Etodolac improves collagen induced rheumatoid arthritis in rats by inhibiting synovial inflammation, fibrosis and hyperplasia

Abstract

Synovial hyperplasia is the main cause of chronic rheumatoid arthritis (RA), but the mechanism of synovial hyperplasia is still unclear. Etodolac (ETD) is a selective COX-2 inhibitor for relieving pain and stiffness in RA, but the disease modifying effect is still lack of evidence. Proteomics method was used to study the differential proteome of synovial tissue in collagen induced arthritis (CIA) in rats. With the help of STRING analysis, the upregulated proteins enriched in the cluster of complement and coagulation cascades and platelet degranulation were highlighted, these proteins with fibrogenic factors Lum, CIV, CXI and Tgfbi participated in the synovial inflammation, fibrosis and hyperplasia in CIA. Based on KOG function class analysis, the proteins involved in the events of the central dogma was explored. They might be hyperplasia related proteins for most of them are related to the proliferation of cancer. ETD significantly attenuated synovial inflammation, fibrosis and hyperplasia in CIA rats by downregulating these proteins. Several proteins have not been observed in RA so far, such as Tmsb4x, Pura, Nfic, Ruvbl1, Snrpd3, U2af2, Srrm2, Srsf7, Elavl1, Hnrnph1, Wars, Yars, Bzw2, Mcts1, Eif4b, Ctsh, Lamp1, Dpp7, Ptges3, Cdc37 and Septin9, they might be potentials targets for RA. Blood biochemistry tests showed the safety of 7 months use of ETD on rats. In conclusion, present study displayed a comprehensive mechanism of synovial hyperplasia in CIA rats, on this basis, the clinical value of ETD in the treatment of RA was well confirmed.

Keywords: Etodolac; Proteomics; Rheumatoid arthritis; Synovial hyperplasia.

Fig. 1

Effects of ETD on CIA rats. Vehicle-CIA rats had obvious symptoms of arthritis measured as increased total scores (a) and the right heel width (b) through 7 months after collagen injection compared with saline-injected controls, and the paw withdrawal thresholds was significantly decreased (c). The total scores and right heel width were significantly decreased, the paw withdrawal thresholds were significantly increased in ETD-CIA rats, **p < 0.01 compared with Vehicle-CIA rats (n = 8). d The appearance of both hind limbs 7 months after modeling, the right hind foot was significantly swollen and deformed, the secondary lesions of contralateral foot were serious in vehicle-treated group, while in ETD-CIA rats, the swelling and deformation of the feet were mild (n = 8). e Representative micrographs depicting HE stained and Masson synovial sections observed under 40 × objectives. Scale bars, 50 μm. The synovial histopathology of control group showed the normal structure of the synovial lining with clear and smooth features, without hyperplasia. The rats in the vehicle-treated model group showed infiltration of plasma cells, macrophages and lymphocytes as well as proliferative synovial cells, and synovial lining showed significant thickening and positive staining of Masson. The lesions in ETD-CIA rats were alleviated significantly. C: Control rats; V: Vehicle-CIA rats; E: ETD-CIA rats

Fig. 2

Visualizations of Go functional enrichment and interactome analysis results. a The cluster heat map of 367 differentially expressed proteins between Vehicle-CIA rats and Control rats. Among them, 261 upregulated ones and 106 downregulated ones. The cluster heat map of 203 differentially expressed proteins between ETD-CIA rats and Vehicle-CIA rats. Among them, 41 upregulated ones and 162 downregulated ones. Red is upregulated protein; blue is downregulated protein. The clustering is based on the log standard abundance of the significant differential expressed proteins (P value<0.05). b GO enrichment analysis of the top10 bar chart of 261 upregulated proteins in Vehicle-CIA rats compared with Control rats. c GO enrichment analysis of the top10 bar chart of 162 downregulated proteins in ETD-CIA rats compared with Vehicle-CIA rats. C: Control group; V: Vehicle-CIA rats; E: ETD-CIA rats. d Metascape visualization of the interactome network formed by 245 with gene names in 261 up-regulated proteins where the Molecular Complex Detection MCODE complexes are colored according to their identities. Nine MCODE complexes automatically identified in Metascape, colored by their identities. e The complement and coagulation cascades, and platelet degranulation cluster abstained by STRING online analysis. The local network clusters were colored by different colors

Fig. 3

Visualizations of KEGG pathway classification and KOG function class analysis results. a Comparison of the distribution of differentially expressed proteins (DEP) and all proteins (ALL) at KEGG Level 2. The pathway to enrich the largest number of proteins is genetic information processing pathway. There are 111 proteins in upregulated proteins in Vehicle-CIA rats compared with control group, and 71 ones in downregulated proteins in ETD-CIA rats compared with Vehicle-CIA rats. b KOG function class analysis of upregulated proteins in Vehicle-CIA rats and downregulated proteins in ETD-CIA rats. C: Control group; V: Vehicle-CIA rats; E: ETD-CIA rats

Fig. 4

The proteins enriched in cluster of complement and coagulation cascades, and platelet degranulation and their role in RA. a The heatmap of the proteins enriched in cluster of complement and coagulation cascades, and platelet degranulation as well as immunoglobulins. b Schematic diagram of up-regulated proteins involved in complement system. The upregulation of immunoglobulins, Colecl2, Apoe, Klkb1 as well as a series of complement compartments C1s, C1qb, C3, C5, C7, C8g, C9 represented the activation of complement cascade in present study, and those down-regulated proteins in ETD treatment group were marked with green borders. c The expression of ApoE and C1q were verified by PRM, the results were consistent with that of DIA. d The upregulation of Fgb, Fgg, Tgfbi, Lum, CIV, CXI contributed to the synovial fibrosis, and ETD reduced synovial fibrosis by downregulating the expressions of Fgb, Fgg, Tgfbi, Lum and CXI. e The expression of Tgfbi were verified by PRM, the result was consistent with that of DIA. # p < 0.05 compared with Control rats; *p < 0.05, **p < 0.01 compared with Vehicle-CIA rats. C: Control group; V: Vehicle-CIA rats; E: ETD-CIA rats

Fig. 5

The differential proteins enriched in central dogma. a The heap map of 89 upregulated proteins in synovial tissue of vehicle-CIA rats compared with normal control rats. They were classified in the following events according to the KOG function analysis: “Cell cycle control, cell division, chromosome partitioning”, “Chromatin structure and dynamics”, “Transcription”, “Replication, recombination and repair”, “RNA processing and modification”, “Translation, ribosomal structure and biogenesis”, “Posttranslational modification, protein turnover, chaperones”. b 59 downregulated ones in ETD-CIA rats compared with that in vehicle- treated rats. c The represent proteins in each event of central dogma was selected and verified by PRM method, including Pura, Nfic, Srrm2, Carhsp1 and Top2a, and the results were well consistent with that of DIA, which confirmed the accuracy of DIA proteomics results. # p < 0.05 compared with Control rats; *p < 0.05, **p < 0.01 compared with Vehicle-CIA rats. C: Control group; V: Vehicle-CIA rats; E: ETD-CIA rats