Effect of a small molecule inhibitor of nuclear factor-κB nuclear translocation in a murine model of arthritis and cultured human synovial cells

A small cell-permeable compound, dehydroxymethylepoxyquinomicin (DHMEQ), does not inhibit phosphorylation and degradation of IκB (inhibitor of nuclear factor-κB [NF-κB]) but selectively inhibits nuclear translocation of activated NF-κB. This study aimed to demonstrate the antiarthritic effect of this novel inhibitor of the NF-κB pathway in vivo in a murine arthritis model and in vitro in human synovial cells. Collagen-induced arthritis was induced in mice, and after onset of arthritis the mice were treated with DHMEQ (5 mg/kg body weight per day). Using fibroblast-like synoviocyte (FLS) cell lines established from patients with rheumatoid arthritis (RA), NF-κB activity was examined by electrophoretic mobility shift assays. The expression of molecules involved in RA pathogenesis was determined by RT-PCR, ELISA, and flow cytometry. The proliferative activity of the cells was estimated with tritiated thymidine incorporation. After 14 days of treatment with DHMEQ, mice with collagen-induced arthritis exhibited decreased severity of arthritis, based on the degree of paw swelling, the number of swollen joints, and radiographic and histopathologic scores, compared with the control mice treated with vehicle alone. In RA FLS stimulated with tumor necrosis factor-α, activities of NF-κB components p65 and p50 were inhibited by DHMEQ, leading to suppressed expression of the key inflammatory cytokine IL-6, CC chemokine ligand-2 and -5, matrix metalloproteinase-3, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. The proliferative activity of the cells was also suppressed. This is the first demonstration of an inhibitor of NF-κB nuclear translocation exhibiting a therapeutic effect on established murine arthritis, and suppression of inflammatory mediators in FLS was thought to be among the mechanisms underlying such an effect.     

NF-kappaB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor

Inhibition of NF-kappaB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-kappaB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-kappaB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-kappaB inhibitors to rheumatoid arthritis therapy.     

Geldanamycin induces apoptosis and inhibits inflammation in fibroblast-like synoviocytes isolated from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblasts-like synoviocytes (FLS), major cells in the synovium, together with infiltrated leukocytes, contribute greatly to RA progression. In our study, we hypothesized that geldanamycin (GA), a cancer drug might be able to inhibit RA FLS growth. To test the idea, RA FLS were isolated and cultured for cancer drug test. The results showed that GA can specifically inhibit the growth of RA FLS compared with normal FLS. Essentially, GA was found to promote reactive oxygen species production in RA FLS and induce programmed cell death. The annexinV/propidium iodide and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining confirmed that GA can directly induce apoptosis and subsequently inhibit the growth of RA FLS, which was also confirmed by Western blot assay. In addition, our data demonstrated that inflammation was inhibited by suppressed nuclear factor κB signaling pathway. The therapeutic effect of GA was explored in collagen-induced arthritis mice. In short, GA was a promising drug for the treatment of RA by specifically inhibiting the proliferation and inflammation of RA FLS.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.     

Guggulsterone blocks IL-1beta-mediated inflammatory responses by suppressing NF-kappaB activation in fibroblast-like synoviocytes

Guggulsterone is a plant sterol that is used to treat hyperlipidemia, arthritis, and obesity. Although its anti-inflammatory and anti-hyperlipidemic effects have been well documented, the effect of guggulsterone on fibroblast-like synoviocytes (FLS) has not yet been reported. Therefore, in this study, the effect of guggulsterone on interleukin (IL)-1beta-induced inflammatory responses in the FLS of rheumatic patients was investigated. Treatment of FLS with IL-1beta induced production of chemokines such as RANTES and ENA-78. In addition, Western blot analysis and gelatin zymography revealed that IL-1beta activated matrix metalloproteinase (MMP)-1 and -3 in FLS. However, pre-incubation with guggulsterone completely inhibited the ability of IL-1beta to induce the production of chemokines and to activate MMPs. Although the NF-kappaB binding activity and nuclear p50 and p65 subunit levels, as well as IkappaBalpha degradation in the cytoplasm was greater in cells stimulated with IL-1beta than in unstimulated cells, treatment with guggulsterone abolished all of these increases. Collectively, these results suggest that guggulsterone would be useful as an inhibitor of joint destruction in patients with rheumatoid arthritis.     

PDCD5在类风湿关节炎成纤维样滑膜细胞凋亡中表达上调

为了研究程序化细胞死亡因子5(PDCD5)在类风湿关节炎成纤维样滑膜细胞凋亡中的作用,在不同的时间加入有效剂量100 nmol/L雷公藤内醇酯（triptolide）后,采用实时定量PCR、RT-PCR、Western 印迹和直接免疫荧光染色方法检测体外分离培养的类风湿关节炎成纤维样滑膜细胞中PDCD5在mRNA和蛋白水平的表达及蛋白表达特征.在雷公藤内醇酯诱导类风湿关节炎成纤维样滑膜细胞凋亡的过程中,PDCD5mRNA表达水平明显地渐次增加,呈现一种明确的时间依赖性递增表达模式,而PDCD5蛋白有时间依赖性表达上调持续16 h,并维持在相对恒定水平.直接免疫荧光染色结果显示,在正常体外培养的类风湿关节炎成纤维样滑膜细胞中,PDCD5蛋白的表达较弱,且主要分布在细胞浆.经雷公藤内醇酯处理4 h后,大多数细胞有PDCD5蛋白的聚集,直至12 h,细胞核周围PDCD5蛋白聚集显著增强.36 h后,PDCD5蛋白以核固缩的形式存在于凋亡的RA FLS中,细胞核染色质明显浓缩,片段化并出现了凋亡小体.上述结果表明,在类风湿关节炎成纤维样滑膜细胞凋亡的过程中,PDCD5表达上调并在凋亡早期出现核转位,PDCD5蛋白核转位要早于凋亡小体形成.PDCD5蛋白核转位是类风湿关节炎成纤维样滑膜细胞凋亡的早期事件,PDCD5不仅参与了类风湿关节炎成纤维样滑膜细胞的凋亡过程,而且在类风湿关节炎滑膜增生的凋亡调节中起到重要调节作用.     

Interleukin-35 (IL-35) inhibits proliferation and promotes apoptosis of fibroblast-like synoviocytes isolated from mice with collagen-induced arthritis

Rheumatoid arthritis (RA) is an inflammatory disorder of the joints that affects 0.5–1 % of adults. Excessive growth of the fibroblast-like synoviocytes (FLS) promotes hyperplasia of synovial tissues and causes its invasion into the bone and cartilage, which eventually causes deformity and dysfunction of affected joints. Interleukin 35 (IL-35) was shown to suppress the inflammatory responses to collagen-induced arthritis (CIA) via upregulation of T regulatory cells and suppression of T helper type 17 cells in a mouse model. To study the effects of IL-35 on the proliferation and apoptosis frequency of cultured FLS isolated from mice with CIA as well as to examine the effects of IL-35 on CIA in vivo. Thirty DBA/1 J mice, which are used as an animal model for RA, were divided randomly (ten mice per group) to a CIA group (collagen treatment), a CIA + IL-35 group (collagen and IL-35 treatments), and a control group (no treatment). Starting on the 24th day after collagen administration, IL-35 was injected intraperitoneally into mice of the CIA + IL-35 group once per day for 10 days. An arthritis index was calculated, and pathological analysis of synovial tissue was performed. FLS isolated from CIA mice were treated with various concentrations of IL-35 (12.5–100 ng/ml). The MTT assay was used to examine FLS proliferation, and apoptosis frequency of FLS was detected by flow cytometry. On day 24, the CIA mice began to exhibit arthritis symptoms, and the symptoms rapidly progressed with time. Treatment with IL-35 significantly alleviated arthritis symptoms and reduced the synovial tissue inflammation. In addition, IL-35 treatment inhibited proliferation and promoted apoptosis in cultured FLS from CIA mice in a dose-dependent manner. IL-35 could ameliorate the symptoms of arthritis in the CIA mouse model in vivo and inhibited FLS proliferation while promoting FLS apoptosis in vitro, thereby exhibited the potential in inhibiting the progression of RA.     

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a major negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.     

Local treatment with the selective IkappaB kinase beta inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-kappaB is a key regulator of synovial inflammation. We investigated the effect of local NF-kappaB inhibition in rat adjuvant arthritis (AA), using the specific IkappaB kinase (IKK)-beta blocking NF-kappaB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-beta-induced IkappaB alpha phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-alpha and IL-1-beta in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-beta-induced TNF-alpha production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-alpha-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-kappaB blockade using a small peptide inhibitor of IKK-beta has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-beta-targeted NF-kappaB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.