Rheumatoid Arthritis Synovial Fibroblasts Research Articles

Correction: Influence of Extracellular RNAs, Released by Rheumatoid Arthritis Synovial Fibroblasts, on Their Adhesive and Invasive Properties.

Extracellular RNA (exRNA) has been characterized as a molecular alarm signal upon cellular stress or tissue injury and to exert biological functions as a proinflammatory, prothrombotic, and vessel permeability-regulating factor. In this study, we investigated the contribution of exRNA and its antagonist RNase1 in a chronic inflammatory joint disease, rheumatoid arthritis (RA). Upon immunohistochemical inspection of RA, osteoarthritis (OA), and psoriatic arthritis synovium, exRNA was detectable only in the RA synovial lining layer, whereas extracellular DNA was detectable in various areas of synovial tissue. In vitro, exRNA (150-5000 nt) was released by RA synovial fibroblasts (RASF) under hypoxic conditions but not under normoxia or TNF-α treatment. RNase activity was increased in synovial fluid from RA and OA patients compared with psoriatic arthritis patients, whereas RNase activity of RASF and OASF cultures was not altered by hypoxia. Reduction of exRNA by RNase1 treatment decreased adhesion of RASF to cartilage, but it had no influence on their cell proliferation or adhesion to endothelial cells. In vivo, treatment with RNase1 reduced RASF invasion into coimplanted cartilage in the SCID mouse model of RA. We also analyzed the expression of neuropilins in synovial tissue and SF, as they may interact with vascular endothelial growth factor signaling and exRNA. The data support the concepts that the exRNA/RNase1 system participates in RA pathophysiology and that RASF are influenced by exRNA in a prodestructive manner.

MiR-650 inhibits proliferation, migration and invasion of rheumatoid arthritis synovial fibroblasts by targeting AKT2

This study was aimed to investigate the effects of miR-650 on the proliferation, migration, invasion and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs). Synovial tissue samples were collected from 16 rheumatoid arthritis (RA) patients and 13 patients with joint trauma undergoing joint replacement surgery. The RASFs were isolated and cultured. MiR-650 and AKT2 expressions in both synovial tissues and cells were detected using the quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry (IHC). Dual luciferase reporter gene assay was employed to evaluate the effect of miR-650 on the luciferase activity of AKT2. Then RASFs were transfected with miR-650 mimics, miR-650 inhibitors and pLenti6/V5-AKT2 respectively. And changes in cellular proliferation, migration, invasion and apoptosis were detected through MTT assay, wound healing assay, Transwell invasion assay and flow cytometry analysis, respectively. MiR-650 was significantly down-regulated in RASFs than normal cells, whereas AKT2 was up-regulated in RASFs. Dual luciferase reporter gene assay showed that miR-650 could specifically bind to the 3'UTR of AKT2 and significantly repress the luciferase activity. MiR-650 significantly decreased the expression of AKT2. Down-regulation of miR-650 or up-regulation of AKT2 could increase proliferation, migration, invasion of RASFs, and decrease RASFs apoptosis. The conversed results were observed, when miR-650 was up-regulated in RASFs. MiR-650 could inhibit the proliferation, migration and invasion of RASFs through targeted regulation of AKT2 expression.

Glucose-6-Phosphate Isomerase (G6PI) Mediates Hypoxia-Induced Angiogenesis in Rheumatoid Arthritis

The higher level of Glucose-6-phosphate isomerase (G6PI) has been found in both synovial tissue and synovial fluid of rheumatoid arthritis (RA) patients, while the function of G6PI in RA remains unclear. Herein we found the enrichment of G6PI in microvascular endothelial cells of synovial tissue in RA patients, where a 3% O2 hypoxia environment has been identified. In order to determine the correlation between the high G6PI level and the low oxygen concentration in RA, a hypoxia condition (~3% O2) in vitro was applied to mimic the RA environment in vivo. Hypoxia promoted cellular proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), and induced cell migration and angiogenic tube formation of human dermal microvascular endothelial cells (HDMECs), which were accompanied with the increased expression of G6PI and HIF-1α. Through application of G6PI loss-of-function assays, we confirmed the requirement of G6PI expression for those hypoxia-induced phenotype in RA. In addition, we demonstrated for the first time that G6PI plays key roles in regulating VEGF secretion from RASFs to regulate the hypoxia-induced angiogenesis in RA. Taken together, we demonstrated a novel pathway regulating hypoxia-induced angiogenesis in RA mediated by G6PI.

Cinnamomulactone, a new butyrolactone from the twigs of Cinnamomum cassia and its inhibitory activity of matrix metalloproteinases.

Cinnamomum cassia (Lauraceae) has long been used as one of the most frequently used traditional oriental medicines for the treatment of gastritis, diabetes, blood circulation disturbance and inflammatory diseases. Cinnamomulactone (1), a new butyrolactone was isolated from the twigs of C. cassia together with nine known compounds, coumarin (2), trans-cinnamic acid (3), cinnamaldehyde (4), 2-hydroxycinnamaldehyde (5), 2-methoxycinnamaldehyde (6), 2-hydroxy-cinnamyl alcohol (7), benzoic acid (8), (+)-syringaresinol (9) and phenethyl (E)-3-[4-methoxyphenyl]-2-propenoate (10). The planar structure of 1 was elucidated on the basis of spectroscopic data analysis and its configurations were determined by coupling constant (3 J HH) analysis and a comparison with specific rotation data of related compounds on the literatures. The structures of known compounds were confirmed by the comparison of their spectroscopic data to the reported values. Compound 10 was isolated for the first time from this plant. Compounds 1, 2, 4, and 9 showed inhibitory activity against matrix metalloproteinases (MMPs) gene expression. Among them, compound 1 has been revealed to suppress the gene expression of MMP-3 and interleukin (IL)-1β as well as MMP-1 in tumor necrosis factor (TNF)-α stimulated rheumatoid arthritis synovial fibroblasts.

Tumorsuppressive MicroRNA-188-5p Reveals Novel Oncogenes for Hepatocellular Carcinoma

Background & Aims: MicroRNAs are known to be dysregulated in Hepatocellular Carcinoma (HCC) and to play crucial roles in tumor development and progression. In a preliminary study, our group found that miR-188-5p is downregulated in activated synovial fibroblasts in rheumatoid arthritis (RASF). RASF reveal tumor-like features such as enhanced migration and proliferation. Re-Expression of miR-188-5p strongly inhibited migration in RASF (Int J Clin Exp Pathol, 2015). Chloride voltage-gated channel 5 (CLCN5), which is the host gene for miR-188-5p, was shown to be induced by Hepatocyte nuclear factor (HNF) 1 (Am J Physiol Renal Physiol., 2010). Interestingly, our group demonstrated in a previous study that HNF-1 is a lost tumorsuppressor in HCC (Gut, 2008). Therefore, in this project, our aim was to explore a possible role of miR-188-5p in cancer development and progression with a focus on HCC.

TLRs, future potential therapeutic targets for RA.

Toll like receptors (TLR)s have a central role in regulating innate immunity and in the last decade studies have begun to reveal their significance in potentiating autoimmune diseases such as rheumatoid arthritis (RA). Earlier investigations have highlighted the importance of TLR2 and TLR4 function in RA pathogenesis. In this review, we discuss the newer data that indicate roles for TLR5 and TLR7 in RA and its preclinical models. We evaluate the pathogenicity of TLRs in RA myeloid cells, synovial tissue fibroblasts, T cells, osteoclast progenitor cells and endothelial cells. These observations establish that ligation of TLRs can transform RA myeloid cells into M1 macrophages and that the inflammatory factors secreted from M1 and RA synovial tissue fibroblasts participate in TH-17 cell development. From the investigations conducted in RA preclinical models, we conclude that TLR-mediated inflammation can result in osteoclastic bone erosion by interconnecting the myeloid and TH-17 cell response to joint vascularization. In light of emerging unique aspects of TLR function, we summarize the novel approaches that are being tested to impair TLR activation in RA patients.

Expression and Regulation of PIWIL-Proteins and PIWI-Interacting RNAs in Rheumatoid Arthritis.

ObjectiveThe PIWIL (P-element induced wimpy testis like protein) subfamily of argonaute proteins is essential for Piwi-interacting RNA (piRNA) biogenesis and their function to silence transposons during germ-line development. Here we explored their presence and regulation in rheumatoid arthritis (RA).MethodsThe expression of PIWIL genes in RA and osteoarthritis (OA) synovial tissues and synovial fibroblasts (SF) was analysed by Real-time PCR, immunofluorescence and Western blot. The expression of piRNAs was quantified by next generation small RNA sequencing (NGS). The regulation of PIWI/piRNAs, proliferation and methylation of LINE-1 after silencing of PIWIL genes were studied.ResultsPIWIL2 and 4 mRNA were similarly expressed in synovial tissues and SF from RA and OA patients. However, on the protein level only PIWIL4 was strongly expressed in SF. Using NGS up to 300 piRNAs were identified in all SF without significant differences in expression levels between RA and OASF. Of interest, the analysis of the co-expression of the detected piRNAs revealed a less tightly regulated pattern of piRNA-823, -4153 and -16659 expression in RASF. In RASF and OASF, stimulation with TNFα+IL1β/TLR-ligands further significantly increased the expression levels of PIWIL2 and 4 mRNA and piRNA-16659 was significantly (4-fold) induced upon Poly(I:C) stimulation. Silencing of PIWIL2/4 neither affect LINE-1 methylation/expression nor proliferation of RASF.ConclusionWe detected a new class of small regulatory RNAs (piRNAs) and their specific binding partners (PIWIL2/4) in synovial fibroblasts. The differential regulation of co-expression of piRNAs in RASF and the induction of piRNA/Piwi-proteins by innate immune stimulators suggest a role in inflammatory processes.

Bone Morphogenetic Protein 2 and Transforming Growth Factor β1 Inhibit the Expression of the Proinflammatory Cytokine IL-34 in Rheumatoid Arthritis Synovial Fibroblasts

IL-34 is a proinflammatory cytokine implicated in rheumatoid arthritis (RA). The current study aimed to assess the IL-34 expression in response to two members of the transforming growth factor (TGF)-β family, TGF-β1 and bone morphogenetic protein (BMP)-2, in synovial fibroblasts from RA patients. IL-34, TGF-β1, and BMP-2 productions were measured in patient synovial fluids by enzyme-linked immunosorbent assay. IL-34 mRNA levels were quantified by real-time quantitative PCR in human synovial fibroblasts and murine mesenchymal stem cells. Pharmacologic inhibitions were used to determine the involvement of activin receptor-like kinase 1 (ALK1) and ALK5 downstream TGF-β1 and BMP-2. IL-34, TGF-β1, and BMP-2 were expressed in synovial fluids from RA patients. We found a significant correlation between IL-34 and TGF-β1 expressions. Levels of both IL-34 and TGF-β1 were thus correlated with the total leukocyte counts in the synovial fluids. TGF-β1 and BMP-2 decreased IL-34 expression in the synovial fibroblasts or in murine mesenchymal stem cells in a dose- and time-dependent manner through ALK5 and ALK1 pathways, respectively. In addition, TGF-β1 and BMP-2 antagonized tumor necrosis factor α-induced IL-34 gene expression. This work identifies TGF-β1 and BMP-2 as potent inhibitors of IL-34 expression in RA synovial fibroblasts. These cytokines, as upstream inhibitors of IL-34, may thus contribute to antagonize inflammation and bone erosions in RA.

Expression and Functions of Immediate Early Response Gene X-1 (IEX-1) in Rheumatoid Arthritis Synovial Fibroblasts.

In rheumatoid arthritis (RA), synovial fibroblasts (RA-SFs) accumulate in affected joints, where they play roles in inflammation and joint destruction. RA-SFs exhibit tumor-like proliferation and are resistant to apoptosis. Although RA-SF activation is well described, negative regulators of RA-SF activation are unknown. We previously reported that histone deacetylase (HDAC) inhibitors facilitate apoptosis in RA-SFs. Here we found that RA-SFs treated with the HDAC inhibitor Trichostatin A (TSA) exhibited an upregulation of the immediate early response gene X-1 (IEX-1). IEX-1 has roles in apoptosis sensitivity, cell-cycle progression, and proliferation, and is reported to be involved in immune responses, inflammation, and tumorigenesis, and to have anti-arthritic properties. To investigate IEX-1’s role in RA-SFs, we used in vitro-cultured synovial fibroblasts from RA and osteoarthritis (OA) patients. We confirmed that TSA upregulated the IEX-1 protein and mRNA expressions in RA-SFs by western blotting and quantitative RT-PCR. Inhibiting HDAC1, 2, and 3 (but not 6 or 8) also upregulated IEX-1. The IEX-1 mRNA levels were higher in RA-SFs than in OA-SFs, and were further upregulated in RA-SFs by the pro-inflammatory cytokines TNFα and IL-1β. The staining of surgical specimens showed that IEX-1 was present in the pannus from affected RA joints. Si-RNA-mediated IEX-1 knockdown upregulated the lipopolysaccharide (LPS)-induced expression of TNFα and various chemokine mRNAs, indicating that IEX-1 downregulates TNFα and chemokines. Furthermore, apoptosis analysis showed that IEX-1 knockdown protected RA-SFs from apoptosis induced by TSA or by an anti-Fas mAb, indicating that IEX-1 is pro-apoptotic in RA-SFs. Collectively, our results showed that IEX-1 is induced by TNFα and IL-1β in RA-SFs, in which it suppresses TNFα and chemokine production and induces apoptosis; thus, IEX-1 negatively regulates RA-SF activation. Further investigation of IEX1’s functions in RA-SFs may lead to new therapeutic approaches for RA.

MicroRNA-126 affects rheumatoid arthritis synovial fibroblast proliferation and apoptosis by targeting PIK3R2 and regulating PI3K-AKT signal pathway.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and destruction of the joints as well as an increased risk of cardiovascular disease. RA synovial fibroblasts (RASFs) are involved in the progression of RA and release pro-inflammatory cytokines. On the other hand, microRNAs (miRs) may help control the inflammatory response of immune and non-immune cells. Therefore, our study used lentiviral expression vectors to test the effects of miR-126 overexpression on RASF proliferation and apoptosis. Luciferase experiments verified the targeting relationship between miR-126 and PIK3R2 gene. The co-transfection of anti-miR-126 and PIK3R2 siRNA to RASFs were used to identify whether PIK3R2 was directly involved in proliferation and apoptosis of miR-126-induced RASFs. Real-time polymerase chain reaction (PCR) was used to detect miR-126 and PIK3R2 expressions. MTT assay was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis and cell cycle. Western blotting was used to detect PIK3R2, PI3K, AKT and p-AKT proteins. After Lv-miR-126 infected RASFs, the relative expression of miR-126 was significantly enhanced. MiR-126 promoted RASF proliferation and inhibited apoptosis. Levels of PIK3R2 decreased while total PI3K and p-AKT levels increased in RASFs overexpressing miR-126. Co-transfection of anti-miR-126 and PIK3R2 siRNA also increased PI3K and p-AKT levels as well as RASF proliferation and reduced apoptosis, as compared to anti-miR-126 treatment alone. Finally, luciferase reporter assays showed that miR-126 targeted PIK3R2. Our data indicate that miR-126 overexpression in RASFs inhibits PIK3R2 expression and promotes proliferation while inhibiting apoptosis. This suggests inhibiting miR-126 may yield therapeutic benefits in the treatment of RA.

MicroRNAs interfere with DNA methylation in rheumatoid arthritis synovial fibroblasts

BackgroundThe DNA of rheumatoid arthritis synovial fibroblasts (RASF) is globally hypomethylated; this contributes to an aggressive behaviour. In an attempt to remethylate these cells, we supplemented with methyl donors. We...

Influence of Extracellular RNAs, Released by Rheumatoid Arthritis Synovial Fibroblasts, on Their Adhesive and Invasive Properties.

Extracellular RNA (exRNA) has been characterized as a molecular alarm signal upon cellular stress or tissue injury and to exert biological functions as a proinflammatory, prothrombotic, and vessel permeability-regulating factor. In this study, we investigated the contribution of exRNA and its antagonist RNase1 in a chronic inflammatory joint disease, rheumatoid arthritis (RA). Upon immunohistochemical inspection of RA, osteoarthritis (OA), and psoriatic arthritis synovium, exRNA was detectable only in the RA synovial lining layer, whereas extracellular DNA was detectable in various areas of synovial tissue. In vitro, exRNA (150-5000 nt) was released by RA synovial fibroblasts (RASF) under hypoxic conditions but not under normoxia or TNF-α treatment. RNase activity was increased in synovial fluid from RA and OA patients compared with psoriatic arthritis patients, whereas RNase activity of RASF and OASF cultures was not altered by hypoxia. Reduction of exRNA by RNase1 treatment decreased adhesion of RASF to cartilage, but it had no influence on their cell proliferation or adhesion to endothelial cells. In vivo, treatment with RNase1 reduced RASF invasion into coimplanted cartilage in the SCID mouse model of RA. We also analyzed the expression of neuropilins in synovial tissue and SF, as they may interact with vascular endothelial growth factor signaling and exRNA. The data support the concepts that the exRNA/RNase1 system participates in RA pathophysiology and that RASF are influenced by exRNA in a prodestructive manner.

Calycosin suppresses expression of pro-inflammatory cytokines via the activation of p62/Nrf2-linked heme oxygenase 1 in rheumatoid arthritis synovial fibroblasts

The activation of synovial fibroblasts (SFs) and the subsequent production and expression of pro-inflammatory cytokines play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). In the current study, rheumatoid arthritis synovial fibroblasts (RASFs) isolated from the joint of the patients were used to evaluate the suppressive effects of calycosin (CAL), a compound derived from the Chinese medicinal herb Radix Astragali, on the expression of pro-inflammatory cytokines in RASFs. The results demonstrated that increased mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-25 (IL-25), interleukin-33(IL-33) were significantly inhibited by CAL. Furthermore, the compound obviously suppressed IL-6 and IL-33 secretion. The key inflammatory mediator, cyclooxygenase-2 (COX-2) was significantly attenuated by CAL. A mechanistic study showed that the antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1(NQO1) and Nrf2 of RASFs were markedly activated by CAL. Furthermore, CAL potentiated the accumulation of sequestosome 1 (SQSTM1, p62) and the degradation of Kelch-like ECH-associated protein 1 (Keap1), thereby inducing Nrf2 translocation from the cytoplasm to the nucleus. Thus, CAL suppresses the expression of pro-inflammatory cytokines via p62/Nrf2-linked HO-1 induction in RASFs, which suggests that the compound should be further investigated as a candidate anti-inflammatory and anti-arthritic agent.

Robo3A and Robo3B expression is regulated via alternative promoters and mRNA stability.

BackgroundThe transmembrane receptor family Roundabout (Robo) was described to have an essential role in the developing nervous system. Recent studies demonstrated that Robo3 shows an altered expression in rheumatoid arthritis as well as in melanoma.Context and purpose of the studyUntil today no detailed studies of the two Robo3 isoforms (Robo3A and Robo3B) and their roles in rheumatoid arthritis synovial fibroblasts, respectively malignant melanoma are available. To get a better understanding regarding the role of Robo3A and Robo3B in the molecular process of rheumatoid arthritis and melanoma the exact characterization of expression and regulation is object of this study.ResultsmRNA and protein expression of the transcriptional variants were analyzed by quantitative RT-PCR respectively western blotting and revealed particularly enhanced expression of Robo3B in rheumatoid arthritis and melanoma. Promoter assays and inhibitor studies also disclosed that there is apparently a cell- and isoform-specific regulation of the Robo3 expression. Finally, dissimilar mRNA stabilities of Robo3A and Robo3B are identified as decisive posttranscriptional gene expression control.ConclusionIn summary, this study supported an isotype specific role of Robo3B in disease hinting to different functional roles of each isoform.

MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts.

TNF-α is a major cytokine implicated in rheumatoid arthritis (RA), and its expression is regulated at the transcriptional and posttranscriptional levels. However, the impact of changes in microRNA expression on posttranslational processes involved in TNF-α signaling networks is not well defined in RA. In this study, we evaluated the effect of miR-17, a member of the miR-17-92 cluster, on the TNF-α signaling pathway in human RA synovial fibroblasts (SFs). We demonstrated that miR-17 expression was significantly low in RA serum, SFs, and synovial tissues, as well as in the serum and joints of adjuvant-induced arthritis rats. RNA-sequencing analysis showed modulation of 664 genes by pre-miR-17 in human RA SFs. Ingenuity pathway analysis of RNA-sequencing data identified the ubiquitin proteasome system in the TNF-α signaling pathway as a primary target of miR-17. Western blot analysis confirmed the reduction in TRAF2, cIAP1, cIAP2, USP2, and PSMD13 expression by miR-17 in TNF-α-stimulated RA SFs. Immunoprecipitation assays showed that miR-17 restoration increased the K48-linked polyubiquitination of TRAF2, cIAP1, and cIAP2 in TNF-α-stimulated RA SFs. Thus, destabilization of TRAF2 by miR-17 reduced the ability of TRAF2 to associate with cIAP2, resulting in the downregulation of TNF-α-induced NF-κBp65, c-Jun, and STAT3 nuclear translocation and the production of IL-6, IL-8, MMP-1, and MMP-13 in human RA SFs. In conclusion, this study provides evidence for the role of miR-17 as a negative regulator of TNF-α signaling by modulating the protein ubiquitin processes in RA SFs.

AAA-ATPase p97 suppresses apoptotic and autophagy-associated cell death in rheumatoid arthritis synovial fibroblasts.

Valosin containing protein (p97) is a chaperone implicated in a large number of biological processes including endoplasmic reticulum (ER)-associated protein degradation and autophagy. Silencing of p97 in rheumatoid arthritis (RA) synovial fibroblasts (RASFs) increased the amount of polyubiquitinated proteins, whereas silencing of its interaction partner histone deacetylase 6 (HDAC6) had no effect. Furthermore, silencing of p97 in RASFs increased not only rates of apoptotic cell death induced by TRAIL but also induced an autophagy-associated cell death during ER stress that was accompanied by the formation of polyubiquitinated protein aggregates and large vacuoles. Finally, we demonstrated an anti-arthritic effect of siRNAs targeting p97 in collagen-induced arthritis in rats. Our data indicate that p97 may be a new potential target in the treatment of RA.

Abstract # 1764 Tumor necrosis factor (TNF) increases transient potential receptor type ankyrin (TRPA)-1 expression and function in rheumatoid arthritis synovial fibroblasts: TRPA1 as therapeutic target to reduce joint inflammation in RA?

In collagen-induced arthritis, endocannabinoids, like anandamide, improves synovial inflammation. In previous studies anandamide decreased TNF-induced IL-6, IL-8 and MMP-3 in synovial fibroblasts (SFs) in a TRPA1-dependent manner. Joint pain and inflammation are driven by pro-inflammatory cytokines like TNF, which is also involved in sensitizing TRP-channels. Therefore, the hypothesis was tested that TNF augments TRPA1 mediated effects in SFs. TRPA1 expression after TNF exposure was analyzed by western blotting and cell based ELISA. Cell proliferation was determined by cell titer blue and cell death was monitored by lactate dehydrogenase (LDH) levels. Calcium flux assays were performed with Fura-2. Exposure of SFs to TNF (10 ng/ml) for 72 h increased TRPA1 protein accompanied by an increase in intracellular calcium following TRPA1 agonist allyl isothiocyanate (AITC). While SFs did not respond to AITC without TNF pretreatment, TNF pre-incubation elicited a robust increase in calcium in response to AITC (10 μ M–500 μ M). This was paralleled by increased LDH indicating cell death in response to TRPA1 activation. Cell proliferation was also modulated by TRPA1 activation. While TNF naive cells demonstrated reduced proliferation in response to 100 μ M AITC, TNF pre-treatment lowered the effective concentration to 12.5 μ M. The observed increase in TRPA1 mediated effects by TNF might explain lack of effects of endocannabinoids on basal cytokine production in SFs. Targeting TRPA1 in inflammation might have therapeutic advantages, since it mainly affects SFs pre-activated by pro-inflammatory cytokines.

Dimacrolide Sesquiterpene Pyridine Alkaloids from the Stems of Tripterygium regelii.

Two new dimacrolide sesquiterpene pyridine alkaloids (DMSPAs), dimacroregelines A (1) and B (2), were isolated from the stems of Tripterygium regelii. The structures of both compounds were characterized by extensive 1D and 2D NMR spectroscopic analyses, as well as HRESIMS data. Compounds 1 and 2 are two rare DMSPAs possessing unique 2-(3′-carboxybutyl)-3-furanoic acid units forming the second macrocyclic ring, representing the first example of DMSPAs bearing an extra furan ring in their second macrocyclic ring system. Compound 2 showed inhibitory effects on the proliferation of human rheumatoid arthritis synovial fibroblast cell (MH7A) at a concentration of 20 μM.

Modulation of IL-6 induced RANKL expression in arthritic synovium by a transcription factor SOX5.

Receptor activator of nuclear factor κB ligand (RANKL) is critically involved in bone erosion of rheumatoid arthritis (RA). We previously reported association between younger age at onset of RA and a RANKL promoter SNP that conferred an elevated promoter activity via binding to a transcription factor SOX5. Here we study the regulation of SOX5 levels in relation to RANKL expression in RA synovial fibroblasts (SF) and the development of bone erosion in the collagen-induced arthritis (CIA) mouse. Our data indicated SOX5 levels were higher in synovium and synovial fluid from RA compared to osteoarthritis patients. Pro-inflammatory cytokines upregulated SOX5 and RANKL expression in both primary RA SF and the rheumatoid synovial fibroblast cell line, MH7A. Overexpression of SOX5 resulted in significantly increased RANKL levels, while knockdown of SOX5 resulted in diminished IL-6 mediated RANKL upregulation in MH7A cells. Chromatin immunoprecipitation (ChIP) showed approximately 3-fold enrichment of RANKL-specific DNA in anti-SOX5 immunoprecipitate in IL-6 treated MH7A cells as compared to untreated cells. Locally silencing SOX5 gene significantly diminished RANKL positive cells and bone erosion in CIA mice. These findings suggest SOX5 is an important regulator of IL-6-induced RANKL expression in RA SF.

Analysis of exosomal microRNAs derived from rheumatoid arthritis synovial fibroblasts

Analysis of exosomal microRNAs derived from rheumatoid arthritis synovial fibroblasts