Effects of bee venom on protease activities and free radical damages in synovial fluid from type II collagen-induced rheumatoid arthritis rats

The effect of bee venom acupunture (BVA) (api-toxin) on the development of type II collagen (CII)-induced arthritis (CIA) in rats has been studied. We have compared the levels of activity of a comprehensive range of cytoplasmic, lysosomal and matrix protease types, together with the levels of free radical-induced protein damage (determined as protein carbonyl derivative) in synovial fluid from CIA-treated, BVA-treated and normal rats. Many protease types showed significantly increased activity in CIA compared with normal rats. BVA (5 and 10 μl/100 g) significantly reduced these enzyme activities by some 80% each, but levels of plasma proteases activity (including those enzyme types putatively involved in the immune response, such as dipeptidyl aminopeptidase IV and proline endopeptidase) in CIA, BVA (5 μl/100 g)-treated and normal plasma samples were not significantly different. The level of free radical induced damage to synovial fluid proteins was approximately three-fold higher in CIA compared with normal rats. However, BVA (5 μl/100 g) significantly decreased the level of reactive oxygen free radical species (ROS) induced oxidative damage to synovial fluid proteins. It was concluded that activation of proteolytic enzymes and free radicals are likely to be of equal potential importance as protein damaging agents in the pathogenesis of rheumatoid arthritis (RA), and the development of novel therapeutic strategies for the latter disorder should include both protease inhibitory and free radical scavenging elements. In addition, the protease inhibitory element should be designed to inhibit the action of a broad range of enzymatic mechanistic types (cysteine, serine, metallo proteinases and peptidases). In conclusion, BVA is considered to be an effective RA modulator, inhibiting protease activities and removing ROS.     

类风湿关节炎新靶点药物治疗进展

类风湿关节炎(RA)是一种异质性疾病,多种细胞参与发病并最终导致关节破坏,但其病理生理过程尚不明确,可能涉及遗传和(或)环境因素。近年来,随着基础研究深入,RA治疗有了显著进展,除传统抗风湿药物(DMARDs),生物制剂已扩展了RA治疗手段。笔者主要综述近年来该领域药物治疗现状。     

Honeybee venom induces calcium-dependent but caspase-independent apoptotic cell death in human melanoma A2058 cells.

Honeybee (Apis mellifera) venom (BV) has been reported to exhibit anticancer effects, but its mode of action at the cellular and molecular levels remains largely unknown. We found that honeybee venom induced apoptosis in human melanoma A2058 cells but not in normal skin fibroblast Detroit 551 cells. The BV-induced apoptosis was accompanied by generation of reactive oxygen species and alteration of mitochondrial membrane potential transition. Treatment with antioxidants significantly attenuated BV-induced apoptosis. Although caspase-2 and -3 were slightly activated by BV, inhibitors of caspase-2 and -3 could not block BV-induced apoptosis in A2058 cells. Data from immunostaining indicated that EndoG and AIF were translocated from mitochondria to the cytosol or nucleus, suggesting that BV induces apoptosis in A2058 cells via a caspase-independent pathway. In addition, cJun N-terminal kinases (JNK) and ERK were rapidly activated after a 5min incubation with BV, while p38 and AKT were inactivated after 30min administration of BV. Inhibition of JNK significantly attenuated BV-triggered apoptotic death. Moreover, BV induced a rapid and marked increase in cytosolic calcium ion. Incubation of cells under calcium-free conditions effectively diminished BV-induced apoptosis. Furthermore, when the calcium-free treatment was combined with ouabain, the recovery of cellular calcium fluctuation protected A2058 cells against BV-induced apoptosis. Finally, treatment of A2058 cells with melittin, the major component of BV, resulted in similar elevation of calcium levels and cell killing effects, suggesting that melittin is the major determinant in BV-triggered cell death. These observations provide a molecular explanation for the antiproliferative properties of BV, and suggest that this agent may be useful in treating melanoma.     

Binding of piroxicam to synovial fluid and plasma proteins in patients with rheumatoid arthritis

Summary The protein binding of piroxicam in synovial fluid and plasma from patients with rheumatoid arthritis was studied in vitro by equilibrium dialysis. The binding parameters were calculated from the experimental data with the Scatchard model, assuming binding to two classes of sites. Each plasma sample was diluted to an albumin concentration equal to that of synovial fluid from the same patient. The association constants for primary and secondary binding sites in the concentration range of piroxicam 4.5–90·10^–5 mol/l were similar in synovial fluid and in plasma. For synovial fluid K₁=2.38·10⁵ l/mol and K₂=2.29·10³ l/mol; for plasma K₁=1.93·10⁵ l/mol and K₂=2.08·10³ l/mol. The number of binding sites was also the same in the two fluids. Although the concentration of piroxicam in synovial fluid was about half that in plasma, the binding of piroxicam to protein in synovial fluid was the same as in plasma.     

Bee venom induced cytogenetic damage and decreased cell viability in human white blood cells after treatment in vitro: a multi-biomarker approach

The aim of this study was to evaluate cytogenotoxic effects of bee venom to human lymphocytes and take a look into the mechanisms behind them. Bee venom was tested in concentrations ranging from 0.1 μg/ml to 20 μg/ml over different lengths of time. Cell viability, type of the cell death, and morphological alterations were evaluated using phase-contrast and fluorescent microscopy in addition to DNA diffusion assay, whereas cytogenotoxic effects were assessed with the micronucleus test. DNA damage and its relation to oxidative stress were evaluated combining the standard alkaline and the Fpg-modified comet assay. Our results showed lower cell viability, morphological cell alterations, cytogenotoxicity, and dominantly necrotic type of cell death in human lymphocytes after treatment with bee venom. All the effects were time- and dose-dependent. These results provide an insight into the effects of bee venom on the cell structure that could be relevant for therapeutic purposes.     

类风湿关节炎灭活滑膜液单个核细胞对非灭活滑膜液单个核细胞分泌IL-10水平的影响

目的观察类风湿关节炎灭活滑膜液单个核细胞对非灭活滑膜液单个核细胞分泌IL-10水平的影响。方法抽取类风湿关节炎(RA)患者关节液,分离关节液单个核细胞,分为对照组:2×10~6的非灭活单个核细胞,培养条件为37℃,5%CO_2孵育箱培养48h;实验组:细胞数为2×10~6个非灭活单个核细胞与甲醛灭活后的单个核细胞按1:10混合培养.用ELISA法测定培养上清中IL-10的水平.结果实验组较对照组IL-10水平明显升高(P<0.01).结论灭活滑膜液单个核细胞对非灭活滑膜液单个核细胞IL-10分泌有促进作用,提示未经克隆选择的RA滑膜液混合细胞可能具有诱导分泌IL-10CD_4调节性T细胞的生成作用.     

Excitatory amino acid transporters expressed by synovial fibroblasts in rats with collagen-induced arthritis

Although previous studies have demonstrated increased levels of the brain neurotransmitter glutamate (Glu) in the synovial fluid from patients with arthritis, not much attention has been paid to the possible role of Glu in joint synovial tissues to date. Constitutive expression of mRNA was for the first time shown with glutamate aspartate transporter, glutamate transporter-1 and excitatory amino acid carrier-1 (EAAC1), in addition to with particular ionotropic and metabotropic Glu receptors, in cultured synovial fibroblasts prepared from knee joints of male Lewis rats. Immunohistochemical analysis revealed high localization of immunoreactive EAAC1 at synovial tissues. The accumulation of [³H]Glu occurred in a temperature- and sodium-dependent manner in cultured synovial fibroblasts, with a K_m of 23.1 ± 1.1 μM and a V_max of 237.1 ± 31.1 pmol/(mg protein min), respectively. In rats with arthritis induced by immunization to type-II collagen, marked increases were seen in hind paw volume, cytokine mRNA expression and Glu levels in synovial tissues, in addition to histological erosion. In cultured synovial fibroblasts prepared from these arthritic rats, [³H]Glu accumulation was drastically increased with biochemical and pharmacological profiles similar to those seen in normal synovial fibroblasts. The exposure to Glu at 500 μM doubled the incorporation of 5-bromo-2′-deoxyuridine in cultured synovial fibroblasts of arthritic but not normal rats, without significantly affecting mRNA expression of different cytokines in both synovial fibroblasts. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation through particular transporters functionally expressed by synovium in rheumatoid arthritis.     

Plasma and synovial fluid meclofenamic acid concentrations in patients with rheumatoid arthritis of the knee

Summary We have measured plasma and synovial fluid concentrations of meclofenamic acid at 2, 4, 8, and 12 h during steady-state administration (100 mg three times daily for 4–7 days). Paired plasma and synovial samples were obtained pre-treatment and at one of the above times in twelve patients with a diagnosis of rheumatoid arthritis. In addition, the extent of protein binding of meclofenamic acid was assessed in vitro in the pre-treatment plasma and synovial fluid specimens.Peak total concentrations of 1.73 and 0.86 µg·ml^–1 were observed in plasma (at 2 h) and synovial fluid (at 4 h) respectively. The extent of protein binding was 99.7 and 99.6% (not significantly different) in plasma and synovial fluid respectively.The results of this study are compared to those from similar reported studies of other nonsteroidal anti-inflamatory compounds.     

Characterization of salicylate binding to synovial fluid and plasma protein in patients with rheumatoid arthritis

Summary Protein binding of salicylate in synovial fluid and plasma from patients with rheumatoid arthritis was studied by equilibrium dialysis. Protein binding in the synovial fluid was considerably lower at all salicylate concentrations studied (0.07 – 2.2 mM). Scatchard plots of the data were analyzed assuming binding to two classes of binding sites, each plasma sample being diluted to an albumin concentration equal to that in synovial fluid from the same patient. Binding to the primary binding sites was considerably decreased in synovial fluid in comparison with plasma. The affinity of the secondary binding sites was slightly lower. Thus, at a low therapeutic drug concentration, the decreased binding of salicylate to synovial fluid protein in patients with rheumatoid arthritis could mainly be accounted for by decreasing affinity of binding to the primary binding sites.     

Effects of clarithromycin in patients with active rheumatoid arthritis

ABSTRACT

Objective: To evaluate the clinical efficacy, safety, and tolerability of clarithromycin in patients with rheumatoid arthritis.

Research design and methods: This was a 6-month, monocenter, randomized, double-blind, placebo-controlled study. A total of 81 patients with early rheumatoid arthritis were treated with either once-daily oral clarithromycin (500?mg) or daily oral placebo for 6 months.

Main outcome measures: The primary efficacy variable was the percentage of patients who had a 20% improve­ment according to American College of Rheumatology (ACR) criteria (an ACR 20 response) at 6 months. Secondary outcome measures were 50% improvement and 70% improvement according to ACR criteria (an ACR 50 response and an ACR 70 response, respectively).

Results: A significantly greater percentage of patients treated with 500?mg clarithromycin met the ACR 20 response at 6 months compared with patients who received placebo (59 vs. 33%; p < 0.001). Greater percentages of patients treated with 500?mg clarithromycin also achieved ACR 50 responses (34 vs. 10%; p < 0.001) and ACR 70 responses (20 vs. 3%; p = 0.003) compared with patients who received placebo, respectively. Clarithromycin was well tolerated. There were no dose-limiting toxic effects.

Conclusions: In patients with early active rheumatoid arthritis, treatment with clarithromycin significantly improved the signs and symptoms of rheumatoid arthritis. Clarithromycin has been shown to be effective against rheumatoid arthritis.     

Naringenin induces apoptosis through downregulation of Akt and caspase-3 activation in human leukemia THP-1 cells

Naringenin (NGEN), one of the most abundant flavonoids in citrus fruits, has been shown to inhibit in vitro growth of in human cancer cells, although the mechanism of action is poorly understood. Herein, we investigated NEGN’s pro-apoptotic effect on human leukemia THP-1 cells. NGEN treatment inhibited THP-1 cells’ growth a concentration-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies and the accumulation of cells in the sub-G1 phase. NGEN-induced apoptosis was accompanied by increased hyperpolarization of the mitochondrial membrane potential, downregulation of Bcl-2, upregulation of Bax, activation of caspases and subsequent poly(ADP-ribose)polymerase (PARP) cleavages. z-DEVD-fmk, a caspase-3 inhibitor, significantly inhibited both the cytotoxic effect and apoptotic characteristics induced by NGEN treatment demonstrating caspase-3’s important role in the observed cytotoxic effect. The induction of apoptosis was also associated with the inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt, and PI3K inhibitor LY29004 significantly increases NGEN-induced cell death. These findings provide evidence that NEGN’s pro-apoptotic effect is mediated by the activation of caspases and mitochondria dysfunctions that correlate with the inactivation of the PI3K/Akt pathway in THP-1 cells. Therefore, NGEN has a strong potential as a therapeutic agent for preventing cancers such as leukemia.     

两种方法分离培养类风湿性关节炎成纤维样滑膜细胞的比较

目的比较体外分离培养成纤维样滑膜细胞（FLS）的方法。方法滑膜组织来自关节镜清洗术的活动期类风湿关节炎患者,分别采用消化酶培养法、组织块培养法分离培养FLS。采用倒置相差显微镜以及流式细胞术对第3～4代FLS进行鉴定。结果 2种原代分离培养的方法均可成功培养出FLS。传代可使FLS达到形态学上的纯化要求,倒置相差显微镜观察均符合FLS的形态结构特征,流式细胞术检测示CD44以及CD90呈强阳性,CD55呈弱阳性。组织块法培养法FLS成功率较高,细胞游出时间较早,细胞损伤小。结论组织块培养法适合于FLS的培养,第3～4代细胞的数目、活性及纯度符合进一步实验的要求。     

Transsynovial kinetics of piroxicam in patients with rheumatoid arthritis

Summary Twenty-four patients suffering from rheumatoid arthritis, who required articular puncture, participated in this open label study. Following a washout period of 7 days piroxicam 20 mg was administered once daily for 7 days.The average drug concentration in a dose interval in plasma fluctuated between 4.45 µg/ml and 8.02 µ/ml, and in synovial fluid it ranged between 4.16 µg/ml and 5.55 µg/ml. Piroxicam was eliminated from plasma with a half-life of 41.7 h and 43.2 h from synovial fluid as calculated from measured and interpolated data from all patients.Despite the short treatment period, clinical improvement could clearly be demonstrated.     

心理护理对类风湿关节炎患者的影响

目的分析心理护理对类风湿性关节炎患者的影响。方法将52例类风湿性关节炎患者随机分为治疗组和对照组各26例。对照组给予常规护理,治疗组在此基础上给予针对性的心理护理。观察2组复发率及患者满意度。结果 2组均获随访0.5～2年,治疗组复发率低于对照组,满意度高于对照组,差异均有统计学意义(P<0.05或P<0.01)。结论对类风湿性关节炎患者进行心理护理,可降低致残率,提高患者的生活质量,缩短康复时间,值得临床推广应用。     

类风湿性关节炎患者间质性肺疾病的临床概况

目的了解类风湿性关节炎肺间质病变的发生情况、特征及相关因素分析,以便早期发现类风湿性关节炎的肺部病变。方法类风湿性关节炎患者45例,免疫比浊法检测类风湿因子、C反应蛋白、免疫球蛋白及补体;间接免疫荧光法和免疫印迹法检测抗核抗体及亚类。血气分析测定氧分压、动脉血二氧化碳分压和肺泡-动脉血氧分压差。肺功能仪测定潮气容积、最大肺活量、用力肺活量、第1秒用力呼气容积、最大呼气中段流量、最大通气量和一氧化碳弥散功能。放射学检查包括胸部正侧位X线片、双手像和肺高分辨率CT扫描。结果45例类风湿关节患者中,14例存在肺间质病变,其中10例有呼吸道症状。肺功能检测异常主要为弥散功能降低和限制性通气障碍。8例胸部X线片存在异常,14例高分辨率CT发现异常。肺高分辨率CT在发现类风湿性关节炎肺间质病变病变时优于普通胸部X线片。结论类风湿性关节炎肺间质病变的发生与疾病活动性和严重性相关,肺弥散功能、高分辨率CT在早期发现病变时有诊断意义。     

Quinaldic acid in synovial fluid of patients with rheumatoid arthritis and osteoarthritis and its effect on synoviocytes in vitro

Background

Previously, we have demonstrated that kynurenic acid (KYNA), an endogenous metabolite of tryptophan formed along kynurenine pathway, is present in synovial fluid of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. In this study, the goal was to investigate the presence of quinaldic acid (QUDA), a putative metabolite of KYNA, in synovial fluid of RA and OA patients.

英夫利昔单抗治疗类风湿关节炎的临床研究

目的探讨英夫利昔单抗治疗类风湿关节炎的临床疗效,为临床合理用药提供参考。方法回顾性分析我院风湿免疫科2013年10月至2015年6月收治的30例患者应用英夫利昔单抗治疗类风湿关节炎的情况,观察患者治疗前后症状、体征及实验室检查等情况。结果治疗后患者的血沉和CRP明显降低,病情活动情况明显减轻。30例患者中,1例出现局部注射反应,2 d内可自动消失。其余患者未发现明显不良反应。结论英夫利昔单抗能明显改善RA等风湿免疫疾病的炎症反应,抑制关节破坏进展,改善患者的临床表现及预后。     

microRNA-155在类风湿关节炎外周血中的表达及临床意义

目的：通过研究microRNA-155（miR-155）在类风湿关节炎（RA）患者外周血的表达水平,分析miR-155与疾病临床特征的相关性,探讨其在RA发病及疾病进展中的意义。方法抽取50例初发RA患者及36例健康对照组外周血分离PBMC（外周血单个核细胞）,提取和纯化miRNA,实时定量PCR检测miR-155的表达,以小分子RNA U6作为内对照,对miR-155的表达量进行相对定量分析。同时收集RA患者有关的临床参数,统计分析miR-155的表达水平与RA合并临床参数的相关性。对RA活动组中,随机分为甲氨蝶呤（MTX）单药治疗组,肿瘤坏死因子（TNF）-α抑制剂单药治疗组,两组随访观察12周,收集患者治疗前后外周血。实时荧光定量PCR法检测RA患者治疗前后外周血miR-155的表达水平变化。结果 RA患者中PBMC中miR-155的表达水平较健康对照组明显升高,(P<0.01);同时RA患者miR-155的表达与患者活动度有关,与肺间质病变等无明显相关,RA患者TNF-α抑制剂单药治疗组中,miR-155的表达较治疗前下降,而在甲氨蝶呤治疗组中无明显下降。结论 miR-155在RA的PBMC中异常高表达,且与RA的活动度相关,提示miR-155可能可作为RA诊断及RA活动的一个预测指标。 miR-155在RA的发病机制中可能参与了TNF-α的致炎过程。     

类风湿性关节炎患者D-二聚体测定及其临床意义

目的：了解类风湿性关节炎患者血浆D-二聚体水平及其临床意义。方法采用免疫比浊法测定56例类风湿性关节炎患者血浆D-二聚体水平,按其数值将患者分为D-二聚体水平升高组和正常组,分别比较两组间患者晨僵时间、关节肿胀数、关节疼痛数、红细胞沉降率、C反应蛋白、类风湿因子、抗CCP抗体及关节外表现,并通过Spearman相关分析,分析D-二聚体与疾病活动度（DAS28评分）的相关性。结果D-二聚体水平增高组患者ESR、CRP、肿胀关节侧数均高于D-二聚体水平正常组,且D-二聚体水平增高组患者关节外症状的发生率（血管炎、转氨酶升高、贫血）也高于对照组,D-二聚体水平与DAS28评分呈正相关。结论血浆D-二聚体水平与类风湿性关节炎病情活动相关,临床可作为类风湿性关节炎病情活动的依据。     

Chromene induces apoptosis via caspase-3 activation in human leukemia HL-60 cells

In this study, the potent anti-tumor effects of brown algae on human leukemia HL-60 cells were investigated. The Sargassum siliquastrum extract among the 14 species of brown algae exhibited profound growth inhibitory effect on HL-60 cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, therefore, S. siliquastrum was selected for use in further experiments. The highest inhibitory activity of S. siliquastrum on HL-60 cells was detected in the chloroform fraction, and the active compound was identified as a kind of chromene, sargachromanol E (SE). SE treatment showed significant growth inhibitory effects on HL-60 cells in a dose-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies, fragmented DNA ladder, and the accumulation of DNA in the sub-G₁ phase of cell cycle. SE induced apoptosis was accompanied by downregulation of Bcl-xL, upregulation of Bax, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, z-DEVD-fmk, a caspase-3 inhibitor, significantly inhibited cell cytotoxicity, apoptotic characteristics such as apoptotic bodies, sub-G₁ DNA content, and cleavage of PARP induced by SE. These results suggest that SE exerts its growth inhibitory effects on HL-60 cells through caspase-3-mediated induction of apoptosis. Therefore, SE offers promising chemotherapeuric potential to prevent cancers such as human leukemia.