Rheumatoid Arthritis Synovial Fibroblast Cells Research Articles

The Association of Myostatin on the Expression of Inflammatory Cytokines and Chemokines in Rheumatoid Arthritis Synovial Fibroblasts

Rheumatoid arthritis (RA) is a chronic autoimmune disease initiated and progressed by RA synovial fibroblast cells (RASFs) of the synovium. Inflammatory cytokines/chemokines play a crucial role in disease development. Myostatin (MSTN) is widely comprehended as a significant regulator of pro-inflammatory cytokines/chemokines in many other autoimmune diseases. However, its role during the progression of RA is still unclear. We hypothesized that MSTN regulates the inflammatory cytokines/chemokines expression in RASFs. The immortalized RASF cells (MH7A) were treated with MSTN or MSTN inhibitor (0, 10, and 20 ng/mL) for 0, 24, and 48 h. Then, the secretion and expression of inflammatory cytokines (IL-8, IL-17, TNF-α, IL-6, IL-23, IFN-γ, IFN-β) and chemokines (CCL-2, CCL-20, CXCL-13, CXCL-1) were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, MH7A cells were treated with 20 ng/mL of TNF-α, IL-17, IFN-γ IFN-β, CCL-2, and CXCL-1 for 24 h to examine their effects on MSTN expression and secretion. Data were analyzed by Kruskal-Wallis rank sum test, followed by Dunn’s multiple comparison test. The MSTN expression and secretion were measured by RT-qPCR, ELISA, and Western blot. Results showed that 20 ng/mL MSTN treatment significantly (p<0.05) increased the secretion of most inflammatory cytokines/chemokines at 24 or 48 h. CXCL-1 showed the highest secretion (30-fold in average) in response to MSTN treatment, followed by CCL-2 (13-fold in average) at 24 h and IL-23 (3-fold in average) at 48 h. Similar to ELISA results, MSTN significantly upregulated the mRNA expression of most inflammatory cytokines/chemokines at 24 or 48 h. The highest expression was TNF-α (60-fold in average) at 48 h, followed by CXCL-1 (15-fold in average) and CCL-2 (13-fold in average) at 24 h. The effect of MSTN inhibitor on cytokines/chemokines secretion and mRNA expression was inconsistent. Among the six cytokines/chemokines used to investigate their effects on MSTN expression in MH7A cells, IFN-γ significantly increased MSTN secretion (13-fold in average) and mRNA expression (100-fold in average) but other cytokine/chemokines showed no significant response. Western blot results showed that MSTN secretion was significantly increased by IFN-γ treatment, supporting the ELISA results. Current results demonstrated that MSTN upregulated the mRNA expression and secretion of various inflammatory cytokines/chemokines and IFN-γ significantly increased MSTN expression and secretion, indicating a cross-stimulation between MSTN and inflammatory cytokines/chemokines in RA. This study was funded by NIH 5R01AR043521-29. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

New biflavonoids isolated from Xylia kerrii leaves extract with selective cytotoxicity against MH7A human rheumatoid arthritis synovial fibroblast cells.

Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9μM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100μM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.

Successful Transfection of MicroRNA Mimics or Inhibitors in a Regular Cell Line and in Primary Cells Derived from Patients with Rheumatoid Arthritis.

The transfection of microRNA (miRNA) mimics and inhibitors can lead to the gain and loss of intracellular miRNA function, helping us better understand the role of miRNA during gene expression regulation under specific physical conditions. Our previous research has confirmed the efficiency and convenience of using liposomes to transfect miRNA mimics or inhibitors. This work uses miR-424 as an example, to provide a detailed introduction for the transfection process of miRNA mimics and inhibitors in the regular SW982 cell line and primary rheumatoid arthritis synovial fibroblasts (RASF) cells from patients by using lipofection, which can also serve as a reference to miRNA transfection in other cell lines. Key features • MiRNA mimics and inhibitors transfection in regular SW982 cell line and primary RASF cells. • Treatment and culture of RASF primary cells before transfection. • Using liposomes for transfection purposes.

Tofacitinib enhances IGF1 via inhibiting STAT6 transcriptionally activated-miR-425-5p to ameliorate inflammation in RA-FLS.

Rheumatoid arthritis (RA) is a systemic autoimmune disease, which has been reported closely associated with the dysfunction of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. This study aims to explore the potential therapeutic effect of Tofacitinib, a putative JAK/STAT inhibitor, in RA. Tofacitinib suppressed proliferation and accelerated apoptosis of rheumatoid arthritis synovial fibroblasts (RA-FLS) as confirmed by CCK-8, EdU and Western blot assays. Tofacitinib significantly inhibited expression of pro-inflammatory factors including tumor necrosis factor-α (TNF-α), vascular endothelial growth factor A, matrix metalloproteinase 1, matrix metalloproteinase 3, interleukin-6 and interferon gamma in RA-FLS cells. mechanistically, tofacitinib decreased signal transducer and activator of transcription 6 (STAT6), which transcriptionally activates miR-425-5p, and thus increased insulin like growth factor 1 (IGF1) expression, a target of miR-425-5p in RA-FLS. Overexpression of STAT6 restored the expression of pro-inflammatory factors and proliferation inhibited by Tofacitinib in RA-FLS. Overall, Tofacitinib exerted inhibitory effect on proliferation and inflammation of RA-FLS through modulating STAT6/miR-425-5p/IGF1 signal axis. These findings shed light on the novel strategies for improving RA.

Jin-Wu-Jian-Gu Formulation Attenuates Rheumatoid Arthritis by Inhibiting the IL33-ST2 Signaling Pathway.

The present research attempted to investigate the molecular mechanism of Jin-Wu-Jian-Gu Formulation (JWJGF) in inhibiting rheumatoid arthritis (RA) in a pharmacological approach for analysis and experimental validation. First, the potential targets and pathways of JWJGF for RA were predicted by network pharmacology. Second, the effect of JWJGF on RA was observed by hematoxylin-eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA). Further, we observed the effects of JWJGF on the IL33-ST2 signaling pathway by Western blot and quantitative real-time PCR (qPCR) experiments, and finally, we studied the effects of Liquiritigenin on rheumatoid arthritis synovial fibroblast (RASF) cells and the IL33-ST2 signaling pathway. Network pharmacology results showed that the key component of JWJGF was Liquiritigenin and the core target of JWJGF was IL-33. The results of HE and ELISA showed that JWJGF could alleviate RA. Western blot and qPCR findings indicated that JWJGF could inhibit the IL33-ST2 signaling pathway. Furthermore, JWJGF could inhibit the proliferation of RASF cells and the IL33-ST2 signaling pathway. In conclusion, this study revealed that JWJGF attenuated RA by inhibiting the IL33-ST2 signaling pathway.

MicroRNA-449 targets histone deacetylase 1 to regulate the proliferation, invasion, and apoptosis of synovial fibroblasts in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic joint disease. The study aimed to explore the effects of microRNA (miR)-449 and histone deacetylase 1 (HDAC1) on the proliferation, invasion, and apoptosis of synovial fibroblasts in rheumatoid arthritis. Synovial tissue was collected from 20 patients with RA and 20 patients with osteoarthritis (OA) who underwent joint replacement surgery. RA synovial fibroblasts (RASFs) and OA synovial fibroblasts (OASFs) were isolated and cultured. Real-time quantitative PCR was used to detect the expression levels of miR-449 and HDAC1 in synovial tissues and cells. Western blot was performed to detect the cellular expression levels of HDAC1 protein, and apoptosis and invasion-related proteins. The proliferation, invasion, and apoptosis of RASFs were detected by MTT assay, Transwell assay, and flow cytometry. The dual-luciferase reporter gene was used to test the targeting relationship between inflammatory miR-449 and HDAC1. Compared with normal synovial tissue and OASFs, the levels of HDAC1 messenger RNA in RA synovial tissue and RASF cells were significantly increased (P<0.01), while the expression levels of miR-449 were significantly decreased (P<0.01). The dual-luciferase reporter gene experiment confirmed that miR-449 could specifically bind to the 3' untranslated region of HDAC1 to inhibit its luciferase activity (P<0.05). HDAC1 inhibition or miR-449 overexpression significantly inhibited the proliferation and invasion of RASFs (P<0.001), while inducing their apoptosis (P<0.001). HDAC1 overexpression reversed the biological effects of miR-449 on RASFs (P<0.001). miR-449 inhibits the proliferation and invasion of RASFs and induces their apoptosis by targeting HDAC1, thereby exerting a protective effect against RA.

Loss of microRNA-147 function alleviates synovial inflammation through ZNF148 in rheumatoid and experimental arthritis.

MicroRNA-147 (miR-147) had been previously found induced in synoviocytes by inflammatory stimuli derived from Tcells in experimental arthritis. This study was designed to verify whether loss of its function might alleviate inflammatory events in joints of experimental and rheumatoid arthritis (RA). Dark Agouti (DA) rats were injected intradermally with pristane to induce arthritis, and rno-miR-147 antagomir was locally administrated into individual ankle compared with negative control or rno-miR-155-5p antagomir (potential positive control). Arthritis onset, macroscopic severity, and pathological changes were monitored. While in vitro, gain or loss function of hsa-miR-147b-3p/hsa-miR-155-5p and ZNF148 was achieved in human synovial fibroblast cell line SW982 and RA synovial fibroblasts (RASF). The expression of miRNAs and mRNAs was detected by using RT-quantitative PCR, and protein expression was detected by using Western blotting. Anti-miR-147 therapy could alleviate the severity, especially for the synovitis and joint destruction in experimental arthritis. Gain of hsa-miR-147b-3p/hsa-miR-155-5p function in TNF-α stimulated SW982 and RASF cells could upregulate, in contrast, loss of hsa-miR-147b-3p/hsa-miR-155-5p function could downregulate the gene expression of TNF-α, IL-6, MMP3, and MMP13. Hence, such alteration could participate in synovial inflammation and joint destruction. RNAi of ZNF148, a miR-147's target, increased gene expression of TNF-α, IL-6, MMP3, and MMP13 in SW982 and RASF cells. Also, mRNA sequencing data showed that hsa-miR-147b-3p mimic and ZNF148 siRNA commonly regulated the gene expression of CCL3 and DEPTOR as well as some arthritis and inflammation-related pathways. Taken together, miR-147b-3p contributes to synovial inflammation through repressing ZNF148 in RA and experimental arthritis.

Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

BackgroundIn this study, we examined the effect of oxidative stress on cellular energy metabolism and pro-angiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC).MethodsPrimary RASFC and HUVEC were cultured with the oxidative stress inducer 4-hydroxy-2-nonenal (4-HNE), and extracellular acidification rate, oxygen consumption rate, mitochondrial function and pro-angiogenic/pro-inflammatory mechanisms were assessed using the Seahorse analyser, complex I–V activity assays, random mutation mitochondrial capture assays, enzyme-linked immunosorbent assays and functional assays, including angiogenic tube formation, migration and invasion. Expression of angiogenic growth factors in synovial tissue (ST) was assessed by IHC in patients with rheumatoid arthritis (RA) undergoing arthroscopy before and after administration of tumour necrosis factor inhibitors (TNFi).ResultsIn RASFC and HUVEC, 4-HNE-induced oxidative stress reprogrammed energy metabolism by inhibiting mitochondrial basal, maximal and adenosine triphosphate-linked respiration and reserve capacity, coupled with the reduced enzymatic activity of oxidative phosphorylation complexes III and IV. In contrast, 4-HNE elevated basal glycolysis, glycolytic capacity and glycolytic reserve, paralleled by an increase in mitochondrial DNA mutations and reactive oxygen species. 4-HNE activated pro-angiogenic responses of RASFC, which subsequently altered HUVEC invasion and migration, angiogenic tube formation and the release of pro-angiogenic mediators. In vivo markers of angiogenesis (vascular endothelial growth factor, angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]) were significantly associated with oxidative damage and oxygen metabolism in the inflamed synovium. Significant reduction in ST vascularity and Ang2/Tie2 expression was demonstrated in patients with RA before and after administration of TNFi.ConclusionsOxidative stress promotes metabolism in favour of glycolysis, an effect that may contribute to acceleration of inflammatory mechanisms and subsequent dysfunctional angiogenesis in RA.

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.

MicroRNA-26b inhibits cell proliferation and cytokine secretion in human RASF cells via the Wnt/GSK-3β/β-catenin pathway.

BackgroundRheumatoid arthritis (RA) is a chronic systemic auto- immune disease characterized by joint synovitis. Recent evidence suggests that rheumatoid arthritis synovial fibroblasts (RASFs) promote joint destruction. In this study, we investigated the role of microRNA-26b (miR-26b) in cell proliferation and inflammatory cytokine secretion using patient-derived Rheumatoid arthritis fibroblast-like synoviocyte (RAFLS) to understand pathways influencing rheumatoid arthritis.MethodsRAFLS were cultured in vitro and transfected with miR-26b mimics (experimental group) and negative sequence (control group). The protein levels of Wnt4, Wnt5ɑ, GSK-3β, CyclinD1, Ser9-GSK-3β and β-catenin were detected by western blot analysis. Tumor Necrosis Factor-ɑ (TNF-ɑ), IL- 1β, and IL-6 levels were quantified by Enzyme-linked Immunosorbent Assay (ELISA). RAFLS proliferation and apoptosis were measured by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively.ResultsGSK-3β and CyclinD1 expression levels were lower in miR-26b mimic group compared to Mock group and negative control (NC) group. Conversely, GSK-3β and CyclinD1 expression levels were markedly higher in the miR-26b inhibitor group compared to Mock and NC group (P < 0.05). Transfection of miR-26b mimics significantly increased the, levels of Ser9-GSK-3β and β-catenin in comparison to Mock and NC groups, while transfection of miR-26b inhibitors showed the opposite effect. In miR-26b mimic group, TNF-α, IL- 1β and IL-6 levels were lower than the Mock and NC groups, while in miR-26b inhibitor group, these cytokine levels were higher than the Mock and NC groups (P < 0.05). Transfection of miR-26b mimics significantly reduced the cell proliferation of RAFLS, compared to the Mock and NC groups, and miR-26b inhibitors increased the proliferative capacity of RAFLS compared to Mock and NC groups (P < 0.05). The miR-26b mimic group exhibited higher RAFLS apoptosis rate compared to Mock and NC group and miR-26b inhibitor group showed significantly lower RAFLS apoptosis rate compared to Mock and NC groups (P < 0.05).ConclusionsMiR-26b regulates β-catenin and CyclinD1 levels by inhibiting GSK-3β expression, which in-turn alters the Wnt/GSK-3β/β-catenin pathway to lower RAFLS proliferation and elevate cell apoptosis and the secretion of TNF-α,IL-1β and IL-6 cytokines. Therefore, our results show that miR-26B plays a central role in inhibiting the inflammation associated with rheumatoid arthritis.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9063056861547150

Anti-inflammatory effects of a novel ricinoleic acid poloxamer gel system for transdermal delivery

Our previous study showed that the use of ricinoleic acid as an oil phase resulted in the formation of a stable pluronic lecithin organogel (PLO gel) with better thixotropic properties and higher permeation of ketoprofen than the isopropyl palmitate PLO gel. This study aims to evaluate and compare the in vitro and in vivo anti-inflammatory effects of the ricinoleic acid PLO gel system with isopropyl palmitate PLO gel. Ketoprofen was used as a model drug. In vitro anti-inflammatory activity and cell viability tests were performed in human rheumatoid arthritis synovial fibroblast cell line using a blank ricinoleic acid PLO gel and compared to that of the isopropyl palmitate PLO gel. In vivo anti-inflammatory activity of ricinoleic acid PLO gel containing 10% ketoprofen was evaluated and compared with the isopropyl palmitate PLO gel in a carrageenan-induced rat paw edema model. The results from the in vitro study showed that the blank ricinoleic acid PLO gel possessed significantly higher anti-inflammatory activity than isopropyl palmitate PLO gel at 1mM concentration (p<0.05), while both the gel formulations had no significant cytotoxic activity. Further in vivo testing of the formulation showed that the ricinoleic acid PLO gel formulation was significantly more effective in reducing pain and edema when compared to the isopropyl palmitate PLO gel. In addition, the ricinoleic acid PLO gel formulation markedly inhibited the synthesis of prostaglandin E2. In conclusion, the efficacy of PLO gels used in pain management may be enhanced by using ricinoleic acid instead of isopropyl palmitate as an oil phase.

CD38 and E2F transcription factor 2 have uniquely increased expression in rheumatoid arthritis synovial tissues

The purpose of the current study was to find novel rheumatoid arthritis (RA)-specific gene expression by simultaneously comparing the expression profiles of the synovial tissues from patients with RA, osteoarthritis (OA) and ankylosing spondylitis (AS). The Illumina Human HT-12 v4 Expression BeadChip was used to investigate the global gene expression profiles in synovial tissues from RA (n = 12), OA (n = 14) and AS (n = 7) patients. By comparing the profiles in synovial tissues from RA, OA and AS, we identified the CD38, ankyrin repeat domain 38 (ANKRD38), E2F transcription factor 2 (E2F2), craniofacial development protein 1 (CFDP1), cluster of differentiation (CD)7, interferon-stimulated exonuclease gene 20 kDa (ISG20) and interleukin-2 receptor gamma (IL)-2RG genes as differentially expressed gene expression in RA synovial tissues. The increased expression of CD38, E2F2 and IL-2RG, as revealed using real-time polymerase chain reaction (PCR) with synovial tissues from RA (n = 30), OA (n = 26) and AS patients (n = 20), was in agreement with the microarray data. Immunohistochemistry revealed significant CD38 expression and E2F2 in synovial membranes from RA patients (n = 5). The CD38(+) cells had high a percentage in the RA patients' blood (n = 103) and in the CD3(+) and CD56(+) subsets. The CD38(+) cell percentage was correlated significantly with RF level (P = 0·026) in RA patients. The IL-1α and IL-β levels were depressed significantly in the culture medium of RA synovial fibroblast cells (n = 5) following treatment with siRNAs targeting the E2F2 or CD38 genes. This study suggests that the uniquely increased expression of CD38 and E2F2 in RA synovial tissues contribute to the immunoactivation of the disease.

FRI0005 Signal transducer and activator of transcription 3 regulates synovial inflammation through notch/hif1a pathways

BackgroundAngiogenesis is one of the primary events in the pathogenesis of inflammatory arthritis. Notch/HIF signalling interactions regulate angiogenesis and endothelial cell (EC) fate in Rheumatoid Arthritis (RA), however the precise...

AB0040 Il-29 induces production of il-6 and il-8 in rheumatoid arthritis synovial fibroblasts via jak/stat3 and mapk/nf-kb signaling pathways

BackgroundIL-29 (Interferon Lambda 1)is a newly described member of the interferon family, which binds to its receptor to activate JAK-STAT, MAPK or Akt signaling pathways to induce antiviral, antiproliferative, antitumor...

Mitochondrial mutagenesis correlates with the local inflammatory environment in arthritis

BackgroundTo examine the association between mitochondrial mutagenesis and the proinflammatory microenvironment in patients with inflammatory arthritis.MethodsFifty patients with inflammatory arthritis underwent arthroscopy and synovial tissue biopsies, synovial fluid and clinical...

Acute serum amyloid A regulates cytoskeletal rearrangement, cell matrix interactions and promotes cell migration in rheumatoid arthritis

ObjectiveSerum amyloid A (A-SAA) is an acute-phase protein with cytokine-like properties implicated in the pathogenesis of rheumatoid arthritis (RA), atherosclerosis, diabetes and Alzheimer's disease. This study characterises the mechanism of...

POSH promotes cell survival in Drosophila and in human RASF cells

In Drosophila, Eiger, a tumor necrosis factor α (TNFα) superfamily ligand, induces cell death by activating the c-Jun N-terminal kinase (JNK) pathway. Here, we report that overexpression of Plenty of SH3s ( POSH) suppresses Eiger-induced cell death and produces highly deformed tissues. These results imply that high levels of POSH protect tissues from cell death. In humans, rheumatoid arthritis synovial fibroblasts (RASF) are generally resistant to apoptosis. We show that POSH is expressed at relatively high levels in RASF, and its reduction by RNAi sensitizes these cells to Fas-mediated apoptosis. Thus, we demonstrate that POSH promotes cell survival in Drosophila and in human RASF.

Peptidoglycan Enhances IL-6 Production in Human Synovial Fibroblasts via TLR2 Receptor, Focal Adhesion Kinase, Akt, and AP-1- Dependent Pathway

Peptidoglycan (PGN), the major component of the cell wall of Gram-positive bacteria, activates the innate immune system of the host and induces the release of cytokines and chemokines. We investigated the signaling pathway involved in IL-6 production stimulated by PGN in rheumatoid arthritis synovial fibroblasts. PGN caused concentration- and time-dependent increases in IL-6 production. PGN-mediated IL-6 production was attenuated by TLR2 small interfering RNA and nucleotide-binding oligomerization domain 2 small interfering RNA. Pretreatment with PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor, and AP-1 inhibitor (tanshinone IIA) also inhibited the potentiating action of PGN. PGN increased the focal adhesion kinase (FAK), PI3K, and Akt phosphorylation. Stimulation of rheumatoid arthritis synovial fibroblast cells with PGN increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the IL-6 promoter. PGN mediated an increase in the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to AP-1 element was inhibited by Ly294002, Akt inhibitor, and FAK mutant. Our results suggest that PGN increased IL-6 production in human synovial fibroblasts via the TLR2 receptor/FAK/PI3K/Akt and AP-1 signaling pathway.

Down‐regulation of myeloid cell leukemia 1 by epigallocatechin‐3‐gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor α–induced apoptosis

Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor alpha (TNFalpha)-induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin-3-gallate (EGCG) in down-regulating Mcl-1 expression and its mechanism of RA synovial fibroblast sensitization to TNFalpha-induced apoptosis. EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP-ribose) polymerase (PARP), Mcl-1, Bcl-2, Akt, and nuclear translocation of NF-kappaB. In RA synovial fibroblasts, EGCG (5-50 microM) inhibited constitutive and TNFalpha-induced Mcl-1 protein expression in a concentration- and time-dependent manner (P<0.05). Importantly, EGCG specifically abrogated Mcl-1 expression in RA synovial fibroblasts and affected Mcl-1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl-1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down-regulation of the TNFalpha-induced Akt and NF-kappaB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF-kappaB inhibitors. Interestingly, Mcl-1 degradation by EGCG sensitized RA synovial fibroblasts to TNFalpha-induced PARP cleavage and apoptotic cell death. Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFalpha-induced apoptosis by specifically blocking Mcl-1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.

Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1β-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice

IntroductionSecretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis.MethodsGene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1β stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)α, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively.ResultsPIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1β-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1β-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice.ConclusionsOur results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.