Proteomics analysis of lung tissue reveals protein makers for the lung injury of adjuvant arthritis rats.

Lung injury is one of the common extra­articular lesions in rheumatoid arthritis (RA). Due to its insidious onset and no obvious clinical symptoms, it can be easily dismissed in the early stage of diagnosis, which is one of the reasons that leads to a decline of the quality of life and subsequent death of patients with RA. However, its pathogenesis is still unclear and there is a lack of effective therapeutic targets. In the present study, tandem mass tag­labeled proteomics was used to research the lung tissue proteins in RA model (adjuvant arthritis, AA) rats that had secondary lung injury. The aim of the present study was to identify the differentially expressed proteins related to RA­lung injury, determine their potential role in the pathogenesis of RA­lung injury and provide potential targets for clinical treatment. Lung tissue samples were collected from AA­lung injury and normal rats. The differentially expressed proteins (DEPs) were identified by tandem mass spectrometry. Bioinformatic analysis was used to assess the biological processes and signaling pathways associated with these DEPs. A total of 310 DEPs were found, of which 244 were upregulated and 66 were downregulated. KEGG anlysis showed that 'fatty acid degradation', 'fatty acid metabolism', 'fatty acid elongation', 'complement and coagulation cascades', 'peroxisome proliferator­activated receptor signaling pathway' and 'hypoxia­inducible factor signaling pathway' were significantly upregulated in the lung tissues of AA­lung injury. Immunofluorescence staining confirmed the increased expression of clusterin, serine protease inhibitors and complement 1qc in lung tissue of rats with AA lung injury. In the present study, the results revealed the significance of certain DEPs (for example, C9, C1qc and Clu) in the occurrence and development of RA­lung injury and provided support through experiments to identify potential biomarkers for the early diagnosis and prevention of RA­lung injury.

Expression of AIM2 in Rheumatoid Arthritis and Its Role on Fibroblast-Like Synoviocytes.

OBJECTIVES: To determine differences in AIM2 inflammasome expression levels between rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the role of AIM2 in RA fibroblast-like synoviocytes (RA-FLS). METHODS: Serum AIM2 levels among health controls (HC, n = 20), OA (n = 25), and RA (n =49) patients were compared via ELISA. The different expression levels of AIM2, ASC, caspase-1, and IL-1ß between RA and OA synovium were semiquantified by qRT-PCR and immunohistochemical (IHC) staining. IHC staining was recorded by H scores, and its correlation with the ESR and CRP levels of RA patients was determined. SiRNA AIM2 was transferred to RA-FLS and its effects on the proliferation and migration via CCK-8 assay and Transwell test, respectively. RESULTS: In RA sera, the HC expressed higher level of AIM2 than OA and RA patients, and ASC, caspase-1, and IL-1ß expressed higher in RA patients than HC; no significant differences were observed between sera of OA and RA patients. However, in affected knee synovium, AIM2, ASC, caspase-1, and IL-1ß were expressed higher in RA than that of OA. Moreover, the H scores of AIM2, ASC, and IL-1ß were positively correlated with the ESR and CRP levels in RA patients. The proliferation of FLS was significantly inhibited after transferring with AIM2 siRNA to FLS. There were no differences in apoptosis and migration assay between the si-AIM2 group and the control group. CONCLUSION: AIM2 inflammasome pathway involves in the pathogenesis of RA. si-AIM2 inhibits the proliferation of RA-FLS, which may be a promising therapeutic strategy for the treatment of RA.

[Increased level of plasma AMPK is positively correlated with disease activity in patients with rheumatoid arthritis].

Objective To explore the relationships of plasma adenylate-activated protein kinase α1 (AMPKα1) and phosphorylated AMPK (p-AMPK) levels of rheumatoid arthritis (RA) with inflammatory indexes. Methods The study recruited 15 health controls (HCs), 17 patients with osteoarthritis (OA) and 61 patients with RA[including 22 cases of very active (VRA), 20 cases of moderate active (MRA) and 19 cases of inactive (IRA) based on the disease activity score in 28 joints-erythrocyte sedimentation rate (DAS28-ESR)]. The levels of ESR and C-reactive protein (CRP) in the patients were measured; the levels of plasma AMPKα1 and p-AMPK as well as interleukin-17 (IL-17) and tumor necrosis factor-α (TNF-α) were detected by ELISA. Pearson r test was used to compare the correlations of AMPKα1 and p-AMPK levels with the DAS28 score, inflammation indexes and inflammatory cytokines. Results Compared with the HCs, the plasma AMPKα1 and p-AMPK levels of the OA patients and RA patients with different disease activities were higher in a certain extent. P-AMPK levels showed a moderately positive correlation with DAS28 score (r=0.27, 95% CI: 0.01-0.49). AMPKα1 showed a moderately positive correlation with TNF-α levels (r=0.46, 95% CI: 0.24-0.64) in the RA patients. Conclusion The p-AMPK level may increase in the plasma of RA patients, and it has a positive correlation with clinical disease activity and inflammation indicators.

Metformin, an AMPK Activator, Inhibits Activation of FLSs but Promotes HAPLN1 Secretion.

AMP-activated protein kinase (AMPK) is essential for maintaining energy balance and has a crucial role in various inflammatory pathways. In this study, AMPK levels positively correlated with many inflammatory indexes in rheumatoid arthritis (RA) patients, especially in the affected synovium. In RA sera, a positive correlation between phosphorylated (p-)AMPK-α1 levels and DAS28 (disease activity score 28) activity (r = 0.270, p < 0.0001) was found. Similarly, a positive correlation was observed between AMPK-α1 and tumor necrosis factor α (TNF-α) levels (r = 0.460, p = 0.0002). Differentially expressed genes between osteoarthritis (OA) and RA synovium from NCBI GEO profiles and our RNA sequencing data suggested activation of metabolic pathways specific to RA-fibroblast-like synoviocytes (FLSs). AMPK-α1 was highly expressed in the synovium of RA but not OA patients. An AMPK activator, metformin, inhibited FLS proliferation at higher but not lower concentrations, whereas the inhibitor dorsomorphin promoted the proliferation of RA-FLSs. Interestingly, both metformin and dorsomorphin inhibited the migration of RA-FLSs. After metformin treatment, expression of interleukin 6 (IL-6), TNF-α, and IL-1ß were significantly downregulated in RA-FLSs; however, increased expression of p-AMPK-α1, protein kinase A (PKA)-α1, and HAPLN1 (hyaluronan and proteoglycan link protein 1) was observed. Increased levels of HAPLN1 in RA-FLSs by an AMPK activator could potentially be beneficial in protecting the joints. Hence, our results demonstrate the potential of an AMPK activator as a therapeutic for RA.

[Pannus does not occur only in rheumatoid arthritis: a pathological observation of pannus of knee osteoarthritis].

OBJECTIVE: To compare the histopathological features of the synovium between rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: We retrospectively analyzed the synovial specimens obtained after synovial surgery in 72 cases of RA and 24 cases of OA. Two independent pathologists reviewed the sections of the synovial tissues with HE staining, quantitatively scored the degree of fibroblast-like synoviocyte (FLS) hyperplasia, vascular hyperplasia, fibroplasia, and lymphocyte infiltration, and examined the presence plasma cell infiltration. The pathological morphology of the synovial tissues was evaluated in relation with the clinical data of the patients. RESULTS: Pannus formation was also detected in the synovium of OA patients, which showed a lesser degree of OA-FLS hyperplasia, fibrosis and lymphocyte infiltration and a significantly lower rate of plasma cell infiltration compared with the pannus in RA patients. Vascular proliferation was also milder in the pannus of OA patients than in RA pannus, but the difference was not statistically significant. In OA patients, the pannus could be observed under a microscope and was difficult to distinguish from that in RA patients. CONCLUSIONS: Pannus formation occurs also in the synovium of OA patients but with milder FLS hyperplasia, fibrosis and lymphocyte infiltration and a lower rate of plasma cell infiltration compared with the pannus in RA patients. These differences in the pannus between OA and RA can be of potential value in the diagnosis and treatment of the patients.

[Effect of sanshui baihu decoction on the proliferation of fibroblast-like synoviocytes and its secretion of IL-6 and IL-17].

OBJECTIVE: To observe the effect of Sanshui Baihu Decoction (SBD) containing serum on the proliferation of in vitro cultured fibroblast-like synoviocytes (FLS) derived from rheumatoid arthritis (RA) and osteoarthritis (OA) and its secretion of interleukin-6 (IL-6) and IL-17, and to explore the pharmacological mechanism of SBD. METHODS: The FLS obtained from cultured RA and OA patients' synovial tissue were cultured and passaged in vitro in a routine way. The cultured medium was changed to DMEM with 20% SBD containing serum and cultured for 72 h after cultured for 3 to 6 generations. The proliferation rate of FLS was detected by MTT assay. Levels of IL-6 and IL-17 in the supernatant were detected by ELISA. Leflunomide and saline containing serum were used as positive and negative control respectively. RESULTS: SBD containing serum significantly inhibited the proliferation of RA-FLS and OA-FLS, and decreased the secretion of IL-17 in RA-FLS. Its inhibition efficiency of SBD was equivalent to that of Leflunomide. No obvious inhibition on the secretion of IL-6 in RA-FLS was observed. It had no significant effect on the secretion of IL-17 and IL-6 in OA-FLS. CONCLUSION: SBD could inhibit the proliferation of FLS and the secretion of IL-17 in RA-FLS, which might be one of its pharmacological mechanisms for treating RA.

[Methanol extract of Celastrus orbiculatu suppresses synovial hyperplasia and cartilage erosion and degradation in rheumatoid arthritis].

OBJECTIVE: To investigate the effects of methanol extract of Celastrus orbiculatu (MECO) on synovial hyperplasia and cartilage erosion and degradation in rheumatoid arthritis (RA), and explore the possible mechanisms to provide clues for new drug development for RA treatment. METHODS: The articular synovium from patients with RA and normal articular cartilage were co-implanted into the back of severe combined immunodeficient (SCID)mice to establish the chimeric model SCID- HuRAg. Four weeks later, the mice were given MECO intragastrically at 30 mg/day, leflunomide at 500 microg/day or distilled water, respectively, for 4 consecutive weeks. After completion of the treatments, the histological scores of the grafts for synovial hyperplasia, cartilage invasion by synoviocyte and cartilage degradation around the chondrocytes were evaluated, and serum level of tumor necrosis factor-alpha (TNF-alpha) was measured with radioimmunoassay. The expression of TNF-alpha mRNA and the cell apoptosis in the synovium were detected with in situ hybridization (ISH) and TUNEL, respectively, and the results were analyzed with the image analysis system. RESULTS: The grafts survived in the mice till the end of experiment. MECO and leflunomide, in comparison with distilled water, significantly lowered the scores for synovial hyperlasia (2.00+/-0.76 and 2.25+/-0.89 vs 3.63+/-0.52), cartilage erosion (1.69+/-0.80 and 2.00+/-1.36 vs 3.75+/-0.53), cartilage degradation (1.88+/-0.83 and 2.13+/-0.83 vs 3.63+/-0.74) and serum TNF-alpha level (0.84+/-0.09 and 0.83+/-0.12 vs 0.99+/-0.11 ng/ml). Cell apoptosis of the synovium increased significantly with MECO and leflunomide treatments, but the expression of TNF-alpha mRNA in the synovium decreased significantly in MECO group. CONCLUSION: MECO can effectively suppress synovial hyperplasia and cartilage erosion and degradation SCID-HuRAg mice by reducing TNF-alpha production in the synovium and promoting synovial apoptosis. MECO can be comparable with leflunomide in their effect, but the former is more effective in suppressing TNF-alpha expression in the synovium.