Mechanism of Hip Arthropathy in Ankylosing Spondylitis: Abnormal Myeloperoxidase


 Ankylosing spondylitis (AS) is a chronic inflammation of the joints and spine. Its pathogenesis has not been elucidated, especially involving hip joint disease. The purpose of this study was to analyze the proteome of diseased hip in AS and to identify key protein biomarkers. We used label-free quantification combined with liquid chromatography mass spectrometry (LC–MS/MS) to screen for differentially expressed proteins in hip ligament samples between AS and No-AS groups. Key protein was screened by Bioinformatics methods and verified by in vitro experiments. There were 3755 identified proteins, of which 92.916% were quantified. 193 DEPs (49 up-regulated proteins and 144 down-regulated proteins) were identified according to P < 0.01 and Log|FC| > 1. DEPs were mainly involved in cell compartment, including the vacuolar lumen, azurophil granule,primary lysosome, etc. The main KEGG pathway included Phagosome, Glycerophospholipid metabolism, Lysine degradation, Pentose phosphate pathway. Myeloperoxidase (MPO) was identified as a key protein participated in Phagosome pathway, which induces hip lesions in ankylosing spondylitis. Further experiments confirmed that MPO promoted the inflammatory response of fibroblasts in AS-affected hip joint.


Introduction
Ankylosing spondylitis (AS) is a chronic systemic in ammatory disease with an incidence of 0.1% ~ 1.4% [1]. This disease mainly occurs in young males and is mainly characterized by spinal arthritis and sacroiliac arthritis, and associated with a number of other features including peripheral arthritis, psoriasis, anterior uveitis, in ammatory bowel disease, etc [1][2][3][4][5]. Main pathological structures of arthritis are cartilage junctions and attachment points [6,7]. Due to the slow progression, it usually takes 5 to 10 years after the onset of symptoms to be diagnosed. In the later stages, loss of activity of spinal and sacroiliac joint results in poor quality of life, increased nancial burden, and more serious mental illness [6,8,9].
There are no effective radical drugs at present. Previous researches focused on the overall pathogenesis of AS have involved multiple elds: genetics, intestinal diseases, intracellular environmental barriers, hormones, etc [1,2,4,10,11]. Multiple Genes abnormally express proteins and involve different pathways in different diseased tissues, which may be the main factor leading to AS. However, it is complex. The overall pathogenesis of AS has not been fully elucidated. In particular, there are few reports on the mechanism of hip arthropathy in patients with AS.
Proteins are the end products of gene expression and can better and directly illuminate the processes behind the manifestation of diseases. Comparative proteomics methods are widely used to analyze the relative amounts of proteins in two or more biological samples to screen for biomarker proteins for judgment and prognosis [12][13][14]. Label-free quantitative (LFQ) analysis is one of proteomics methods [12,13]. Compared to label quantitative analysis, LFQ can avoid the drawbacks of labeling quanti cation techniques: additional sample processing, sample complexity caused by incomplete labeling, di culty in detecting low abundance peptides, limited number of labeled samples, etc [13,15].
In this study, LFQ combined with bioinformatics technology was used to screen potential biomarkers. It was found that the protein changes in hip joint during the development of AS to speculate the signal transduction pathway. This provides a preliminary insight into the molecular characteristics of hip arthropathy in patients with AS, which is conducive to a further comprehensive understanding of the biological characteristics of AS and to provide new molecular targets for personalized treatment.

Patients and Samples Collection
From December 2018 to August 2019, a total of 12 ligament samples were collected from patients undergoing hip replacement in the First A liated Hospital of Guangxi Medical University. The study population included the AS group (n = 6) and No-AS group (n = 6). The AS group included patients with AS combined with hip arthropathy. The No-AS group included 6 patients with Aseptic femoral head necrosis (AFHN).
Inclusion criteria: (1) patients with AS combined with hip arthropathy or patients with AFHN; (2) hip replacement. Exclusion criteria: (1) combined with other connective tissue diseases such as rheumatoid, systemic lupus erythematosus; (2) combined with tumor, trauma and other diseases. All tissues collected during the operation were cleaned with phosphate buffered solution (PBS, Solarbio, Beijing, China) and put into the labeled cryopreservation tubes respectively. Cryopreservation tubes were stored in a -80 ℃ refrigerator until they were used.
General data (age, gender, Body mass index (BMI), etc.) and preoperative examination data (erythrocyte sedimentation rate (ESR), blood routine, human leukocyte antigen (HLA)-B27, etc) were collected. The diagnosis of AS was based on the revised New York standard in 1984 [16]. The diagnosis of AFHN was based on a pathological examination of femoral head [17]. We performed label free Protein analysis with the assistance of KangChen Bio-tech (Shanghai, China). All procedures were performed in compliance with the resolution of Helsinki and approved by the Local Ethics Committee. All participants received written informed consent. According to the instructions of the BCA (Bicin-choninic Acid) Protein Assay Kit (Kangchen Bio-tech, Shanghai, China), reagent A and reagent B were mixed at A 50:1 ratio and added to A 96-well plate ( 160 µL/per well, 5 wells for calibration curve and 1 well for blank). 20 µL sample (dilute 5~10 times) or calibration standard protein (5 different concentration levels) was respectively added into the well. The plate were shook and incubated at 37 °C for 30min, and were read with 562 nm wave length. Protein concentration of each sample was calculated the according to the calibration curve.

Acetone Precipitation
For each sample, 100 µg protein was took into ep tube and diluted to 1 mg/ml by RIPA buffer. 4~6 fold volumes of pre-chilled acetone (-20 °C, Sangon Biotech, Shanghai, China) was added into the ep tube (alkylated protein), which was shook on ice for 30min or incubated at -20 °C overnight. After centrifugation (speed 10,000g, 4 °C), the supernatant was carefully discarded without disturbing the pellet. The sample was wash twice with 200 µL 80% chilled acetone. which was incubated and mixed at 55 °C for 10 min. Ten mmol IAA (iodoacetamide) (Sigma-Aldrich, St. Louis, MO, USA) was added after samples cooling down to room temperature(RT).The ep tube incubated in the dark for 15min. Trypsin(sequence grade)(Promega, Madison, WI, USA) was re-suspended with resuspension buffer to 0.5 µg / µL and incubated at RT for 5 min. Trypsin solution(protein : trypsin = 50 : 1) was added into the ep tube. They were mixed well and spun down, incubated at 37 °C with thermomixer for about 8hr or overnight. Louis, MO, USA) column was equilibrated with added 500 µL ACN. ACN was washed out with 500 µL 0.1% FA 2 times. The peptide solution was added into the C18 column, After centrifugation at low speed, and liquid(A) was collected. The steps are repeated again. Peptide was eluted with 400 µL 70% ACN, and liquid(A) was collected. The desalting step (Equilibrate… to Elute…) was repeated by liquid(A) once again. Two liquid were merged and dried by vacuum under 4 °C or RT. Buffer A was added to redissolve the polypeptide to 1 buffer g/buffer L for lc-ms /MS detection or -80℃ storage.

Separation via Nano-UPLC
For each sample, 2 µg peptide were separated and detected with a nano-UPLC (EASY-nLC1200, Thermo Scienti c, MA, USA) coupled to Q-Exactive mass spectrometry (Thermo Scienti c, MA, USA). Analysis was performed using a reversed-phase column (100 µm, ID × 15 cm, Reprosil-Pur 120 C18-AQ, 1.9µm, Dr. Math). The mobile phases solution include phase A solution (0.1 % FA, 2 % ACN) and phase B solution(80 % ACN, 0.1 % FA ). The chromatographic column is balanced by 100% phase A solution. Through an automatic sampler, the samples were directly delivered to the chromatographic column to be separated at a ow rate of 300 nL/min and a gradient of 120 min. Phase B solution is used in sequence: 8 to 30 % for 92 min, 30 to 40 % for 20 min, 40 to 100 % for 2 min, 100 % for 2 min, 100 to 2 % for 2 min and 2 % for 2 min.

LC-MS/MS
Data dependent acquisition was performed in pro le and positive mode with Orbitrap analyzer at a resolution of 70,000 (@200 m/z) and m/z range of 350-1600 for MS1; For MS2, the resolution was set to 17,500 with a dynamic rst mass. The automatic gain control (AGC) target for MS1 was set to 3.0 E*6 with max IT 50ms, and 5.0 E*4 for MS2 with max IT 100ms. The top 20 most intense ions were fragmented by HCD with normalized collision energy (NCE) of 27 %, and isolation window of 2 m/z. The dynamic exclusion time window was 30 s.

MaxQuant Database Search
Raw MS les were processed with MaxQuant (Version 1.5.6.0). The protein sequence database (Uniprot_organism_2016_09) was downloaded from UNIPROT. This database and its reverse decoy were then searched against by MaxQuant software. The quanti cation type was LFQ with match between run and intensity-based absolute quanti cation (iBAQ); Trypsin was set as speci c enzyme with up to 3 miss cleavage; Oxidation [M] and Acetyl [protein N-term] were considered as variable modi cation (max number of modi cations per peptide is 3), Carbamidomethyl [C] was set as xed modi cation; Both peptide and protein of false discovery rate (FDR) should be less than 0.01. Only unique & razor peptides were used for quanti cation. All the other parameters were reserved as default.

Differentially Expressed Proteins (DEPs)
Differentially expressed proteins (DEPs) were de ned as: P < 0.01, the ratio of | AS group / No-AS group | > 2 or |Log Fold change ( FC ) | 1, unique peptide > = 2 protein. Relative protein expression values between 2 groups were compared. There were 6 biological replications in this experiment, which were tested by the t-test. False Discovery Rate (FDR) <0.05.

GO and KEGG Enrichment Analysis
DEPs were analyzed for GO (Gene Ontology) and KEGG(Kyoto Encyclopedia of genes and genomes) enrichment by the plugin ClueGO [18] in the Cytoscape software(version: 3.6.1) [19]. There are three categories of GO annotation: Biological process (BP), Cell compartment (CC) and Molecular function (MF) . The standard setting with statistically signi cant difference was P < 0.05 and Kappa score was 0.4. Bonferroni corrected P < 0.05 is considered to be valid.

PPI Network Construction
We integrated the DEPs into PPI network by String [20](version 11.0) to evaluate the interaction between DEPs. A composite score > 0.4 was considered to be a statistically signi cant interaction. Analysis data of the PPI network was loaded into Cytoscape software for visual adjustment.

Identi cation of Key Protein
Top 30 hub proteins were screened by the plug-in CytoHubba [21] in Cytoscape software from PPI networks. Key protein was selected from the correlations of hub proteins and KEGG pathways.

Cell Culture and Transfection
Fibroblasts from the two groups were separated and cultured. The broblasts were cultured in speci c medium DMEM (10% FBS, 100μg/ mL streptomycin and 100 IU/mL penicillin) at 37℃ in a 5% CO2 incubator. Cells in the AS group were transfected with Lipofectamine 3000 (L3000015, Thermo sher Scienti c, MA, USA) according to the manufacturer's instructions. The siRNA-MPO treated cells were siRNA-MPO group. As a negative control, cells treated with The siRNA-NC group were siRNA-NC group. The transfection e ciency was evaluated by Western blotting and Quantitative Real-time PCR (QPCR).

2.5Western Blotting Analysis
Western blot analysis was performed between groups. Total proteins were extracted from cell samples. BCA protein concentration determination kit (Servicebio, Wuhan, China) was used to determine the total protein concentration. The proteins are separated by 10% SDS-PAGE (Servicebio, Wuhan, China) and transferred to cellulose nitrate membrane. The membrane was sealed in 5% degreased dry milk in 37°C TBS buffer (Servicebio, Wuhan, China) for 1 hour and incubated overnight with primary antibodies (MPO antibodies, Abcam, Europe) at 4°C. Then, the membranes were washed 3 times in TBST and incubated with secondary antibodies at room temperature for 30 minutes. A scanner (EPSON V300, Japan) was used to sweep membrane and upload band data. Adobe PhotoShop (Adobe, the United States) software was used to process the color, and Alpha Innotech (Alpha Innotech, the United States) software was used to analyze the gray values of band. Relative expression level of protein = (gray value of the target protein band)/(gray value of the GAPHD protein band).

Quantitative Real-time PCR QPCR
According to the manufacturer's instructions, Tripure reagent (Roche, Switzerland) was applied to extract total RNA from cells. The cDNA was synthesized according to the reaction system. Next, quantitative realtime PCR QPCR was performed using a 2×UltraSYBR Mixture. The qPCR condition was set as follows:95°C, 10 min, 95°C, 15 sec, 60°C, 20 sec, 72°C, 25 sec, 40 cycles, followed by a 5-minute nal extension at 72 °C. The 2-ΔΔCt method (Ct of target genes minus the Ct of GAPDH) [22] was used to calculate the relative expression levels of mRNA were calculated. The primer sequence of MPO is as follows: TCGGTACCCAGTTCAGGAAG (forward) and CCAGGTTCAATGCAGGAAGT (reverse).

Enzyme-linked Immunosorbent Assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA) was used to detect in ammatory markers in cells before and after transfection. The concentrations of interleukin 6 (IL-6), interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) in the cell culture medium were determined by ELISA sandwich method according to the instructions of ELISA assay kit (MLBIO, Shanghai, China). The collected cell culture medium was centrifuged at 4℃ for 10 min at 1000 RPM to obtain the supernatant. The absorbance value (OD value) of each culture medium was determined at 450nm using a microplate analyzer (Mlbio, Shanghai, China).
Finally, the concentrations of IL-6, IL8 and TNF-α in each sample were obtained through the standard curve equation.

Statistical Analysis
The data were expressed as the mean ± sd, and differences between 2 groups were analyzed with Student's t-test. P < 0.05 was considered statistically signi cant. Bonferroni or Benjamini-Hochberg adjusted FDR, which was <0.05. Statistical analysis was performed using the Statistical Program for Social Sciences (SPSS) software 25.0 (IBM, USA).

Clinical Data of Patients
In this study, ligament tissue from 12 patients undergoing hip arthroplasty was collected and divided into AS (n=6) and NO-AS groups (n=6)(Figue 1 and Table 1). Except for HLA-B27, there were statistically signi cant differences in age and gender between 2 groups. The characteristics of AS patients involving hip arthropathy were younger and HLA-B27 positive.

Differentially Expressed Proteins
A total of 3755 proteins were identi ed, among which 3489 proteins had quantitative information. There were 193 DEPs (49 up-regulated proteins and 14 down-regulated proteins) between AS and No-AS groups (P.value 0.05) ( Table 2). Boths volcano map and heat map shown signi cant differences in proteins between 2 groups (Figure 2 and Figure 3).

GO Functional Enrichment Analysis
DEPs were subjected to GO enrichment analysis, and items were screened according to P.adj.value (P.adj.value<0.05). It was found that there were a large number of DEPs enriched in BP, CC and MF. Among them, the DEPs enriched in BP and CC are slightly more than those enriched in MF. In BP, most proteins were mainly involved in golgi vesicle transport, endosomal transport, endoplasmic reticulum to Golgi vesicle-mediated transport, etc. In terms of CC, most proteins were involved in vacuolar lumen, azurophil granule,primary lysosome, etc. In MF, most proteins were involved in vitamin binding, heparin binding, tetrapyrrole binding, etc. The enrichment results of DEPS showed common characteristics between BP and CC, including procollagen-lysine 5-dioxygenase activity, peptidyl-lysine 5-dioxygenase activity, 2-oxoglutarate-dependent dioxygenase activity. (Figure 4).

KEGG Pathway Enrichment Analysis
KEGG pathway enrichment analysis of DEPs was performed by the Gluego. 4 pathways were identi ed, including Phagosome, Glycerophospholipid metabolism, Lysine degradation, Pentose phosphate pathway (P. adj. value<0.05). Nine proteins were involved in the Phagosome. Three proteins were involved in the Pentose phosphate pathway. Five proteins were involved in the Glycerophospholipid metabolism. Four proteins were involved in the Lysine degradation. The P value of the Phagosome pathway is the smallest and the number of DEPs involved in it is the largest. The number of DEPs involved in the Pentose phosphate pathway is the lowest. These DEPs are involved in the abnormal operation of the Phagosome, which may be the most important pathogenetic pathway. And key proteins may play important roles in this pathway. (Figure 5).

PPI NetworkC construction
String database was used to evaluate the interaction between 2 groups of DEPs, and PPI network was constructed. Data was entered into the Cytoscape for visual adjustment. DEPs visual network has 188 nodes and 332 edges ( Figure 6). As can be seen from the gure, the number of down-regulated DEPs was higher than that of up-regulated DEPs. However, the absolute value of FC in up-regulated DEPs seemed to be generally larger than that in down-regulated DEPs. DEPs, whether up-regulated or down-regulated, are closely linked to each other and together constitute a complex protein-protein interaction network.

Identi cation of Key Proteins
Top 30 hub proteins were obtained respectively by the Degree and Closeness method in CytoHubba ( Figure 7A and 7B). Top 10 hub proteins in 2 methods and proteins in KEGG pathways were screened (Table 3). Finally,1 hub protein was identi ed as the key protein by intersection of KEGG pathways and 2 method, which was Myeloperoxidase (MPO) (Figure 8). The expression of MPO protein was relatively high in AS group. Its related KEGG pathway is Phagosome pathway (Figure 9). The up-regulation of MPO protein may be involved in the pathogenesis of AS-affected hip joint through the Phagosome pathway.

Cell Culture and Veri cation of In ammation
The results of primary broblast culture were shown in Figure 9A and 9B. Western blot analysis shown that the expression of MPO in AS group was higher than that in NO-AS group (P < 0.05). (Figures 9C).
After transfection, the mRNA and protein expressions of MPO in siRNA-MPO group were lower than those in siRNA-NC group (P < 0.05). (Figures 9D). This suggested that siRNA-MPO treatment of AS broblasts resulted in 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 NO-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). (Figures 9E).
This suggested that the high expression of MPO in AS cells may promote the high expression of in ammatory factors such AS IL-6, IL-8 and TNF-α. However, after siRNA-MPO treatment, the expression of MPO decreased, and resulted in the down-regulation of IL-6, IL-8 and TNF-α.
Hip arthropathy is very common in patients with AS. AS with chronic sacroiliac arthritis may lead to severe hip joint lesions, including malformation of the femur head [23,24]. This particular hip lesion prevented many young adults from walking normally and requires hip arthroplasty to improve quality of life [25,26,27]. The molecular mechanism of hip arthropathy in AS has been rarely reported in the past. Therefore, we analyzed the hip ligament tissue between the AS group and the NO-AS group by combining proteomics with bioinformatics, and obtained 193 DEPs. GO functional analysis showed that there were abundant DEPs in BP, CC and MF. Among them, the number of DEPs enriched in BP and CC was slightly more than that enriched in MF. It was found that MF and BP share common characteristics, including: procollagen-lysine 5-dioxygenase activity, peptidyl-lysine 5-dioxygenase activity and 2-oxoglutaratedependent dioxygenase activity. MPO was identi ed as a key protein involved in the phagosome pathway.
In terms of sample collection, we tried our best to control the basic data of the two groups of study population to achieve consistency. However, patients with AS who undergo hip replacement tend to be younger men. This clinical phenomenon is unique and leads to a statistically signi cant difference in age comparison between AS and non-AS groups. Therefore, we could not control for the difference in age between the two groups, which may be one of the limitations of the study. It was found that there was a high degree of consistency between the two groups of ligament samples, indicating that the collection and preservation of ligament samples was successful. This provides a preliminary guarantee for the accuracy of subsequent experiments. Although DEPs slightly tended to enrich in BP and CC, the difference was not obvious. In PPI network, two different algorithms are calculated by Degree method and Closay method, which may be conducive to improving the accuracy of key proteins. Finally, MPO related to Phagosome pathway was screened out from DEPs related to KEGG pathway. The up-regulated expression of MPO in the Phagosome pathway may be a mechanism of AS hip lesions.
MPO is a member of the heme -containing peroxidase subfamily. It is usually expressed in large amounts in various immune cells such as neutrophils, lymphocytes, monocytes, macrophages and so on [28,29,30,31,32]. MPO is usually stored in eosinophilic particles inside the cell. When pathogenic bacteria invade the body, these particles will enter the extracellular space through degranulation or exocrine [28,29]. Neutrophils are a line of defense against pathogens. When the pathogen enters the tissue, the neutrophils engulf the pathogen and place it on the phagosome. Most pathogens are killed and digested in the phagosomes [33,34]. In neutrophils, eosinophilic granules containing MPO fuse with the phagosome. When the endoplasmic membrane is ruptured due to pathogen invasion, substances in particles including MPO will be released [35,36]. Imequilibrium of cellular degranulation mechanisms is a common feature of many in ammatory diseases, such as acute lung injury, rheumatoid arthritis, psoriasis, etc. [37,38,39]. However, the mechanism controlling cell degranulation is not clear. Some studies suggest that cellular degranulation may depend on the activation of intracellular signaling pathways, including the Src family of tyrosine kinases, β-arrestin, et al. [40,41].
The neutrophil degranulation results in an increase in MPO protein concentration. MPO catalyzes various substrates and disproportionates superoxides. Under the condition of su cient chlorine, H2O2 is catalyzed to effectively convert to HOCl [42,43]. Klebanoff SJ[44] found that the antimicrobial system formed in the phagosomes included MPO, H2O2 and halides (especially chlorides). The initial product of MPO-H2O2-Cl system is HOCl, followed by the formation of chlorine, chloramine, hydroxyl radical, singlet oxygen and ozone. These toxic substances can be released outside the cell, where they may attack normal tissue and trigger disease. Green JN et al. [45] believe that HOCL can react with granulosin and produce 3-chlorotyrosine, protein carbonyl group and a large amount of chloramines. HOCL may also kill ingested bacteria through indirect mechanisms involving protein oxidation and monochloramine formation. Odobasic D et al. [33] pointed out that substances generated by the reaction include HOCl, hypothiocyanate, tyrosine free radicals and active nitrogen intermediates, etc., which can have a profound impact on cell functions by modifying proteins, lipids and DNA. Mayadas TN et al. [46] found that neutrophils play an important role as effector cells in many in ammatory and autoimmune diseases through phagocytosis, release of reactive oxygen species, degranulation, extracellular trap, proin ammatory cytokines and protease mediation. These diseases include atherosclerosis, cardiovascular disease, in ammatory diseases of the lungs and kidneys, and rheumatoid arthritis. Van der Veen BS et al.
[47] also pointed out that excessive production of MPO-derived oxidants is associated with tissue damage in many diseases, especially those characterized by acute or chronic in ammation. MPO may play a role beyond its oxidative properties, which may be independent of its catalytic activity and affect various processes of cellular signaling and cell-cell interactions, thus regulating the in ammatory response. Pitanga TN et al. [48] believe that the corpuscule-associated MPO-H2O2-Cl system may be involved in extensive endothelial cell injury under the condition of neutrophil activation. Panagopoulos V et al. [49] found that MPO inhibits the differentiation of osteoclasts in in ammatory sites, participates in the formation of extracellular matrix, angiogenesis, and bone mineralization, and plays an important role in bone integrity. Past studies have shown that MPO is an active disease biomarker associated with a variety of diseases, such as cardiovascular disease, cancer, kidney disease, lung injury, rheumatoid arthritis, and multiple sclerosis [28,29,50,51,52]. MPO is an important participant in in ammation and immune response in the body. It has a variety of functions in tissues, such as participating in sterile in ammatory response, maintaining normal immune response, remodeling local microvessels, and enhancing the activity of osteoblasts and osteoclasts. Abnormal expression of MPO can lead to a range of diseases. The phagocytes are generally considered to be important structures in the processing of antigens by various immune cells. The recognition and processing of exogenous antigens by neutrophils and macrophages is an important part of the non-speci c immune response [53]. Normal patients have no exogenous antigens in the hip joint. In AS-affected hip patients, overexpression of MPO may result in abnormal cell activity, including immune cells. These abnormalities activate the phagosome pathway and treat the tissue as an antigen, inducing aseptic in ammatory response and autoimmune response. Figure 11 shows the role of MPO in the phagosome pathway. The phagosomes contain MPO, which catalyzes H2O2 and produces HClO. They participate in antigen processing and produce a series of cellular effects. In our study, MPO overexpression may be one of the factors leading to aseptic in ammatory and immune responses in the hip joint.
AS is considered as a chronic in ammatory autoimmune disease, which can cause spinal stiffness and brosis, AS well AS various degrees of damage to muscles, bones, lungs, eyes and other parts. Ligament attachment site is one of the main pathological sites of AS. And broblasts are the main components of ligaments. Abnormal expression of MPO may contribute to changes in the physiological activity of broblasts. On the basis of cell culture, we explored whether up-regulated MPO led to the in ammatory response of AS broblasts by interfering with the overexpression of MPO. As a result, we further con rmed that MPO was up-regulated in AS hip broblasts. By inhibiting the overexpression of MPO, the expression of in ammatory factors such AS IL-6, IL-8 and TNF-α in AS hip broblasts could be downregulated. Therefore, overexpression of MPO may induce up-regulation of in ammatory cytokines such as IL-6, IL-8 and TNF-α in broblasts. This can be one of the factors that contribute to the autoin ammatory response of the AS-diseased hip joint.
The mechanism of MPO synthesis has not been fully elucidated [54]. The gene encoding human MPO may contain 12 exons and 11 introns, located on chromosome 17q22 [55]. Lin Km et al.
[56] indicated that there are three different MPO promoters in the 5 ' anking region of human MPO gene, and they may regulate the transcription of MPO. Zhao WG et al. [57] believed that only one of the three identi ed promoter regions of human MPO was active. But all three played a role in the expression of MPO in mice.
Some studies indicate that MPO biosynthesis begins with PreMPO, which is a primary translated product produced in the endoplasmic reticulum. The PreMPO is processed to form apoproMPO. ApoPrompo binds returnably with the molecular chaperone and obtains heme to become the enzymatic active ProMPO.
After leaving the endoplasmic reticulum, proMPO enters the secretion pathway, most of which is formed into mature MPO through proteolysis and dimerization, and a small part is secreted out of the cell in the form of single proMPO. [30,58]. The synthesis and processing of MPO is very complex. Many factors are interrelated and play a controlling role in the expression of MPO in an organization. Nauseef WM [30] suggestted that there is a need to explore the possibility that nutritional factors produced in a speci c in ammatory environment may promote the revival of MPO gene expression in cells that do not normally produce MPO. Nagra RM et al. [59] believed that macrophages do not actively produce MPO, but MPO is present in microglia/macrophages in and around the lesion of multiple sclerosis. MPO is closely associated with in ammatory response and may promote the production of in ammatory cytokines through multiple pathways [28,29,31]. In the experiment, the overexpression of MPO and in ammatory cytokines (IL-6, IL-8, TNF-α) in broblasts may indicate that broblasts are present in the posture of in ammatory response.