Connective tissue growth factor, a regulator related with 10-hydroxy-2-decenoic acid down-regulate MMPs in rheumatoid arthritis.

10-hydroxy-2-decenoic acid (10H2DA) is suggested to be a potential medication for rheumatoid arthritis (RA) by activation of matrix metalloproteinases (MMPs) via mitogen-activated protein kinase signaling pathways. The aim of the present work was to seek differentially expressed proteins in rheumatoid arthritis synovial fibroblasts (RASFs) treated with 10H2DA by comparative proteomics analysis. Two-dimensional electrophoresis (2-DE) and LC-MS/MS were performed to identify changes in protein expression after 24-h 10H2DA treatment. Differentially expressed proteins were identified by real-time PCR and Western blot analysis. Influence of down-regulation of connective tissue growth factor (CTGF) expression on MMPs was studied by RNAi. Ten proteins were up-regulated and 9 proteins were down-regulated after 24-h 10H2DA treatment. A total of 19 differentially expressed proteins were identified and found to be associated with glycolysis, lipid metabolism, cell adhesion, ATP synthesis, oxidation reduction, and anti-apoptosis. CTGF, a member of the C-terminal cystein-rich proteins (CCN) family, was down-regulated after 24-h 10H2DA treatment. MMPs were down-regulated after RNAi (CTGFi). These results suggest that CTGF is a regulator factor in the expression of MMPs, and 10H2DA down-regulate the concentration of MMPs probably by down-regulating the expression of CTGF.

Up-regulation of fatty acid oxidation in the ligament as a contributing factor of ankylosing spondylitis: A comparative proteomic study.

OBJECTIVES: The present study first utilized a standardized shotgun proteomic analysis method to determine differences in protein expression of fibroblasts in the ligament between AS patients and healthy controls. METHODS: Proteins extracted from primarily cultured FLLs from 35 AS patients and 10 normal subjects were analyzed by automated 2D-Nano-LC-ESI-MS/MS. Differentially expressed proteins were screened by 2-sample t-test and fold change. Bioinformatics analysis of differentially expressed proteins was based on the IPA. Fatty acid ß-oxidation-related proteins and INSR pathway-related proteins in the ligament were confirmed by real-time PCR and Western blot. RESULTS: A total of 556 differential proteins were screened in AS. Of them, 322 proteins were up-regulated and the remaining 234 proteins were down-regulated. GO and pathway analyses showed that six fatty acid ß-oxidation-related proteins (HADHB, ECHS1, ACSL4, ACADM, ACSL1 and HADH) were up-regulated in FLL cells, which was consistent with the results obtained from real-time PCR, Western blot and MS, while INSR pathway-related proteins (INSR, IRS1, PI3K and PKC) was low in the ligament of AS as compared with that in healthy controls. CONCLUSION: The lower body fat level in AS maybe due to up-regulation of fatty acid ß-oxidation-related enzymes regulated by INSR/PI3K/PKC pathway. BIOLOGICAL SIGNIFICANCE: Ankylosing spondylitis (AS), a common spondyloarthropathy, is an inflammatory rheumatic disease with a predilection for the axial skeleton. Clinical hallmarks of AS include sacroiliitis, uveitis, enthesitis and persistent spinal inflammation. The pathogenic mechanism of disease causation and perpetuation remains poorly understood. In this study, we primarily cultured fibroblast cells from ligament biopsies, knowing that fibroblast cells are dominant cells in the diseased ligament. One of the characteristic pathologic changes in AS is inflammation of the attachment points, including the muscle, ligament and bone or joint capsule. Inflammation of the tendon attachment point is usually non-bacterial and can lead to pain and swelling of the tendon ligament. To obtain more information, we used Shotgun proteomic analysis based on multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS). we firstly mixed the lysates of FLL cells derived from the ligaments of 35 AS patients and 10 normal subjects, identified proteins by automated 2D-Nano-LC-ESI-MS/MS method, GO and pathway analyses showed that six fatty acid ß-oxidation-related proteins (HADHB, ECHS1, ACSL4, ACADM, ACSL1 and HADH) were up-regulated in the ligament, which was consistent with the results obtained from real-time PCR, Western blot and MS, while INSR pathway-related proteins (INSR, IRS1, PI3K and PKC) was low in the ligament of AS as compared with that in healthy controls. We also find that AS subjects had significantly lower body mass index (BMI) and BMI Z-scores compared with that in healthy controls. The results remind us that up-regulation of fatty acid ß-oxidation-related proteins lower the body fat content, which is a new discovery contributing to the progression of AS. This is the first report on fatty acid oxidation in AS. It was found that the body fat level was low in AS due to high fatty acid oxidation, suggesting that insulin signaling may play an important role in the metabolic switch from predominant to fatty acid metabolism that characterizes the ligament of AS. One mechanism for this transition is increased expression of genes that regulate the rate of fatty acid oxidation. This effect may be mediated by PI3K, a downstream mediator of many receptor tyrosine kinases, including the INSR. This is a newly discovered factor contributing to the progression of AS.

10-Hydroxy-2-decenoic acid from Royal jelly: a potential medicine for RA.

AIM OF THE STUDY: Rheumatoid arthritis synovial fibroblasts (RASFs) are known to produce matrix metalloproteinases (MMPs) and cause joint destruction. The purpose of this study is to develop a potential medicine for rheumatoid arthritis (RA). MATERIALS AND METHODS: To this end, first, the MMPs inhibition factor was purified from an alkali-solubilized fraction of RJ (Apis mellifera) by C18 reverse-phase column chromatography and identified as 10-hydroxy-2-decenoic acid (10H2DA) by LTQ XL analysis. Next, Experimental test 10H2DA how to inhibited the activities of MMPs: with RASFs isolated from rheumatoid tissues by enzymatic digestion, cultures in monolayers were treated with 10H2DA (0.5mM, 1mM, and 2mM) or PBS for 2h followed by stimulation with TNF-alpha (10 ng/ml) for 2h, mRNA. Protein levels of MMP-1 and MMP-3 were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), the DNA-binding activity of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) by electrophoretic mobility shift assay (EMSA), and the protein kinase activity of p38, ERK and JNK by kinase assay. RESULTS: The molecular investigation revealed that the 10H2DA-mediated suppression was likely to occur through blocking p38 kinase and c-Jun N-terminal kinase-AP-1 signaling pathways. In contrast, 10H2DA had no effect on extracellular signal-regulated kinase activity, NF-kappaB DNA-binding activity and IkappaBalpha degradation. CONCLUSION: These results suggest that 10H2DA may be of potential therapeutic value in inhibiting joint destruction in RA.