late apoptosis cells percentage were significantly increased(14.24%,5.41% respectively)in the 0.7 mM UA group.Meanwhile,the abnormal expressions of STK3,Cleaved CASP3,CANX and HSP B1 invovled in apoptosis were also found.These results were verified by Western blot assay.2.Abnormal expression of plasma proteins and its biological significance in gout patients.(1)The detection rates of hypertension,hyperlipidemia and metabolic syndrome in gout group were significantly higher than that in the control group.(2)Compared with the control group,the levels of SUA,DBP,TG,CHO and ALT in gout group were all high.But the ALT is in the normal range.(3)Compared with the gout group,the Scr,BUN,and HOMA-IR levels of gout with kidney damage group were all higher.The eGFR of gout with kidney damage group was lower than that of the simple gout group.(4)Compared plasma proteins of normal control group with gout patients,a total of 32 differentially expressed proteins were identified,of which10 proteins in gout were up-regulated and 22 proteins were down-regulated.These proteins are divided into five groups according to functions and their interactions netrworks showed that they are closely related.(5)Within plasma proteins of simple gout and gout with kidney damage patients,a total of 10 differential proteins were identified.The plasma complement C4 A,C4B and SERPINF1 of gout with kidney damage patients were upregulated.(6)Correlation analysis found that multiple correlation relationship between the abnormal plasma proteins and clinical biochemical indexes,which indicates their potential in biomarker.Conclusion: 1.High level of uric acid caused protein molecular network abnormal in HRMC cell,involving in multiple cell behaviors,of which apoptosis was obviously increased,and its mechanism may involve in the activation of the endoplasmic reticulum stress and corresponding the activation of the Caspase pathway.2.We developed the analytical approach based on proteomics technique for the identification of protein expression in plasma in gout patients,and identified the abnormal abundance of proteins,which is associated with clinical biochemical indicators and associated diseases;the immune inflammatory response proteins were significant,which indicating the relationship between these proteins and goutt.Additionally,after analyzed with biological significance,GSN and others were suggested to have the potential as biomarker of gout patients.