Overexpression Of Myeloperoxidase Promotes Hip Lesions In Ankylosing Spondylitis

Part Ⅰ Dfferentially expressed proteins in the diseased hip of AS were screened by Label-Free Quantification(LFQ)Objective: to establish the biological data of hip joint with AS by screening the differentially expressed proteins between AS group and non-AS group.A total of 12 ligament tissue specimens were collected from patients undergoing hip arthroplasty,including the AS group(n = 6)and the non-AS group(n = 6).Ligament tissue specimens were cleaved and proteins were extracted.After label-free Quantification(LFQ),the relative expression levels of the two groups of proteins were compared and analyzed.The equipment and technologies involved include: Nano-UPLC,LC-MS/MS mass spectrometry,Max Quant database,etc.Differentially expressed proteins in the standards are set: P < 0.01,the Log2 |Fold change(FC)| > 1,unique peptide> = 2.Material and Methods: after LFQ,there was no significant difference in the expression of most proteins between the two groups,and the consistency between the samples was very high.A total of 36,639 polypeptides,including3755 proteins,were identified.Among them,there were 3,489 quantifiable proteins.The number of differentially expressed proteins between AS group and non-AS group was 193,including 49 up-regulated proteins and 144down-regulated proteins.Conclusion: There are a certain number of differentially expressed proteins between AS group and non-AS group.These differentially expressed proteins are involved in the pathogenesis of AS hip joint through some pathways.Screening the differentially expressed proteins of AS hip joint by LFQ technique provides a valuable data basis for subsequent studies on the pathological mechanism of AS hip joint.Part Ⅱ Key proteins and related pathways were analyzed by bioinformatics Objective: Combined with the differentially expressed proteins between AS group and non-AS group,bioinformatics method was used to analyze the biological information of AS-affected hip joint,and to search for the key proteins and related pathways that may lead to AS-affected hip joint.Materials and Methods: Go function(biological process,molecular function and cell component)enrichment analysis,KEGG pathway analysis and protein-protein interaction analysis of differentially expressed proteins were performed by Cytoscape software.Through Cytohubba plug-in,the Degree method and Closay method are adopted to screen out common hub proteins respectively.Key proteins and related pathways were screened from the related networks between common hub proteins and KEGG pathways.Results: GO function enrichment analysis showed that in terms of biological processes,most of the differentially expressed proteins were involved in Golgi vesicle transport,vesicle-mediated transport to the plasma membrane,endoplasmic reticulum to Golgi vesicle-mediated transport,etc.In terms of cell composition,most of the differentially expressed proteins are involved in vacuolar lumen,primary lysosome,azurophil granule,etc.In terms of molecular functions,most of the differently-expressed proteins were involved in procollagen-lysine 5-dioxygenase activity,peptidyl-lysine 5-dioxygenase activity,2-oxoglutarate-dependent dioxygenase activity,etc.Four KEGG pathways were identified,including Phagosome pathway,Glycerophospholipid metabolism pathway,Lysine degradation pathway and Pentose phosphate pathway.The first 10 HUB proteins of the two methods intersect with each other to obtain a common HUB protein.The network of common HUB protein and KEGG pathway was established.An up-regulated key protein,myeloperoxidase(MPO),was obtained.Its related KEGG pathway is Phagosome.Conclusion: Overexpressed MPO participates in the pathogenesis of AS hip joint through the Phagosome pathway.Part Ⅲ Up-regulated expression of MPO contributes to the inflammatory response of the AS hipObjective: AS is a chronic inflammatory autoimmune disease associated with severe hip joint disease.Through the experiment of interfering with the expression of MPO protein,to explore whether the overexpression of MPO contributes to the inflammatory response of AS-affected hip joint.Materials and methods: the human ligament tissue samples were collected during surgery and the fibroblasts from the tissue were isolated and cultured.Vimentin expression was detected by flow cytometry to confirm the cells.Before transfection,the expression of MPO protein in fibroblasts between the AS group and the non-AS group was compared.After the fibroblasts were transfected with siRNA-MPO,the expression of MPO and the concentrations of interleukin 6(IL-6),interleukin 8(IL-8)and tumor necrosis factor α(TNF-α)in cell culture medium were compared between siRNA-MPO group and siRNA-NC group.The experimental methods included Western Blot,Quantitative real-time PCR(q PCR)and enzyme-linked immunosorbent assay(ELISA).Results: Vimentin expression was positive in both fibroblasts.Western blot analysis showed that the expression of MPO protein in AS group was higher than that in non-AS group(P < 0.05).After transfection,the m RNA and protein expressions of MPO in siRNA-MPO group were lower than those in siRNA-NC group(P < 0.05).Si RNA-MPO treatment of AS fibroblasts may contribute to the down-regulation of MPO expression.ELISA analysis showed that the expression levels of IL-6,IL-8 and TNF-α in AS group were higher than those in non-AS group(P < 0.05).The expression levels of IL-6,IL-8 and TNF-α in siRNA-MPO group were lower than those in siRNA-NC group(P < 0.05).Therefore,the high expression of MPO may promote the high expression of inflammatory cytokines such as IL-6,IL-8 and TNF-α.As fibroblasts treated with siRNA-MPO decreased the expression of MPO,and finally contributed to the down-regulation of the expression of inflammatory factors such AS IL-6,IL-8 and TNF-α.Conclusion: The overexpressed MPO may contribute to the up-regulation of IL-6,IL-8 and TNF-α in AS hip fibroblasts,which may be a factor of the autoinflammation of the AS-diseased hip joint.