The cyclooxygenase-2/thromboxane A2 pathway: a bridge from rheumatoid arthritis to lung cancer?

Patients with rheumatoid arthritis (RA) appear to be at a higher risk of lung cancer (LC). Although the connection between RA and LC has been an active area of research for many years, the molecular pathogenesis of the disease process remains unclear. The cyclooxygenase (COX)-2/thromboxane A2 (TxA2) pathway has been shown to play a potential role in LC development through an auto-regulatory feedback loop. An increased level of TxA2 has been found in RA patients, and intriguingly, the positive feedback loop for the COX-2/TxA2 pathway was shown to have a potential function in RA fibroblast-like synoviocytes (RA-FLS). Thus, the molecular basis of LC development in patients with RA has been at least in partly described. It is possible that COX-2-derived TxA2 could be monitored for the early detection of LC in RA patients, and targeting this molecular pathway may decrease the risk of LC in patients with RA.     

佐剂诱导关节炎大鼠模型的建立及成纤维样滑膜细胞的分离

目的建立类风湿性关节炎(RA)动物模型;从炎性关节滑膜中分离成纤维样滑膜细胞(FLS)。方法应用热灭活结核杆菌H37Ra菌株与矿物油混合制备改良的佐剂,尾根部皮内注射Lewis大鼠诱导关节炎;剪取成功诱导关节炎大鼠的病变踝关节,从中剥离滑膜组织,充分剪碎后采用胶原酶消化法分离成纤维样滑膜细胞。结果成功诱导了大鼠关节炎,发病率为100%,发病时间有规律,组织学表现与RA相似;成功在体外培养了FLS并对其进行了鉴定,掌握了其生长形态和特征。结论成功制备RA动物模型并获得FLS,为今后RA发病机制探索和药物评价提供了良好的体内动物模型和体外细胞模型。     

甘草附子汤对佐剂性关节炎小鼠滑膜成纤维样细胞增殖的影响

目的:观察佐剂性关节炎(AA)小鼠滑膜组织成纤维样细胞(FLS)增殖的变化及甘草附子汤对其的影响。方法:将48只雄性Balb/c小鼠随机分为正常组,模型组,甘草附子汤组(7.8 g·kg~(-1)),雷公藤多苷组(10 mg·kg~(-1))。采用AA小鼠模型,模型成功后,治疗组小鼠给予相应给药灌胃18 d,1次/d,正常组和模型组小鼠灌胃等体积生理盐水。采用足肿胀法评价药物治疗作用;苏木素-伊红(HE)染色观察各组踝关节组织病理变化;Vimentin免疫组化法检测滑膜组织成纤维样增殖;免疫荧光法检测肿瘤坏死因子-α(TNF-α)表达水平以及蛋白免疫印迹法(Western blot)检测滑膜组织中细胞周期蛋白D_1(Cyclin D_1),增殖细胞核抗原(PCNA),p53和p21蛋白表达。结果:与正常组比较,模型组足肿胀度增加,滑膜增生和骨侵蚀明显,滑膜组织成纤维样细胞增殖率升高(P0.05),滑膜组织中TNF-α,Cyclin D_1,PCNA蛋白表达明显升高(P0.05),p53和p21蛋白表达降低(P0.05)。与模型组比较,甘草附子汤组小鼠足肿胀,滑膜增生和骨侵蚀有所改善,滑膜组织成纤维样细胞增殖率降低(P0.05),滑膜组织中TNF-α水平,Cyclin D_1,PCNA蛋白表达显著降低,而p53和p21蛋白表达升高(P0.05)。结论:甘草附子汤对AA小鼠关节和滑膜损伤具有明显改善作用,可能与降低局部慢性炎症,调节AA小鼠滑膜Cyclin D_1,PCNA,p53和p21的蛋白异常表达,抑制FLS增殖有关。     

Suppression of IL-6 biological activities by activin A and implications for inflammatory arthropathies

Activin A is a cytokine whose multiple functions have yet to be fully determined. In this study, the role of proinflammatory cytokines in regulatory control of activin A production was shown in synoviocytes and chondrocytes. Additional facets of functional inflammation-related activities of activin A were also determined. Results showed that activin A concentrations in the synovial fluid of patients with rheumatoid arthritis and gout were elevated relative to those in patients with osteoarthritis. Further studies showed that production of activin A by synoviocytes and chondrocytes in culture was stimulated by cytokines such as IL-1, transforming growth factor-beta (TGF-β), interferon-gamma (IFN-γ), and IL-8, consistent with previous studies in regard to the control of activin A production in marrow stromal cells and monocytes by cytokines, glucocorticoids and retinoic acid. In addition, the relationship of activin A to IL-6-induced biological activities was investigated. Three major IL-6 activities involved in inflammatory responses were found to be suppressed by activin A. In a dose-dependent manner, activin A efficiently suppressed IL-6-induced proliferation of 7TD1 B lymphoid cells, phagocytic activity of monocytic M1 cells, and fibrinogen production in HepG2. Therefore, it is likely that activin A serves as a suppressor for IL-6, dampening inflammatory responses, and has the potential to perform some previously unrecognized roles in inflammation.     

Dexmedetomidine inhibits the invasion,migration, and inflammation of rheumatoid arthritis fibroblast-like synoviocytes by reducing the expression of NLRC5

Rheumatoid arthritis (RA) is a chronic, autoimmune disease characterized by inflammatory synovitis, but its pathogenesis remains unclear. NLRC5 is a newly discovered member of the NLR family that is effective in regulating autoimmunity, inflammatory responses, and cell death processes. Dexmedetomidine (DEX) has been reported to have a variety of pharmacological effects, including anti-inflammatory and analgesic effects. However, the role of DEX in RA has not been explored. In adjuvant-induced arthritis (AA) rat models, DEX (10 μg/kg and 20 μg/kg) reduced the pathological score, the arthritis score, paw swelling volume, and the serum levels of IL-1β, IL-6, IL-17A, and TNF-α. Moreover, by using Western blot and real-time quantitative PCR (RT-qPCR), it was demonstrated that DEX can inhibit the expression of IL-1β, IL-6, MMP-3, MMP-9 and P-P65 in the synovial tissue of AA rats. In human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs), DEX (250 nM and 500 nM) was found to inhibit the expression of IL-1β, IL-6, MMP-3, MMP-9, and P-P65 following stimulation with TNF-α. Moreover, DEX can inhibit the invasion and migration of RA-FLSs stimulated by TNF-α. Finally, the expression of NLRC5 in RA-FLSs and AA rat models was also reduced by DEX. After silencing NLRC5 in RA-FLSs, the expression of IL-1β, IL-6, MMP-3, MMP-9, and P-P65, as well as the invasion and migration of cells, were significantly reduced. These results indicate that DEX inhibits the invasion, migration, and inflammation of RA-FLSs by reducing the expression of NLRC5 and inhibiting the NF-κB activation.     

程氏蠲痹汤通过抑制PI3K/Akt/mTOR信号轴促进类风湿关节炎滑膜成纤维细胞的自噬

目的 研究程氏蠲痹汤（JBT）通过PI3K/Akt/mTOR信号轴调控类风湿关节炎滑膜成纤维细胞（RA-FLS）自噬的影响及相关机制。方法 实验分为4组：FLS组、FLS+JBT组、FLS+RAPA组、FLS+RAPA+JBT组。CCK8检测程氏蠲痹汤抑制RA-FLS的最佳浓度和时间,透射电镜观察细胞自噬小体的变化,自噬双标腺病毒实验观察细胞内自噬体、自噬溶酶体数量,RT-qPCR和Western blot检测相关指标表达水平。结果 CCK8实验筛选结果表明JBT冻干粉浓度在0.5 mg/mL,培养12 h效果最佳。电镜结果与自噬双标腺病毒实验结果趋势相同,FLS组中自噬体少量存在,JBT加入能显著增加自噬体,自噬溶酶体较少。RT-qPCR结果显示,FLS+RAPA+JBT组较FLS组、FLS+JBT组、FLS+RAPA组相比,PI3K、Akt和mTOR的mRNA水平降低更明显（P<0.01）。WB分析显示,FLS+RAPA+JBT组较FLS组、FLS+JBT组、FLS+RAPA组相比,PI3K、Akt、mTOR蛋白的水平无明显差异（P>0.05）,p-PI3K、p-Akt...     

青蒿琥酯联合甲氨蝶呤对类风湿关节炎滑膜细胞增殖和凋亡的影响

目的观察不同浓度的青蒿琥酯（ART）和甲氨蝶呤（MTX）对类风湿关节炎（RA）滑膜细胞增殖和凋亡的影响。方法收集RA关节液标本25例,体外分离培养滑膜细胞,以生长良好的3～5代滑膜细胞进行试验,调整细胞浓度,分为对照组和不同质量浓度ART组、MTX组。用四甲基偶氮唑盐比色法测定不同质量浓度的MTX和ART单独或联合作用于滑膜细胞对滑膜细胞增殖的影响;用流式细胞术检测不同质量浓度的MTX和ART单独或联合作用于滑膜细胞对滑膜细胞凋亡的影响。结果ART对滑膜细胞的增殖具有显著的抑制作用。ART质量浓度在3.125～50μg/mL范围内均可抑制RA滑膜细胞生长,且抑制作用随质量浓度增大而增强,具有剂量依赖性;ART12.5μg/mL＋MTX20μg/mL组的抑制作用最强,与其他药物组比较,P〈0.05。各药物作用于滑膜细胞24h后,早期凋亡细胞率与对照组比较,有显著差异（P〈0.05）;ART质量浓度在3.125～25μg/mL范围内作用于滑膜细胞24h后,早期凋亡细胞率与未用ART处理的对照组比较,有显著差异（P〈0.05）,并呈剂量依赖性;当ART的质量浓度达到50μg/mL时,早期凋亡细胞率降低,坏死细胞率却升高（P〈0.05）;ART12.5μg/mL＋MTX5μg/mL组的早期凋亡细胞率较其他药物组显著升高（P〈0.05）,而坏死细胞率却较低（P〈0.05）。结论ART和MTX单独或联合作用均可显著抑制RA滑膜细胞的增殖,诱导其凋亡。     

丹参体外抑制类风湿关节炎成纤维样滑膜细胞增殖的研究

目的 检测丹参体外对类风湿关节炎（RA）成纤维样滑膜细胞（FLSs）增殖的影响。方法 用不同浓度丹参处理RA FLSs 24 h后,采用MTT法检测RA FLSs的增殖水平;用流式细胞仪检测其凋亡百分率。结果 加入不同浓度丹参处理RA FLSs 24 h后,细胞增殖受到抑制（P＜0.05）,并在一定丹参浓度范围内（0～1.56 mg/ml）呈现剂量相关性,当丹参浓度达到1.56 mg/ml时,细胞增殖抑制达到平台期。对照组与不同浓度丹参处理组凋亡细胞百分率比较,在丹参浓度为0.195 mg/ml时差异不明显,当丹参浓度为0.39 mg/ml时差异有显著统计学意义（P＜0.05）。结论 丹参在体外抑制RA FLSs增殖,可诱导细胞凋亡。     

RICTOR在肿瘤坏死因子-α参与的类风湿关节炎成纤维样滑膜细胞活化中的作用

目的 初步探讨RICTOR在RA-成纤维样滑膜细胞(FLS)活化机制中的作用.方法 收集3例RA患者滑膜组织,应用组织块法培养FLS.分别以浓度为10、20 ng/ml的TNF-α处理RA-FLS,利用蛋白印迹法检测RICTOR的蛋白表达水平.应用阳离子脂质体把特异性RICTOR siRNA转染RA-FLS后,再以10 ng/ml的TNF-α处理RA-FLS,使用蛋白印迹法检测RICTOR的蛋白表达水平,四甲基偶氮唑盐(MTT)法检测RA-FLS活性抑制率.统计学处理采用单因素方差分析及LSD-t检验.结果 TNF-α处理RA-FLS 48 h后,RICTOR的蛋白表达较阴性对照组明显升高(均P＜0.05).转染RICTOR siRNA后,RICTOR蛋白表达:对照组＜TNF 10 ng/ml组＜TNF 20 ng/ml组,吸光度相对值依次为0.35±0.06、0.60±0.09、1.10±0.12,组间两两比较差异有统计学意义(均P＜0.05).转染RICTOR siRNA后,阴性对照组、RICTOR(-)/TNF-α(-)组、RICTOR (-)/TNF-α(+)组RICTOR蛋白吸光度相对值分别为0.498 4±0.140 1、0.012 8±0.002 0、0.042 5±0.027 3,MTT检测阴性对照组、RICTOR(-)/TNF-α(-)组、RICTOR(-)/TNF-α(+)组、RICTOR(+)/TNF-α(+)组细胞活性抑制率分别为(0.7±2.0)％、(13.3±4.5)％、(15.5±4.9)％、(-7.7±5.2)％,显示无论是否加入TNF-α刺激,RA-FLS中RICTOR的蛋白表达水平均较转染阴性对照siRNA组明显下降,且RA-FLS活性抑制率较转染阴性对照siRNA组明显增高(均P＜0.05).结论 沉默RICTOR所引起的RA-FLS活性抑制不能被TNF-α逆转,提示RICTOR可能在TNF-α参与的RA-FLS增殖活化机制中起着重要作用.     

白头翁皂苷对佐剂性关节炎大鼠FZD8表达的影响

研究中药白头翁有效成分白头翁皂苷(pulchinenoside,PULC)对佐剂性关节炎(adjuvant arthritis,AA)大鼠Frizzled(FZD)表达的影响。采用足跖注射完全弗氏佐剂制备AA大鼠,原代培养AA大鼠滑膜成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS),Real-time qPCR检测PULC灌胃治疗对AA大鼠FLS FZD8表达的影响,MTT,ELISA检测FZD8敲除对FLS增殖和IL-1,IL-6,IL-8表达的影响,Real-time qPCR检测miR-375在FZD8异常表达中的作用。检测发现经PULC灌胃治疗后,AA大鼠FLS中FZD8表达明显降低,FZD8敲除后FLS增殖明显减弱,IL-1,IL-6,IL-8表达明显减少,PULC灌胃治疗后miR-375表达明显升高,上调表达的miR-375能够抑制FZD8的表达。PULC可能通过上调miR-375表达抑制FZD8的表达。