Trichostatin A cooperates with Fas-mediated signal to induce apoptosis in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To clarify the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on the growth and survival of rheumatoid arthritis synovial fibroblasts (RA-SF). METHODS: Cell viability was assessed using a WST-8 assay and direct cell counting. Apoptosis was detected by annexin V staining on a flow cytometer. Protein and mRNA expression was determined by Western blotting, flow cytometry, and RT-PCR. RESULTS: TSA suppressed cell growth of RA-SF in a dose-dependent manner, as determined by WST-8 assay and direct cell counting. Other histone deacetylase inhibitors also showed inhibitory effects on RA-SF proliferation. TSA upregulated p21(WAF1/CIP1) cell cycle inhibitor, suggesting that cell cycle arrest is involved in the reduction of cell numbers. In addition, TSA cooperated with Fas-induced pathway to induce cell death, determined by WST-8 assay and annexin V staining. TSA reduced FLICE inhibitory protein (FLIP) expression but not Bcl-2, Bcl-XL, and Fas expression, indicating that the synergistic effect may be through downregulation of FLIP. CONCLUSION: TSA has antirheumatic effects on RA-SF and might be a potential therapeutic tool for the treatment of RA.     

MiR-155在类风湿关节炎滑膜成纤维细胞中的表达及功能研究

目的 检测miR-155在类风湿关节炎(RA)患者滑膜成纤维细胞(SFs)中的表达,探讨miR-155对RASFs细胞因子分泌、细胞增殖、侵袭能力的影响及其机制.方法 留取RA患者及骨关节炎(OA)患者各9份膝关节置换术后滑膜组织,分离培养滑膜成纤维细胞,取第3～5代细胞用于实验.①提取总RNA,利用实时定量聚合酶链反应(RT-PCR)方法测定miR-155在RASFs和OASFs中固有性表达水平.②Lipo2000脂质体分别转染化学合成的miR-155类似物,miR-155抑制剂及无关序列小RNA对照.③转染48 h后通过酶联免疫吸附试验(E12SA)检测细胞上清基质金属蛋白酶(MMP)-3的分泌;通过3H掺入法检测细胞增殖;通过细胞侵袭试验(transwell)法检测细胞侵袭能力;通过RT-PCR法检测miR-155下游靶标IKBKE的mRNA表达水平.2组比较采用独立样本t检验,多组比较采用方差分析.结果 ①RA患者SFs中miR-155的表达明显高于OA组[分别为(1.79±1.94)和(0.11±0.17),P<0.05];②miR-155可抑制RASFs分泌MMP-3、细胞增殖和侵袭能力;③RASFs过表达miR-155后IKBKE的mRNA水平明显下调(P<0.05).结论 RASFs miR-155的表达上调,可能与滑膜局部的炎症环境相关;miR-155抑制RASFs增殖和降低侵袭能力可能是RASFs分泌MMP-3减少的原因之一,miR-155抑制RASFs分泌MMP-3可能与miR-1.55下调靶标IKBKE mRNA表达水平相关.     

Erythromycin suppresses the expression of cyclooxygenase-2 in rheumatoid synovial cells

OBJECTIVE: To investigate whether erythromycin (EM) can suppress the expression of cyclooxygenase-2 (COX-2) in rheumatoid synovial cells, and determine the mechanisms involved. Methods. Synovial tissues were obtained from 25 patients with rheumatoid arthritis (RA). Rheumatoid synovial cells were cultured with or without EM (0.1-1000 nM) in the presence of interleukin 1beta (IL-1beta) for various times. Protein expression of COX-2, and phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) were detected by Western blot. COX-2 messenger RNA (mRNA) was detected by RT-PCR. DNA binding activity of nuclear factor kappa B (NF-kB) was detected by ELISA. Results. IL-1beta-stimulated synovial cells expressed COX-2 protein. EM suppressed the IL-1beta-induced COX-2 protein expression in a dose-dependent manner and inhibited IL-1beta-induced p38 MAPK phosphorylation, which was correlated with COX-2 expression in synovial cells. In contrast, EM had no effect on DNA binding activity of NF-kB and ERK1/2 expression. CONCLUSION: Our results indicated that EM downregulated COX-2 expression by inhibiting the p38 MAPK cascade, but had no effect on NF-kB or ERK1/2, in rheumatoid synovial cells.     

RNAi抑制类风湿关节炎成纤维细胞环氧合酶-2合成及对炎症因子的影响

目的体外合成和筛选特异性阻断人滑膜成纤维细胞（RASF）环氧合酶-2（hCOX-2）的小分子干扰RNA（siRNA），同时了解COX-2抑制对炎症因子表达水平的影响。方法设计4条针对人COX-2mRNA siRNA（1#-4#siRNA），1条随机序列作为对照。分成A～H组，A组为空白阴性对照，B～F组处理依次为随机siRNA、1#-4#siRNA。应用LipofectAMINE 2000将上述siRNA转染入RASF，各培养孔加入100nmol／L的佛波酯。转染48h后，分别应用反转录聚合酶链式反应（RT-PCR）和Western Blot检测hCOX-2mRNA和蛋白表达水平。采用酶联免疫吸附（ELISA）方法检测各组上清液中前列腺素E2（PGE2）和白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、血管内皮生长因子（VEGF）水平。结果4#siRNA转染的RASF表达hCOX-2mRNA和蛋白水平明显低于其他siRNA干预组和阴性对照，且其上清液PGE2和IL-1β、IL-6、TNF-α、VEGF水平较其他各组明显下降。结论4#siRNA能有效抑制COX-2mRNA表达和COX-2蛋白的合成，且上清液中PGE2和IL-1β、IL-6、TNF-α及VEGF水平最低。     

Cordycepin inhibits IL-1{beta}-induced MMP-1 and MMP-3 expression in rheumatoid arthritis synovial fibroblasts

Objective. MMP is a key enzyme in the degradation of extracellularmatrices, and its expression plays important roles in inflammatorydiseases. Cordycepin (3'-deoxyadenosine), a bioactive compoundof Cordyceps militaris, has been shown to exhibit many pharmacologicalactivities, such as anti-cancer, anti-inflammatory and anti-infectionactivities. In this study, we aimed at the inhibitory effectof cordycepin on IL-1β-induced MMP-1 and MMP-3 expressionas well as the molecular basis using RA synovial fibroblasts(RASFs). Methods. RASFs were isolated from synovial tissue obtained from12 patients with RA and cultured in monolayer. Expression ofMMP-1 and MMP-3 was evaluated using western blotting and real-timePCR. Chemokines were analysed by ELISA. The phosphorylationof mitogen-activated protein kinase was measured by westernblotting. Electrophoretic mobility shift assay was performedto evaluate binding activities of DNA to nuclear factor-B (NF-B)and activator protein-1 (AP-1). Results. Cordycepin inhibited IL-1β-induced MMP-1 and MMP-3expressions in RASFs in a dose-dependent manner. Among variouschemokines [such as monocyte chemoattractant protein-1 (MCP-1),GRO-, regulated upon activation, normal T-cell expressed andpresumably secreted (RANTES) and epithelial neutrophil activatingpeptide 78 (ENA-78)], cordycepin specifically blocked IL-1β-inducedENA-78 production in RASF. Moreover, cordycepin significantlyinhibited IL-1β-induced p38/JNK and AP-1 activation, butnot extracellular signal-regulated kinase (ERK) and NF-B activation. Conclusions. Cordycepin is a potent inhibitor of IL-1β-inducedchemokine production and MMP expression and strongly blocksthe p38/JNK/AP-1 signalling pathway in RASFs. KEY WORDS: Cordycepin, Interleukin-1β, Matrix metalloproteinase, p38 mitogen-activated protein kinase, Activator protein-1