RA is a chronic autoimmune disease that is characterized by inflammation of the joints and the subsequent destruction of cartilage and erosion of the bone . The exact causes of RA are unclear, but some factors are believed to increase the risk of developing the disease, including genetic, environmental, hormonal and psychological factors . Currently, the main drugs for RA treatment are anti-inflammatory drugs and immunosuppressants. Imrecoxib, a COX-2 enzyme inhibitor, has been proven for osteoarthritis treatment . Immunosuppressants are used to treat RA by suppressing the immune response, including MTX and LEF. TGP is reported to show anti-inflammatory and immunomodulatory effects [4, 10]. In our study, proteomics analysis was used to explore DEPs after three months of combined treatment with these four drugs to discover the specific mechanism of the combination drug treatment for RA.
In our study, the results showed that 7 proteins were differentially expressed after combination drug treatment for 3 months through label-free quantitative proteomics. Six proteins (IGKV3-20, TTR, LRG1, SERPINA3, F10 and NMNAT3) were downregulated, and one protein (HBD) was upregulated. Four of the DEPs were directly or indirectly involved in the development of RA. These proteins may be the target of drug combination therapy.
GO enrichment analysis in our present study indicates that the DEPs identified herein are related to transporter activity. Transthyretin (TTR), a transporter of thyroxine, was downregulated after combination drug therapy. TTR is a highly conserved homotetrameric protein and is enriched in human plasma and cerebrospinal fluid [16, 17]. Previous proteomics studies have reported that TTR expression in RA patients was higher than that in healthy people, indicating that TTR might be a potential marker for RA progression [18, 19]. Reactive oxygen species (ROS), as intracellular signaling molecules, are associated with various inflammatory and chronic joint diseases, including RA [20, 21]. In myeloid cells, TTR treatment increases ROS production, suggesting that TTR may promote the development of RA by affecting the production of ROS . Therefore, combination therapy may improve RA by decreasing TTR.
Leucine-rich α-2-glycoprotein 1 (LRG1), a member of the leucine-rich repeat family of proteins, is reported to be involved in a wide variety of pathophysiological processes, such as angiogenesis, tumor formation and osteoarthritis. A previous study showed that the level of serum LRG1 was increased in RA patients compared with normal controls and decreased by anti-TNF treatment. Moreover, serum LRG1 was positively correlated with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and DAS28-CRP score, suggesting that LRG1 might be used as an additional marker for RA . In addition, another study reported that serum LRG1 levels were reduced after treatment with the anti–IL-6 receptor antibody tocilizumab . T cells can differentiate into T helper 17 cells (Th17) or regulatory T cells (Tregs) under different stimulations . The level of Th17 cells is positively associated with the disease activity of RA . Experiments in mice with collagen-induced RA reveal the important role of LRG in RA development and Th17 differentiation. LRG promoted the differentiation of naive CD4 T cells into Th17 cells by activating the TGF-β-smad2 pathway . Angiogenesis is a notable characteristic feature of RA. LRG1 has been found to promote angiogenesis by regulating endothelial cell mitosis . LRG1 may be involved in the process of RA by promoting angiogenesis. Therefore, RA may also be improved by combination therapy by regulating LRG1.
F10 is vitamin K-dependent coagulation factor X of the blood coagulation cascade, which is considered a key factor in the activation of inflammation. A previous study reported that the levels of serum FXa in CFA-treated rats were significantly higher than those in control rats . Moreover, Gang et al. demonstrated that F10 expression was elevated in patients with RA compared with normal subjects by using microarray datasets . RA is an autoimmune disease characterized by inflammation of the synovial membrane. It is well known that the activation of the mitogen-activated protein kinase (MAPK) pathway is closely related to the inflammatory development of RA . Meanwhile, F10 could boost MAPK activation to release chemokines and inflammatory cytokines . F10 also has a proinflammatory action via protease-activated receptor 2 (PAR2) in many cell types . It has been reported that FXa induces JAK2, STAT3 and MAPK phosphorylation through activation of PAR2, PDGF and IL-6, which may have a drastic role in RA progression . Hence, we hypothesize that the reduction in F10 expression may be a target for combination drug therapy to alleviate RA.
SERPINA3, also called alpha-1-antichymotrypsin (ACT), is a member of the serine protease inhibitor family. It is believed that SERPINA3 contributes to the activation of proinflammatory cytokines, pathogen degradation and tissue remodeling via cathepsin G [34, 35]. SERPINA3 is a positive acute-phase reactant in the inflammatory process. When inflammation occurs, SERPINA3 is released into the circulation and inhibits the activities of various serine proteases . In the early stage of RA, the level of SERPINA3 in serum is increased and positively correlated with the levels of CRP, ESR and morning stiffness, suggesting that it may be used as an additional marker for RA . There are several limitations in this study. Due to the small sample size and significant individual differences, only a few DEPs were screened out. In a follow-up study, we will enroll more patients via a multicenter study and examine the details of these DEPs.