| ObjectivesThis study is conducted to explore the proteome of the synovium from RA patients with knee synovitis,compare it with healthy control and analyze the differentially expressed protein.Thus,this study is going to explore pathways and markers with statistical and biological significance,reveal the unique process of RA progression,and guide further exploration on the different etiologies of knee synovitis,to support the clinical practice of synovium biopsy.MethodsThis case-control study enrolls RA patients(Test group)and patients with meniscus injury(Control Group)undergone knee arthroscopic surgery in a single center(Peking Union Medical College Hospital,PUMCH).All patients were interviewed before surgery to collect baseline demographic,clinical and laboratory information.Consent form were obtained from all patients before the interview.Synovium samples were collected during the arthroscopic operation by a well-experienced arthroscopic surgeon.The synovium sample was frozen,grinded and lysed to extract protein with ultrasonication.Primary quality control was performed with electrophoresis.The extracted protein was then digested,filtered by ultrafiltration,separated by reversed-phase chromatography and then injected into Orbitrap Exploris 480 mass spectrometer(Thermo Fisher Scientific,Waltham,MA,USA)to perform LC-MS/MS analysis.The MS data were searched against the UniProt database by MaxQuant software to identify fragment ions,and match with unique peptides in the database(at least one match).Differentially expressed proteins were identified with statistical analysis.Bioinformatic functional analysis was performed on all differentially expressed proteins with GO and KEGG pathway analysis and the result was interpreted with biological significance.ResultsThis study enrolled 5 patients with definite RA diagnosis(according to 2009 ACR/EULAR classification criteria)and 5 patients with meniscus injury during the enrollment period.No significant difference was observed on the baseline demographic characteristics.However clinically,RA patients tend to have more joints involved and a higher ESR,which indicated multi-system inflammation process.A label-free comparative proteomic analysis was performed on the acquired synovium samples.After PCA analysis,one sample was excluded from each group due to intra-group differences.Totally 2,370 proteins were identified and quantified in both group with 284 differentially expressed protein,among which 93 proteins were up-expressed in the test group,and 185 proteins were down-expressed.Functional clustering analysis on the differentially expressed proteins indicated frequent involvement in immunological process,like lymphocyte apoptotic process,monocyte activation,complement and coagulation system.Specifically,two proteins in the complement cascade which can be citrullinated,A1AT and a 2AP,and C8,C9 in the complement cascade were found to be up-expressed in RA synovium,indicating the involvement of complement system in the pathophysiological process of RA synovitis.Conclusion1.The discarded synovium during arthroscopic surgery on knee meniscus injury can be used as control for study on synovitis with high intra-group consistency.2.Proteins related to immunological process were generally up-expressed in RA synovium compared to control group,especially those related to complement system,which includes A1AT,a 2AP,C8,C9.3.The differentially expressed protein in this study indicates“the release of citrullinated protein,production of ACPA and activation of complement system”process may play a vital role in RA progress4.C8,C9 and citrullinated protein like A1AT,a 2AP may help identify RA related synovitis... |
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