Proteomic Research Of Gender Differences In Knee Osteoarthritis

Background and objective: Knee osteoarthritis(KOA)is a chronic osteoarthrosis involving in degeneration of the cartilage of the knee joint and hyperosteogeny.It is a common and frequently-occurring disease in motor systems.The disease has a high incidence and disability worldwide,which seriously affects the human physical and mental health.The epidemiological surveys of most people found that the prevalence of KOA in female was higher than that in male,and the prevalence of postmenopausal female was significantly higher than that of male in the same age,meanwhile the symptoms of female are relatively severe than that of male.The aim of this study is to analyze the differential expression proteins between male and female KOA serum samples via iTRAQ labeling combination with Nano-HPLC-MS/MS,and establish protein profiles of sex differences in KOA,which will help to explore the pathogenes is and progression of the disease,and to provide basis for the further research of clinical diagnosis and treatment of KOA.Methods: A total of 72 serum samples icluding 31 cases of male and 41 cases of female KOA patients were collected from the Ninth Affiliated Hospital of Guangxi Medical Univers ity from Jan 2017 to April 2017.And 30 age-matched of male and female healthy controls were also enrolled to the study,respectively.Samples were reduced,alkylated,and trypsin digested,and then labeled with iTRAQ followed by analyzed via Nano-HPLC-MS/MS.After proteins identification and quantification of each group,the differentially expressed proteins were further analyzed by Gene Ontology(GO),KEGG pathway and Ingenuity Pathway Analysis(IPA).Simultaneously,part of the differential expression proteins were selected to validate the proteomic results by western blot and ELISA.Results: A total of 571 proteins were identified in KOA patients and healthy controls.The criterion for differentially expressed proteins was the fold change of the proteins > 1.2(up-regulated)or < 0.8(down-regulated),with a P-value < 0.05.There were 60 proteins differentially expressed in male KOA group,with 50 up-regulated and 10 down-regulated when compared to the male healthy controls,while there were 179 proteins differentially expressed in female KOA group,with 110 up-regulated and 69 down-regulated when compared with the female controls.GO analys is suggested that the differentially expressed proteins in the male group were mainly involved in biological processes such as immune response,innate immune response and proteolysis,while the differential proteins in the female group were mainly involved in proteolysis,receptor-mediated endocytosis and complement activation.In terms of cellular components,the differentially expressed proteins in the male and female groups were mainly located in extracellular exosome,extracellular region and extracellular space.In the molecular function category,it was found that the differential proteins in the male and female groups were mostly related to serine-type endopeptidase activity,calcium ion binding,while the female group mainly had activity in protein binding and serine-type endopeptidase.According to the KEGG pathway analys is,the differential espression proteins in the male and female groups were mainly involved in the pathways such as systemic lupus erythematosus,complement and coagulation cascades,and phagosome.In addition,the differentially expressed proteins of the female group were also involved in focal adhesion,PPAR signaling pathway,vitamin digestion and absorption and the other signaling pathways.The results of western blot showed that C9,C4 B and SAA1 were increased in the male KOA group when compared to their control group;C4B and HSP90? were increased in the female KOA group when compared to the female control group,while ITIH4 was reduced.The results of the western blot was consistent with iTRAQ,demonstrating that the reliability of iTRAQ results.Furthermore,the results of ELISA showed that the serum ficolin-2 levels were much lower in KOA patients than that in control groups,while the values of DEF1 were significantly elevalted in KOA patients compared to control groups.Conclusion: 1.The differentially expressed proteins in male and female KOA groups screening by proteomic techniques could be used as potential markers of KOA for further study,which would help to reveal the pathogenes is and the progression of KOA in male and female,and to provide the theoretical basis and new thoughts for the diagnosis and treatment of KOA.2.Ficolin-2 and DEF1 are potential diagnostic biomarkers for male and female KOA.