大鼠急性低氧模型尿液蛋白质组的变化

本研究旨在探究急性低氧对大鼠尿液蛋白质组造成的影响。在该项研究中,大鼠被放置于模拟海拔5 000 m高原环境的低氧舱内24 h。在低氧后0、12、24 h收集尿液样本,并使用液相色谱-串联质谱技术（LC-MS/MS）对尿蛋白进行分析。与低氧0 h相比,低氧12 h组共鉴定到144个差异蛋白,低氧24 h组共鉴定到129个差异蛋白。功能分析显示,差异蛋白参与了一系列与低氧应激有关的生物学通路,如抗氧化应激、糖酵解、补体和凝血级联反应等。研究结果表明,尿液蛋白质组可以反映急性低氧刺激后的显著变化。这些发现可能提供了一种判断机体缺氧状态的方法,有助于辅助检测缺氧状态。     

通过尿蛋白质组探索两种中成药对机体的整体影响

中药学作为一种独特的医学体系,是世界传统医学的重要组成部分.然而,中药组分的复杂性和多味中药的配伍应用使其发挥功效的作用机制尚不完全明确,在一定程度上阻滞了中药的现代化发展.尿液不受体内稳态的调节,这使得其能够敏感反映机体发生的变化.本研究分别建立两种中药(复方丹参滴丸和藿香正气口服液)的大鼠灌胃模型,收集健康大鼠灌胃前后的尿液,并使用液相色谱串联质谱技术(liquid chromatography-mass spectrometry, LC-MS/MS)对提取的尿蛋白进行分析,并对差异蛋白进行生物学通路的分析.与灌胃前比,在复方丹参滴丸灌胃模型组中鉴定到84个差异蛋白,在藿香正气口服液灌胃模型组中鉴定到64个差异蛋白.其中,复方丹参滴丸组差异蛋白富集到的生物学通路与其治疗心血管疾病的作用机制有关,如糖酵解、脂类代谢等.结果表明,尿液蛋白质组学可以直接系统反映中药对机体的整体影响,这提供了一种研究中药作用功效的新方法.     

自拟益气活血通络汤联合西药治疗类风湿关节炎86例

目的:研究自拟益气活血通络汤治疗86例类风湿性关节炎效果。方法:选取本院自2012年1月~2013年12月期间收治的86例类风湿性关节炎患者,对其进行随机分组,分为对照组和观察组,43例/组,对照组采用甲氨蝶呤片和双氯芬酸钠片进行治疗,观察组则采用自拟益气活血通络汤并且辅以甲氨蝶呤片和双氯芬酸钠片进行治疗。对比分析两组治疗的有效率以及在临床症状的改善上的差异。结果:经过本院的分组治疗,对照组的总有效率为69.77%,观察组的总有效率为93.02%;另外两组患者在主要临床症状改善方面,观察组的改善效果较之对照组的临床改善效果更佳,且存在着显著性差异(P0.05),具有统计学意义。结论:采用自拟益气活血通络汤治疗类风湿性关节炎具有非常显著的效果,经实践其临床效果值得医学推广。     

PNAS：彻底治愈类风湿性关节炎的新型疗法

类风湿性关节炎是一种引发机体许多关节疼痛炎症的一种疾病,其会引发关节囊肿胀。这种疾病往往会破坏机体的软骨组织和骨质;在世界范围内类风湿性关节炎影响着0．5％至1％的人群的健康,到目前为止医生们已经可以使用多种药物来缓解或者阻断该疾病的发展。近日,刊登在国际杂志PNAS上的一篇研究论文中,来自苏黎世联邦理工学院的研究人员通过研究开发了一种新型疗法。其在治疗患类风湿性关节炎小鼠的功效上可以达到一个新的水平,即小鼠在接受这种新型疗法治疗后会完全被治愈。     

PNAS:彻底治愈类风湿性关节炎的新型疗法

<正>类风湿性关节炎是一种引发机体许多关节疼痛炎症的一种疾病,其会引发关节囊肿胀,这种疾病往往会破坏机体的软骨组织和骨质;在世界范围内类风湿性关节炎影响着0.5%至1%的人群的健康,到目前为止医生们已经可以使用多种药物来缓解或者阻断该疾病的发展。近日,刊登在国际杂志PNAS上的一篇研究论文中,来自苏黎世联邦理工学院的研究人员通过研究开发了一种新型疗法,其在治疗患类风湿性关节炎小鼠的功效上可以达到一个新的水平,即小鼠在接受这种新型疗法治疗后会完全被治愈。     

科研快讯

《科学》:新蛋白可助治疗类风湿性关节炎来自纽约大学医学院,山东大学医学院,耶鲁大学医学院等处的研究人员研发了一种能用于治疗类风湿性关节炎的蛋白,从而为治疗这种自身免性疾病提供新的治疗靶标。这一研究成果公布在《科学》杂志上。     

应用VAS和KSS分析关节镜滑膜切除术后疼痛及膝关节功能

为了系统的评估膝关节类风湿性关节炎关节镜滑膜切除术后影响疼痛的因素和解决方法,本研究利用VAS和KSS系统评估了所在医院自2011年1月至2015年1月间57例膝关节类风湿关节炎行关节镜下滑膜切除术患者,我们将术后31例患者给予抗类风湿药物治疗6~12个月设为A组,26例病例未进行抗类风湿药物治疗或拒绝药物治疗的设为B组。术后3个月和6个月来院复查检测类风湿因子、血沉、C反应蛋白指标,疼痛模拟视觉评分(VAS)评分评估疼痛情况,膝关节功能KSS评分评估关节功能。所有患者进行多因素Logistic回归分析术后疼痛缓解因素,57例患者均获得术后6~18个月随访。研究结果表明,术后6个月膝前痛缓解率为73.7%(42/57),疼痛模拟视觉评分(VAS)平均(3.3±1.1)分;给予抗类风湿药物治疗的患者术后疼痛缓解率(80.6%,25/31)明显高于未给予药物治疗的患者(65.4%,17/26);单因素分析显示不同性别、年龄、手术时间等因素下患者行术后膝前痛的发生率差异无显著性(p0.05),多因素Logistic回归分析结果显示患者合并抗类风湿药物治疗是缓解膝关节类风湿关节炎关节镜滑膜切除术后膝前痛的独立影响因素(p0.05)。我们的评估分析初步表明:膝关节类风湿性关节炎患者行关节镜下滑膜切除术后发生膝前痛缓解率与术后合并抗类风湿药物有关,与患者年龄、性别、手术时间等无关。     

He-Ne激光结合丹参对类风湿性关节炎大鼠血液流变学指标的影响

目的:研究He-Ne激光结合丹参对类风湿性关节炎大鼠的治疗效果,以便为临床提供治疗风湿病的参考依据。方法:通过对健康大鼠注射鸡Ⅱ型胶原诱导后,成功建立了大鼠类风湿性关节炎的模型,随机分组后给予治疗,分析大鼠体重、血液流变学指标的变化。结果:1.与空白对照组相比,不同处理组大鼠类风湿关节炎体重增长幅度不同(P<0.05),其中模型组最小,丹参结合激光治疗组最高。2.与空白对照组相比,不同处理组的全血还原粘度低切、全血粘度(低、中、高切)、红细胞聚集指数均降低(P<0.05),其中模型组大鼠最高;而激光结合丹参治疗组最低。但全血还原粘度中、高切和红细胞压积没有统计学差异(P>0.05)。结论:丹参结合激光对Ⅱ型胶原诱导类风湿性关节炎大鼠体重、血液流变学指标均有一定程度的改善。     

类风湿性关节炎治疗药物进展

类风湿性关节炎是常见的自身免疫性疾病,可引起关节肿痛、畸形以及不同程度的残疾,影响了患者的生活质量。随着对发病机制的深入研究,出现了越来越多的治疗类风湿性关节炎的药物,依据发展的时间及原理,共分为五代:第一代是非甾体类抗炎药物;第二代是糖皮质激素;第三代是改变病情药(慢作用抗风湿药);第四代是以α肿瘤坏死因子(TNF-α)抑制剂为主的早期生物制剂;第五代为直接针对T细胞发生作用的新型生物制剂。其中第四代和第五代的生物制剂是近年来治疗类风湿性关节炎的新型药物,与传统药物相比体现了明显的优势,具有广泛的应用前景。本文对类风湿性关节炎的发病机制进行简要阐述,重点对类风湿性关节炎治疗药物的新进展进行综述。     

TNF-α/NF-κB信号通路在依那西普缓解大鼠类风湿性关节炎中的作用

目的:探讨依那西普(ETN)对胶原诱导大鼠类风湿性关节炎(CIA)的缓解作用及其机制。方法:实验动物分为3组(n=10):(1)对照组;(2)生理盐水治疗组(NS组);(3)(0.5 mg/kg)依那西普治疗组(ETN组)。其中(2)和(3)组的鼠在炎症分值达到两分及以上时随机分组开始治疗。药物采用皮下注射牛Ⅱ型胶原诱导Wistar大鼠建立类风湿性关节炎模型。治疗前和治疗期间每周测量足趾容积和体重,治疗4周后处死,ELISA检测血清TNFα、Ang-1、IL-1β、IL-6、IL-8水平;取足趾部位肌肉提取胞浆和胞核蛋白,Western blot检测NF-κB含量及其亚细胞定位。结果:依那西普治疗组较之生理盐水治疗组能显著缓解类风湿性关节炎症症状、降低TNFα以及下游炎症因子水平(P0.05);能抑制炎症状况下NF-κB的核内定位增加、胞浆定位减少的状况。结论:依那西普可通过抑制TNFα/NF-κB信号通路缓解大鼠类风湿性关节炎。     

L-carnitine and Zinc supplementation impedes intestinal damage in methotrexate-treated adjuvant-induced arthritis rats: Reinstating enterocyte proliferation and trace elements

BackgroundMethotrexate (MTX), a folic acid analogue, is used as a first-line treatment for rheumatoid arthritis (RA) since it has more therapeutic mechanisms than any other drug. Being an undeniable drug for the treatment of arthritis, even low-dose MTX provokes intestinal toxicity as a primary adverse effect and does not revive an anti-inflammatory element. Thus, our study aims to elucidate the anti-arthritic and prophylactic activity of supplements L-carnitine (L) and zinc (Z) against MTX-mediated intestinal damage in arthritis rats.MethodsThe rats were assessed for arthritic parameters such as body weight, paw volume, x-ray scan, and serum trace elements level. To analyze the toxic effects of MTX in the rats, intestine pH, mucosal weight, digestive enzymes, myeloperoxidase, histopathological, and immunohistochemical analysis were performed.ResultsOur study demonstrated that the arthritic parameters have shown that MTX has an ameliorative effect on arthritic rats. Besides, our findings showed that low-dose MTX (2.5 mg/kg b.w.) given once a week for two weeks during arthritis treatment had toxic effects in the rat's intestine, as evidenced by changes in intestine pH and mucosal weight, decreased digestive enzymes, increased MPO, and degenerative changes in histopathological analysis. Concurrent therapy of LZ with MTX, on the other hand, restored the modifications in these parameters.ConclusionMTX in combination with LZ effectively manages arthritis than monotherapy and significantly prevents MTX-induced intestinal damage in arthritis rats. Thus, LZ could be used as an improved therapeutic and safety for MTX-instigated intestinal damage during arthritis treatments. Therefore, our combination of L-carnitine and zinc with MTX would be promising prophylactic activity for arthritis patients.     

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expression

Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.     

Proteomic Analysis for the Identification of Proteins Related to Methotrexate Resistance

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 μmol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.     

甲氨蝶呤联合羟基氯喹对类风湿关节炎合并糖尿病患者糖化血红蛋白水平的影响

目的:观察类风湿关节炎(rheumatoid arthritis,RA)合并糖尿病(diabetes mellitus,DM)患者使用甲氨蝶呤(methotrexate,MTX)联合羟基氯喹(hydroxychloroquine,HCQ)治疗前后糖化血红蛋白(glycosylated hemoglobin,HbA1C)水平的变化。方法:通过回顾性分析,选取联合使用MTX+HCQ或单独使用MTX治疗的RA合并DM患者,且在治疗前及开始治疗12个月内分别至少有1次HbA1C值,记录其年龄、性别、诊断、体重指数、使用糖皮质激素情况。计算用药前到用药后12个月内HbA1C最低值的变化。结果:40例使用HCQ+MTX和45例使用MTX患者符合入选标准。两组间年龄、性别、体重指数、用药前HbA1C水平相似,MTX+HCQ组糖皮质激素使用比例(40.00%)比HCQ组更多(26.67%)(P=0.25)。MTX+HCQ组治疗后HbA1C有明显下降(0.42%,P=0.00),且MTX+HCQ组HbA1C降低幅度高于MTX组(0.42%,0.12%,P=0.02)。结论:与单独使用MTX相比,RA合并DM患者联合使用HCQ+MTX可明显降低HbA1C水平。     

The naturally occurring flavonoid nobiletin reverses methotrexate resistance via inhibition of P-glycoprotein synthesis

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA). However, after long-term treatment, some patients develop resistance. P-glycoprotein (P-gp), as an indispensable drug transporter, is essential for mediating this MTX resistance. In addition, nobiletin (NOB), a naturally occurring polymethoxylated flavonoid, has also been shown to reverse P-gp–mediated MTX resistance in RA groups; however, the precise role of NOB in this process is still unclear. Here, we administered MTX and NOB alone or in combination to collagen II-induced arthritic (CIA) mice and evaluated disease severity using the arthritis index, synovial histopathological changes, immunohistochemistry, and P-gp expression. In addition, we used conventional RNA-seq to identify targets and possible pathways through which NOB reverses MTX-induced drug resistance. We found that NOB in combination with MTX could enhance its performance in synovial tissue and decrease P-gp expression in CIA mice compared to MTX treatment alone. In vitro, in MTX-resistant fibroblast-like synoviocytes from CIA cells (CIA-FLS/MTX), we show that NOB treatment downregulated the PI3K/AKT/HIF-1α pathway, thereby reducing the synthesis of the P-gp protein. In addition, NOB significantly inhibited glycolysis and metabolic activity of CIA-FLS/MTX cells, which could reduce the production of ATP and block P-gp, ultimately decreasing the efflux of MTX and maintaining its anti-RA effects. In conclusion, this study shows that NOB overcomes MTX resistance in CIA-FLS/MTX cells through the PI3K/AKT/HIF-1α pathway, simultaneously influencing metabolic processes and inhibiting P-gp–induced drug efflux.     

运用蛋白质组学技术研究与氨甲蝶呤耐药性相关的蛋白质

氨甲蝶呤（MTX）是一种重要的常用化疗药物,然而由于肿瘤细胞对其耐药性的增强而经常导致其疗效大大降低。为了寻找并鉴定与MTX耐药性相关的蛋白质从而为进一步闸明MTX的耐药机制提供线索,培养来源于小鼠NIH3T3的小鼠胚胎成纤维细胞系3T3R500与其耐300μmol／L MTX的细胞系MTX300,提取上述两种细胞系的总蛋白质,双向凝胶电泳分离蛋白质组成分,扫描并通过软件分析考马斯亮蓝染色的2-DE凝胶,选取表达差异最显著的点,胶内酶切后MALDI-TOF-MS进行肽指纹图谱（PMF）鉴定。图像分析显示,实验组和对照组的蛋白质组图谱之间,一些蛋白质点的表达有明显的变化。通过MALDI-TOF-MS和数据库查询,成功鉴定了耐药后表达变化最显著的蛋白质点为二氢叶酸还原酶（DHFR）,并通过Western blot验证了该结果,提示DHFR在MTX耐药机制中发挥重要作用。     

Disease-modifying antirheumatic drugs are associated with a reduced risk for cardiovascular disease in patients with rheumatoid arthritis: a case control study

Rheumatoid arthritis (RA) is characterized by inflammation and an increased risk for cardiovascular disease (CVD). This study investigates possible associations between CVD and the use of conventional disease-modifying antirheumatic drugs (DMARDs) in RA. Using a case control design, 613 RA patients (5,649 patient-years) were studied, 72 with CVD and 541 without CVD. Data on RA, CVD and drug treatment were evaluated from time of RA diagnosis up to the first cardiovascular event or the end of the follow-up period. The dataset was categorized according to DMARD use: sulfasalazine (SSZ), hydroxychloroquine (HCQ) or methotrexate (MTX). Odds ratios (ORs) for CVD, corrected for age, gender, smoking and RA duration, were calculated per DMARD group. Patients who never used SSZ, HCQ or MTX were used as a reference group. MTX treatment was associated with a significant CVD risk reduction, with ORs (95% CI): 'MTX only', 0.16 (0.04 to 0.66); 'MTX and SSZ ever', 0.20 (0.08 to 0.51); and 'MTX, SSZ and HCQ ever', 0.20 (0.08 to 0.54). The risk reductions remained significant after additional correction for the presence of rheumatoid factor and erosions. After correction for hypertension, diabetes and hypercholesterolemia, 'MTX or SSZ ever' and 'MTX, SSZ and HCQ ever' showed significant CVD risk reduction. Rheumatoid factor positivity and erosions both increased CVD risk, with ORs of 2.04 (1.02 to 4.07) and 2.36 (0.92 to 6.08), respectively. MTX and, to a lesser extent, SSZ were associated with significantly lower CVD risk compared to RA patients who never used SSZ, HCQ or MTX. We hypothesize that DMARD use, in particular MTX use, results in powerful suppression of inflammation, thereby reducing the development of atherosclerosis and subsequently clinically overt CVD.     

Association between SLCO1A2 genetic variation and methotrexate toxicity in human rheumatoid arthritis treatment

Methotrexate (MTX), one of the important disease‐modifying anti‐rheumatic drugs, is the first‐line drug for rheumatoid arthritis (RA) treatment. However, its adverse drug effects (ADEs) often lead to the abortion of MTX therapy. Human organic anion‐transporting polypeptide 1A2 (OATP1A2, also referred as OATP‐A or OATP1) encoded by SLCO1A2 gene is an important isoform of the solute carrier transporter (SLC) family. It is known to participate in the cellular uptake of MTX. In our previous study, we identified four OATP1A2 natural variants (E184K, D185N, T259P, and D288N) with impaired MTX uptake activity. This study aimed to evaluate the association of the SLCO1A2 genetic variations encoding these OATP1A2 variants and MTX‐related toxicity in RA patients. A total of 60 RA patients were genotyped for these four polymorphisms (G550A, G553A, A775C, and G862A). The association between SLCO1A2 genetic variations and MTX toxicity was analyzed by binary logistic regression analysis. Single nucleotide polymorphisms (SNPs) analysis revealed that A775C and G862A SNPs were not detected in RA patients enrolled in this study, and the presence of 550AA genotype was associated with a high risk of MTX ADEs. Haplotype analysis revealed that H3 (H3 = AG) showed a high risk of MTX ADEs. Furthermore, there was a significant association of 550AA genotype and impaired MTX disposition, which might be the cause of the increased incidence of MTX ADEs in RA patients. Therefore, genetic variations in SLCO1A2 gene are risk factors for MTX toxicity and its information contributes to the prediction of MTX‐related toxicity in RA treatment.     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.