RA患者外周血miR-146a、miR-16表达与疾病活动、中医证型相关性及青藤碱制剂对其干预的研究
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摘要
研究背景
类风湿关节炎(rheumatoid arthritis,RA)是一种常见的以关节组织慢性炎症为主要表现的系统性自身免疫性疾病。病变关节主要表现为炎症细胞浸润、滑膜增生、血管翳形成以及由此引发的软骨和骨的损伤。RA呈全球分布,全世界的发病率平均为1%,在我国的发病率为0.26%～0.5%,男女比例为1：3,可发生于任何年龄,发病的高峰为45～50岁。RA是主要的致残性疾病之一,造成关节活动功能丧失,给社会和家庭带来巨大的压力和负担。
RA的发病机制复杂,其中有多种免疫细胞和细胞因子的参与。RA发生时CD4+T细胞活化并增殖,分泌多种细胞因子,如白介素-1(IL-1)和肿瘤坏死因子(TNF),继之巨噬细胞、B细胞、内皮细胞等被激活,激活的B细胞可以产生类风湿因子(RF)和抗环瓜氨酸肽抗体(anti-CCP)。其中IL-1、TNF-α等炎性或促炎细胞因子在发病中起着“中心犯罪”的作用。最近,有研究表明一类小分子RNA-microRNA(miRNA,即微小RNA)参与T、B淋巴细胞的分化,参与固有免疫应答和适应性免疫应答,可能在自身免疫性疾病如RA中发挥重要作用。
miRNA是一类长度约21～25碱基的非编码蛋白质的单链小分子RNA,广泛存在于多细胞生物和病毒体内,主要通过核酸序列互补匹配结合到特定的靶mRNA上,抑制靶mRNA翻译过程或降解靶mRNA,是一种起负调控作用的分子。预测约有30%的基因编码序列受相关的miRNAs调控。miR-146a和miR-16就是其中的两个。在RA中,miR-146a通过对其靶基因的调控,影响转录因子(NF-κB)依赖的细胞因子如IL-1β、TNF-a等的分泌及滑膜细胞增殖,介导RA患者炎症反应。miR-16能快速降解在3’非编码区含有AU的调控元件,包括TNF-a、IL-8、IL-6等,可抑制过度的炎症反应。研究miR-146a及miR-16在RA患者外周血的表达水平及与疾病活动程度、临床指标的关系,有助于进一步探讨其在RA发病及疾病进展中的意义。
RA属于祖国传统医学“痹证”范畴。痹证的发生主要是由于正气不足,感受风、寒、湿、热之邪所致。内因是基础,素体虚弱、正气不足、腠理不密、卫外不固,是引起痹证的内在因素,如再感受外邪,使肌肉、关节、经络痹阻而形成痹证。痹证的治疗以祛风通络为基本大法,治以祛风、除湿、舒筋活络。
青藤碱制剂(清风痛胶囊)具有祛风化湿、温经散寒、活血通络的功效,既往药理及临床研究显示对急、慢性炎症具有良好的抗炎镇痛效果。甲氨蝶呤(MTX)目前仍是应用最广泛的RA改善病情药物,具有抗炎、免疫抑制作用。二者联合治疗活动性RA具有较好的疗效。研究青藤碱制剂(清风痛胶囊)联合MTX对miR-146a及miR-16表达水平的影响有助于了进一步探讨青藤碱制剂(清风痛胶囊)联合MTX对RA的作用机理。
辨证论治是中医学的基本特点之一,是中医确立治法、谴方用药的依据。中医在诊治疾病的过程中,理法方药必一气贯通,才能体现中医辨证论治的精髓,而证正是理法方药一气贯通的桥梁和关键。目前国内外对RA诊断学研究很多,祖国医学多从整体观念出发,从宏观角度进行辨证论治,而现代医学多从实验室检测方法入手,从微观角度进行研究,二者各有所长亦各有所短。关于miRNAs与中医证型的关系未见研究报道。探讨miRNAs与RA中医证型的相关性,对RA辨证的客观化研究和指导临床治疗均有重要意义。
研究目的
1.研究RA患者外周血miR-146a、miR-16的表达,分析miR-146a、miR-16的表达与RA疾病活动的相关关系；
2.观察青藤碱制剂(清风痛胶囊)联合MTX对RA患者外周血miR-146a及miR-16表达水平的影响；
3.探讨RA患者外周血miR-146a、miR-16的表达与中医证型的关系。
方法
1.研究对象
2009年6月至2010年3月在南方医科大学附属南方医院中医风湿科门诊及病房确诊的RA患者40例,男性8例,女性32例,活动期患者24例,其中男性4例,女性20人。缓解期患者16例,男性4例,女性12例。健康对照组为同期医院门诊健康体检者16人,男性4例,女性12例。
2.研究方法
2.1RA患者外周血miR-146a、miR-16的表达与疾病活动的关系
采用实时荧光定量PCR法检测RA患者外周血单个核细胞miR-146a、miR-16的相对表达,以U6snRNA为内参,采用2-△△CT分析方法对miR-146a、miR-16的表达量进行相对定量。收集患者肿胀关节数(Swollen0-28)、压痛关节数(Tender0-28)、整体健康状况评分、类风湿因子(RF)、C-反应蛋白(CRP)、血沉(ESR)等临床资料,分析RA患者外周血miR-146a、miR-16的表达与疾病活动指标的相关性。
2.2青藤碱制剂(清风痛胶囊)联合MTX对RA患者外周血miR-146a及miR-16表达水平的影响
18例活动期RA患者予青藤碱制剂(清风痛胶囊)联合MTX治疗12周,实时荧光定量PCR法检测RA患者治疗前后外周血miR-146a及miR-16的表达水平。
2.3miR-146a、miR-16的表达与中医证型的关系
根据《中药新药临床研究指导原则(试行)》有关RA的中医证型辨证标准,辨证为：风湿夹瘀型20例,肝肾亏损型16例。
3.统计学处理
所有数据用SPSS13.0统计软件分析。计量资料采用(X±S)表示。两样本计量资料比较采用独立样本t检验,组内前后差异比较用配对t检验,多组样本均数比较采用One-way ANOVA,相关分析采用Spearman或Person相关分析方法,P≤0.05为差异有统计学意义。
结果
1.实时荧光定量PCR法检测miR-146a、miR-16的相对表达及分析miR-146a、miR-16的表达与疾病活动的关系
1.1RA患者外周血单个核细胞总RNA聚丙烯酰胺凝胶电泳可见28S、18S、5S明显的3条带,紫外分光光度仪测定吸光度(A)260/280比值均在1.8～2.0之间。无论是目的miRNAs还是内参U6snRNA,它们的扩增曲线及溶解曲线良好,没有引物二聚体,也没有非特异性的扩增。
1.2 miR-146a及miR-16在RA患者外周血单个核细胞的表达均显著高于健康对照组(P=0.004,P=0.001)。
1.3 RA活动组miR-146a表达水平高于缓解组(P=0.035)和对照组(P=0.005),RA缓解组miR-146a表达水平与对照组相比差异无统计学意义(P=1.000)。RA活动组miR-16表达水平高于缓解组(P=0.037)和对照组(P=0.002),RA缓解组miR-16表达水平与对照组相比差异无统计学意义(P=0.755)。
1.4 RA患者miR-146a的表达水平与肿胀关节数(r=0.354,P=0.007)、压痛关节数(r=0.352, P=0.008)、ESR (r=0.541, P=0.000)、CRP (r=0.402, P=0.010)、DAS28 (r=0.417, P=0.007)呈正相关,与RF(r=0.132,P=0.419)无相关性。
1.5 RA患者miR-16的表达水平与肿胀关节数(r=0.479,P=0.000)、压痛关节数(r=0.474, P=0.000)、ESR (r=0.674, P=0.000)、CRP (r=0.517, P=0.001)、DAS28 (r=0.575,P=0.000)呈正相关,与RF(r=0.239,P=0.137)无相关性。
2.青藤碱制剂(清风痛胶囊)联合MTX对RA患者外周血miR-146a及miR-16表达水平的影响
2.1青藤碱制剂(清风痛胶囊)联合MTX治疗12周后RA患者外周血IniR-146a的表达水平下降(P=0.040)。
2.2青藤碱制剂(清风痛胶囊)联合MTX治疗12周后RA患者外周血miR-16的表达水平下降(P=0.021)。
3.miR-146a、miR-16的表达与中医证型的关系
3.1三组miR-146a表达水平具有显著差异(F=19.911,P=0.000)。风湿夹瘀型miR-146a表达水平高于肝肾亏损型(P=0.000)和对照组(P=0.000),肝肾亏损型miR-146a表达水平与对照组相比差异无统计学意义(P=0.852)。
3.2三组miR-16表达水平具有显著差异(F=9.229,P=0.000)。风湿夹瘀型miR-16表达水平高于肝肾亏损型(P=0.036)和对照组(P=0.000),肝肾亏损型miR-16表达水平与对照组相比差异无统计学意义(P=0.827)。
3.3风湿夹瘀型RA患者ESR及CRP高于肝肾亏损型(P=0.001,P=0.020),差异具有统计学意义。
3.4风湿夹瘀型RA患者RF低于肝肾亏损型,差异具有统计学意义(P=0.014)；风湿夹瘀型RA患者抗CCP抗体水平与肝肾亏损型RA患者相比,差异无统计学意义(P=0.145)。
结论
1.RA患者外周血：miR-146a、miR-16的表达水平上调,可能与RA发病机制有关。
2.miR-146a、miR-16的表达水平与肿胀关节数、压痛关节数、ESR、CRP、DAS28呈正相关,与RF无相关性；检测miR-146a、miR-16的表达水平可能是监测RA疾病活动的有效指标。
3.青藤碱制剂(清风痛胶囊)联合MTX治疗后miR-146a、miR-16的表达水平下调,可能对疗效判定有一定意义。
4.RA风湿夹瘀型和肝肾亏损型之间miR-146a、miR-16、ESR、CRP、RF比较差异显著,提示上述指标可能与中医证型相关,可为中医辨证客观化研究提供一定依据。
Background
Rheumatoid arthritis is an autoimmune disease characterized by persistent and progressive synovitis of peripheral joints,leading to destruction of cartilage and subchondral bone.RA distribute worldwidely,afflicting up to 1% of the population abroad.Initial investigations revealed that the incidence rate of RA ranges from 0.26%-0.5% in our country.Women are three times than men.Onset is most frequent in 45 to 50 years,but no age is immune.It can be a disabling and painful condition,which can lead to substantial loss of function and mobilty.
The etiology of RA is complex.Many immunocytes and cytokins play important role.CD4+T lymph cells activate and proliferate,which will secrete many cytokins,such as interleukin-1(IL-1) and tumor necrosis factor(TNF).Then macrophages,B lymph cells and endothelial cells are activated.The actived B lymph cells will release rheumatoid factor(RF) and anti-Cyclic Citrullinated Peptide(anti-CCP).The inflammatory cytokins including IL-1 and TNF-a play a critical role.
MicroRNAs(miRNA) are a recently discovered class of small,evolution conserved noncoding RNAs that function as posttranscriptional repressors of gene primary transcripts via sequential processing by Drosha enzyme in the nucleus and by Dicer enzyme in the cytoplasm,producing 21-25 nucleotide-long mature,functionally active miRNA.Gene mapping and in silico studies are predicted to regulate 30% of protein-coding genes.Recently many researches found that miRNAs can regulate immunocytes differentiation, signal transduction,innate and adaptive immune response.MiRNA-146a can regulate target genes,influence NF-κB dependenting synovial fibroblasts proliferate and inflammatory cytokins including IL-1 and TNF-a secretion.MiRNA-16,a human miRNA containing an UAAAUAUU sequence that is comlementary to the AU-rich elements(ARE) sequence,therefore,appears to be an essential step in ARE-mediated mRNA degradation,containing TNF-a,IL-8 and IL-6,which can restrain the inflammatory response.It is significant to investigate the expression level of miRNA in peripheral blood of RA patients.
According to Traditional Chinese Medicine,this condition is called Bi Zheng. Deficiency of healthy atmosphere and wind,cold,damp and heat pathogenic factor which are all lead to Bi Zheng. The immanent cause is foundation which contain weakness body, healthy atmosphere deficiency,loosened Cou rationale and Guard outside not solid. If someone was effected by exogenous evil,muscles,joints and main collateral channels would be dyfunction and accerlerate the formation of Bi Zheng.Therapetic principles for incipient Bi-Zheng,expelling pathogenic factors should be the main therapeutic principles,including dispelling wind,eliminating dampness and dredging meridians and collaterals.
Sinomenine preparation (Qingfengtong capsules) has an effect of removing wind and dispersing dampness, clearing meridians and collaterals, expelling pathogenic cold from channel.And methotrexate show anti-inflammatory and immunodepression functons.Combined both can get better therapeutic effect. To study of Qingfengtong Capsules combined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16 is helpful for the further understanding their role and mechanism.
Syndrome differentiation and treatment is one of the outstanding feature of traditional Chinese medicine. And it is the basis of treatment and prescription. During the diagnosis and treatment:determining the cause, mechanism and location of the disease according to the medical theories and principles, then deciding the treatment principle and method, and finally selecting a formula as well as proper medicinals.Syndrome of traditional Chinese medicine is the bridge of principles, methods, formulas and medicinals.At present domestic and external researches are concerned about diagnosis.In traditional Chinese medicine syndrome differentiation and treatments are always macroscopic.But Western medicine treat disease microcosmically.Each one has its good points.It is significant to investigate the relationship between microRNA and traditional Chinese medicine syndrome.
Objectives
1.To detect the expression level of miR-146a and miR-16 in peripheral blood of RA patients and explore the relationship between the expression levels and disease activity.
2.To study of Sinomenine preparation combined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16.
3.To investigate the relationship between the expression levels of miRNAs and TCM syndrome type.
Methods
1. Research subjects
40 RA patients were obtained from department of Traditional Chinese Rheumatology of Nan-fang hospital from July 2009 to July 2010,including 8 males and 32 females;for R A patients cohorts, including active 24 RA cases,4 males and 20 females;including sive 16 RA cases,4 males and 12 females. Healthy controls include 4 males and 12 females, none of cases with other autoimmune diseases or severe heart, kidney and (?)diseases.
2. Research methods
2.1 To study the expression level of miR-146a and miR-16 in peripheral blood of RA patients and the relationship between the expression levels and disease activity
Real-time Polym erase Chain Reaction was used to detect the relative expression levels of miR-146a and miR-16.U6snRNA served as reference gene.
2-△△CT method was used t0 quantitate the expression levels of miR-146a and miR-16.Collecting clinical datas which contain swollen joints(0-28), tender joints(0-28),globle health assessment,rheumatoid factor(RF), C reactive protein(CRP), erythrocyte sedimentation rate(ESR) and so on.
2.2 To study of Sinomenine preparation (Qingfengtong Capsules) combined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16
18 active cases were treated with Qingfengtong capsules combined with MTX for 12 weeks. The Real-time Polymerase Chain Reaction method was used to measure the expression level of miR-146a and miR-16 before and after treatment.
2.3To investigate the relationship between the expression levels of miRNAs and clinical datas or TCM syndrome type
Based on guiding principles for the clinical study of Chinese Medicines and clinical experience to formulate the syndrome diagnostic criteria.Mainly devidived into two syndromes:syndrome of wind-dampness combined with blood stasis (n=20), syndrome of deficiency of liver and kidney(n=16).
3. Statistical Methods
All data were analysed using statistical software SPSS13.0. Measurement data were expressed as means±SD. Independent sample t test was used for difference between two groups. Differences between different dose groups were analyzed by one-Way ANOVA. Correlation analysis using Spearman or Person method. P-value less than 0.05 was considered statistically significant.
Results
1.Real time-PCR method used to detect the relative expression levels of miR-146a and miR-16
1.1 Polyacrylamide gel electrophoresis result show 3 bands. Ultraviolet-visible absorption spectroscopy is between 1.8 and 2.0. Regardless of target or house keeping genes,the augmentation curve and dissolved curve is good, no primer dimers, no non-specific amplification.
1.2 The expression levels of miR-146a and miR-16 in the PBMCs of RA patients increased compared to healthy individuals(P=0.004, P=0.001).
1.3The expression levels of miR-146a in active RA patients was higher than that in inactive RA patients(P=0.035) and healthy individuals(P=0.005). The expression levels of miR-16 in active RA patients was higher than that in inactive RA patients(P=0.037) and healthy individuals(P=0.002).
1.4The expression level of miR-146a was positively correlated with swollen joints counts (r=0.354, P=0.007),tender joints counts (r=0.352, P=0.008),ESR (r=0.541, P=0.000),CRP (r=0.402, P=0.010) and disease activity score in 28 joints(DAS28) (r=0.417, P=0.007),but not correlated with RF(r=0.132, P=0.419).
1.5The expression level of miR-16 were positively correlated with swollen joints counts (r=0.479, P=0.000),tender joints counts (r=0.474, P=0.000),ESR (r=0.674, P=0.000),CRP (r=0.517, P=0.001) and disease activity score in 28 joints(DAS28) (r=0.575, P=0.000),but not correlated with RF (r=0.239, P=0.137)
2. Sinomenine preparation (Qingfengtong Capsules) combinined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16
2.1 The expression level of miR-146a was lower than before treatment (P=0.040)
2.2 The expression level of miR-16 was lower than before treatment (P=0.021)
3. The relationship between the expression levels of miRNAs and TCM syndrome type
3.1 The expression levels of miR-146a in each groups show significant difference (F=19.911, P=0.000).The expression levels of miR-146a in syndrome of wind-dampness combined with blood stasis are higher than that in syndrome of deficiency of liver and healthy control (P=0.000). The expression levels of miR-146a show no significant difference between syndrome of deficiency of liver and healthy control (P=0.852)
3.2 The expression levels of miR-16 in each groups show significant difference (F=9.229, P=0.000).The expression levels of miR-16 in syndrome of wind-dampness combined with blood stasis are higher than that in syndrome of deficiency of liver and healthy control (P=0.036, P=0.000). The expression levels of miR-146a show no significant difference between syndrome of deficiency of liver and healthy control (P=0.827)
3.3ESR and CRP in syndrome of wind-dampness combined with blood stasis RA patients are higher than that in syndrome of deficiency of liver and kidney (P=0.001, P=0.020)
3.4The level of RF in syndrome of wind-dampness combined with blood stasis RA patients are lower than that in syndrome of deficiency of liver and kidney (P=0.014).But the anti-CCP level have no significant difference between two TCM syndrome types (P=0.145)
Conclusion
1.The expression levels of miR-146a and miR-16 in the PBMCs of RA patients are upregulated which can indicate they may involve in the mechanism of RA.
2.The expression levels of miR-146a and miR-16 were positively correlated with swollen joints counts,tender joints counts,ESR,CRP and disease activity score in 28 joints(DAS28),but not correlated with RF.The elevated expression levels of miR-146a and miR-16 are correlated with RA disease activity,which suggest that they are very helpful to detemine the disease activity.
3. Sinomenine preparation (Qingfengtong Capsules) combined with methotrexate can downregulate the expression of miR-146a and miR-16,it suggests that the expression level of miRNAs can reflect therapeutic effect.
4.ESR,CRP,RF,anti-CCP,miR-146a and miR-16 are correlated with TCM syndrome types,and these datas can provide a reference for the objectively research about traditional Chinese medicine syndrome type.
类风湿关节炎(rheumatoid arthritis,RA)是一种常见的以关节组织慢性炎症为主要表现的系统性自身免疫性疾病。病变关节主要表现为炎症细胞浸润、滑膜增生、血管翳形成以及由此引发的软骨和骨的损伤。RA呈全球分布,全世界的发病率平均为1%,在我国的发病率为0.26%～0.5%,男女比例为1：3,可发生于任何年龄,发病的高峰为45～50岁。RA是主要的致残性疾病之一,造成关节活动功能丧失,给社会和家庭带来巨大的压力和负担。
RA的发病机制复杂,其中有多种免疫细胞和细胞因子的参与。RA发生时CD4+T细胞活化并增殖,分泌多种细胞因子,如白介素-1(IL-1)和肿瘤坏死因子(TNF),继之巨噬细胞、B细胞、内皮细胞等被激活,激活的B细胞可以产生类风湿因子(RF)和抗环瓜氨酸肽抗体(anti-CCP)。其中IL-1、TNF-α等炎性或促炎细胞因子在发病中起着“中心犯罪”的作用。最近,有研究表明一类小分子RNA-microRNA(miRNA,即微小RNA)参与T、B淋巴细胞的分化,参与固有免疫应答和适应性免疫应答,可能在自身免疫性疾病如RA中发挥重要作用。
miRNA是一类长度约21～25碱基的非编码蛋白质的单链小分子RNA,广泛存在于多细胞生物和病毒体内,主要通过核酸序列互补匹配结合到特定的靶mRNA上,抑制靶mRNA翻译过程或降解靶mRNA,是一种起负调控作用的分子。预测约有30%的基因编码序列受相关的miRNAs调控。miR-146a和miR-16就是其中的两个。在RA中,miR-146a通过对其靶基因的调控,影响转录因子(NF-κB)依赖的细胞因子如IL-1β、TNF-a等的分泌及滑膜细胞增殖,介导RA患者炎症反应。miR-16能快速降解在3’非编码区含有AU的调控元件,包括TNF-a、IL-8、IL-6等,可抑制过度的炎症反应。研究miR-146a及miR-16在RA患者外周血的表达水平及与疾病活动程度、临床指标的关系,有助于进一步探讨其在RA发病及疾病进展中的意义。
RA属于祖国传统医学“痹证”范畴。痹证的发生主要是由于正气不足,感受风、寒、湿、热之邪所致。内因是基础,素体虚弱、正气不足、腠理不密、卫外不固,是引起痹证的内在因素,如再感受外邪,使肌肉、关节、经络痹阻而形成痹证。痹证的治疗以祛风通络为基本大法,治以祛风、除湿、舒筋活络。
青藤碱制剂(清风痛胶囊)具有祛风化湿、温经散寒、活血通络的功效,既往药理及临床研究显示对急、慢性炎症具有良好的抗炎镇痛效果。甲氨蝶呤(MTX)目前仍是应用最广泛的RA改善病情药物,具有抗炎、免疫抑制作用。二者联合治疗活动性RA具有较好的疗效。研究青藤碱制剂(清风痛胶囊)联合MTX对miR-146a及miR-16表达水平的影响有助于了进一步探讨青藤碱制剂(清风痛胶囊)联合MTX对RA的作用机理。
辨证论治是中医学的基本特点之一,是中医确立治法、谴方用药的依据。中医在诊治疾病的过程中,理法方药必一气贯通,才能体现中医辨证论治的精髓,而证正是理法方药一气贯通的桥梁和关键。目前国内外对RA诊断学研究很多,祖国医学多从整体观念出发,从宏观角度进行辨证论治,而现代医学多从实验室检测方法入手,从微观角度进行研究,二者各有所长亦各有所短。关于miRNAs与中医证型的关系未见研究报道。探讨miRNAs与RA中医证型的相关性,对RA辨证的客观化研究和指导临床治疗均有重要意义。
研究目的
1.研究RA患者外周血miR-146a、miR-16的表达,分析miR-146a、miR-16的表达与RA疾病活动的相关关系；
2.观察青藤碱制剂(清风痛胶囊)联合MTX对RA患者外周血miR-146a及miR-16表达水平的影响；
3.探讨RA患者外周血miR-146a、miR-16的表达与中医证型的关系。
方法
1.研究对象
2009年6月至2010年3月在南方医科大学附属南方医院中医风湿科门诊及病房确诊的RA患者40例,男性8例,女性32例,活动期患者24例,其中男性4例,女性20人。缓解期患者16例,男性4例,女性12例。健康对照组为同期医院门诊健康体检者16人,男性4例,女性12例。
2.研究方法
2.1RA患者外周血miR-146a、miR-16的表达与疾病活动的关系
采用实时荧光定量PCR法检测RA患者外周血单个核细胞miR-146a、miR-16的相对表达,以U6snRNA为内参,采用2-△△CT分析方法对miR-146a、miR-16的表达量进行相对定量。收集患者肿胀关节数(Swollen0-28)、压痛关节数(Tender0-28)、整体健康状况评分、类风湿因子(RF)、C-反应蛋白(CRP)、血沉(ESR)等临床资料,分析RA患者外周血miR-146a、miR-16的表达与疾病活动指标的相关性。
2.2青藤碱制剂(清风痛胶囊)联合MTX对RA患者外周血miR-146a及miR-16表达水平的影响
18例活动期RA患者予青藤碱制剂(清风痛胶囊)联合MTX治疗12周,实时荧光定量PCR法检测RA患者治疗前后外周血miR-146a及miR-16的表达水平。
2.3miR-146a、miR-16的表达与中医证型的关系
根据《中药新药临床研究指导原则(试行)》有关RA的中医证型辨证标准,辨证为：风湿夹瘀型20例,肝肾亏损型16例。
3.统计学处理
所有数据用SPSS13.0统计软件分析。计量资料采用(X±S)表示。两样本计量资料比较采用独立样本t检验,组内前后差异比较用配对t检验,多组样本均数比较采用One-way ANOVA,相关分析采用Spearman或Person相关分析方法,P≤0.05为差异有统计学意义。
结果
1.实时荧光定量PCR法检测miR-146a、miR-16的相对表达及分析miR-146a、miR-16的表达与疾病活动的关系
1.1RA患者外周血单个核细胞总RNA聚丙烯酰胺凝胶电泳可见28S、18S、5S明显的3条带,紫外分光光度仪测定吸光度(A)260/280比值均在1.8～2.0之间。无论是目的miRNAs还是内参U6snRNA,它们的扩增曲线及溶解曲线良好,没有引物二聚体,也没有非特异性的扩增。
1.2 miR-146a及miR-16在RA患者外周血单个核细胞的表达均显著高于健康对照组(P=0.004,P=0.001)。
1.3 RA活动组miR-146a表达水平高于缓解组(P=0.035)和对照组(P=0.005),RA缓解组miR-146a表达水平与对照组相比差异无统计学意义(P=1.000)。RA活动组miR-16表达水平高于缓解组(P=0.037)和对照组(P=0.002),RA缓解组miR-16表达水平与对照组相比差异无统计学意义(P=0.755)。
1.4 RA患者miR-146a的表达水平与肿胀关节数(r=0.354,P=0.007)、压痛关节数(r=0.352, P=0.008)、ESR (r=0.541, P=0.000)、CRP (r=0.402, P=0.010)、DAS28 (r=0.417, P=0.007)呈正相关,与RF(r=0.132,P=0.419)无相关性。
1.5 RA患者miR-16的表达水平与肿胀关节数(r=0.479,P=0.000)、压痛关节数(r=0.474, P=0.000)、ESR (r=0.674, P=0.000)、CRP (r=0.517, P=0.001)、DAS28 (r=0.575,P=0.000)呈正相关,与RF(r=0.239,P=0.137)无相关性。
2.青藤碱制剂(清风痛胶囊)联合MTX对RA患者外周血miR-146a及miR-16表达水平的影响
2.1青藤碱制剂(清风痛胶囊)联合MTX治疗12周后RA患者外周血IniR-146a的表达水平下降(P=0.040)。
2.2青藤碱制剂(清风痛胶囊)联合MTX治疗12周后RA患者外周血miR-16的表达水平下降(P=0.021)。
3.miR-146a、miR-16的表达与中医证型的关系
3.1三组miR-146a表达水平具有显著差异(F=19.911,P=0.000)。风湿夹瘀型miR-146a表达水平高于肝肾亏损型(P=0.000)和对照组(P=0.000),肝肾亏损型miR-146a表达水平与对照组相比差异无统计学意义(P=0.852)。
3.2三组miR-16表达水平具有显著差异(F=9.229,P=0.000)。风湿夹瘀型miR-16表达水平高于肝肾亏损型(P=0.036)和对照组(P=0.000),肝肾亏损型miR-16表达水平与对照组相比差异无统计学意义(P=0.827)。
3.3风湿夹瘀型RA患者ESR及CRP高于肝肾亏损型(P=0.001,P=0.020),差异具有统计学意义。
3.4风湿夹瘀型RA患者RF低于肝肾亏损型,差异具有统计学意义(P=0.014)；风湿夹瘀型RA患者抗CCP抗体水平与肝肾亏损型RA患者相比,差异无统计学意义(P=0.145)。
结论
1.RA患者外周血：miR-146a、miR-16的表达水平上调,可能与RA发病机制有关。
2.miR-146a、miR-16的表达水平与肿胀关节数、压痛关节数、ESR、CRP、DAS28呈正相关,与RF无相关性；检测miR-146a、miR-16的表达水平可能是监测RA疾病活动的有效指标。
3.青藤碱制剂(清风痛胶囊)联合MTX治疗后miR-146a、miR-16的表达水平下调,可能对疗效判定有一定意义。
4.RA风湿夹瘀型和肝肾亏损型之间miR-146a、miR-16、ESR、CRP、RF比较差异显著,提示上述指标可能与中医证型相关,可为中医辨证客观化研究提供一定依据。
Background
Rheumatoid arthritis is an autoimmune disease characterized by persistent and progressive synovitis of peripheral joints,leading to destruction of cartilage and subchondral bone.RA distribute worldwidely,afflicting up to 1% of the population abroad.Initial investigations revealed that the incidence rate of RA ranges from 0.26%-0.5% in our country.Women are three times than men.Onset is most frequent in 45 to 50 years,but no age is immune.It can be a disabling and painful condition,which can lead to substantial loss of function and mobilty.
The etiology of RA is complex.Many immunocytes and cytokins play important role.CD4+T lymph cells activate and proliferate,which will secrete many cytokins,such as interleukin-1(IL-1) and tumor necrosis factor(TNF).Then macrophages,B lymph cells and endothelial cells are activated.The actived B lymph cells will release rheumatoid factor(RF) and anti-Cyclic Citrullinated Peptide(anti-CCP).The inflammatory cytokins including IL-1 and TNF-a play a critical role.
MicroRNAs(miRNA) are a recently discovered class of small,evolution conserved noncoding RNAs that function as posttranscriptional repressors of gene primary transcripts via sequential processing by Drosha enzyme in the nucleus and by Dicer enzyme in the cytoplasm,producing 21-25 nucleotide-long mature,functionally active miRNA.Gene mapping and in silico studies are predicted to regulate 30% of protein-coding genes.Recently many researches found that miRNAs can regulate immunocytes differentiation, signal transduction,innate and adaptive immune response.MiRNA-146a can regulate target genes,influence NF-κB dependenting synovial fibroblasts proliferate and inflammatory cytokins including IL-1 and TNF-a secretion.MiRNA-16,a human miRNA containing an UAAAUAUU sequence that is comlementary to the AU-rich elements(ARE) sequence,therefore,appears to be an essential step in ARE-mediated mRNA degradation,containing TNF-a,IL-8 and IL-6,which can restrain the inflammatory response.It is significant to investigate the expression level of miRNA in peripheral blood of RA patients.
According to Traditional Chinese Medicine,this condition is called Bi Zheng. Deficiency of healthy atmosphere and wind,cold,damp and heat pathogenic factor which are all lead to Bi Zheng. The immanent cause is foundation which contain weakness body, healthy atmosphere deficiency,loosened Cou rationale and Guard outside not solid. If someone was effected by exogenous evil,muscles,joints and main collateral channels would be dyfunction and accerlerate the formation of Bi Zheng.Therapetic principles for incipient Bi-Zheng,expelling pathogenic factors should be the main therapeutic principles,including dispelling wind,eliminating dampness and dredging meridians and collaterals.
Sinomenine preparation (Qingfengtong capsules) has an effect of removing wind and dispersing dampness, clearing meridians and collaterals, expelling pathogenic cold from channel.And methotrexate show anti-inflammatory and immunodepression functons.Combined both can get better therapeutic effect. To study of Qingfengtong Capsules combined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16 is helpful for the further understanding their role and mechanism.
Syndrome differentiation and treatment is one of the outstanding feature of traditional Chinese medicine. And it is the basis of treatment and prescription. During the diagnosis and treatment:determining the cause, mechanism and location of the disease according to the medical theories and principles, then deciding the treatment principle and method, and finally selecting a formula as well as proper medicinals.Syndrome of traditional Chinese medicine is the bridge of principles, methods, formulas and medicinals.At present domestic and external researches are concerned about diagnosis.In traditional Chinese medicine syndrome differentiation and treatments are always macroscopic.But Western medicine treat disease microcosmically.Each one has its good points.It is significant to investigate the relationship between microRNA and traditional Chinese medicine syndrome.
Objectives
1.To detect the expression level of miR-146a and miR-16 in peripheral blood of RA patients and explore the relationship between the expression levels and disease activity.
2.To study of Sinomenine preparation combined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16.
3.To investigate the relationship between the expression levels of miRNAs and TCM syndrome type.
Methods
1. Research subjects
40 RA patients were obtained from department of Traditional Chinese Rheumatology of Nan-fang hospital from July 2009 to July 2010,including 8 males and 32 females;for R A patients cohorts, including active 24 RA cases,4 males and 20 females;including sive 16 RA cases,4 males and 12 females. Healthy controls include 4 males and 12 females, none of cases with other autoimmune diseases or severe heart, kidney and (?)diseases.
2. Research methods
2.1 To study the expression level of miR-146a and miR-16 in peripheral blood of RA patients and the relationship between the expression levels and disease activity
Real-time Polym erase Chain Reaction was used to detect the relative expression levels of miR-146a and miR-16.U6snRNA served as reference gene.
2-△△CT method was used t0 quantitate the expression levels of miR-146a and miR-16.Collecting clinical datas which contain swollen joints(0-28), tender joints(0-28),globle health assessment,rheumatoid factor(RF), C reactive protein(CRP), erythrocyte sedimentation rate(ESR) and so on.
2.2 To study of Sinomenine preparation (Qingfengtong Capsules) combined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16
18 active cases were treated with Qingfengtong capsules combined with MTX for 12 weeks. The Real-time Polymerase Chain Reaction method was used to measure the expression level of miR-146a and miR-16 before and after treatment.
2.3To investigate the relationship between the expression levels of miRNAs and clinical datas or TCM syndrome type
Based on guiding principles for the clinical study of Chinese Medicines and clinical experience to formulate the syndrome diagnostic criteria.Mainly devidived into two syndromes:syndrome of wind-dampness combined with blood stasis (n=20), syndrome of deficiency of liver and kidney(n=16).
3. Statistical Methods
All data were analysed using statistical software SPSS13.0. Measurement data were expressed as means±SD. Independent sample t test was used for difference between two groups. Differences between different dose groups were analyzed by one-Way ANOVA. Correlation analysis using Spearman or Person method. P-value less than 0.05 was considered statistically significant.
Results
1.Real time-PCR method used to detect the relative expression levels of miR-146a and miR-16
1.1 Polyacrylamide gel electrophoresis result show 3 bands. Ultraviolet-visible absorption spectroscopy is between 1.8 and 2.0. Regardless of target or house keeping genes,the augmentation curve and dissolved curve is good, no primer dimers, no non-specific amplification.
1.2 The expression levels of miR-146a and miR-16 in the PBMCs of RA patients increased compared to healthy individuals(P=0.004, P=0.001).
1.3The expression levels of miR-146a in active RA patients was higher than that in inactive RA patients(P=0.035) and healthy individuals(P=0.005). The expression levels of miR-16 in active RA patients was higher than that in inactive RA patients(P=0.037) and healthy individuals(P=0.002).
1.4The expression level of miR-146a was positively correlated with swollen joints counts (r=0.354, P=0.007),tender joints counts (r=0.352, P=0.008),ESR (r=0.541, P=0.000),CRP (r=0.402, P=0.010) and disease activity score in 28 joints(DAS28) (r=0.417, P=0.007),but not correlated with RF(r=0.132, P=0.419).
1.5The expression level of miR-16 were positively correlated with swollen joints counts (r=0.479, P=0.000),tender joints counts (r=0.474, P=0.000),ESR (r=0.674, P=0.000),CRP (r=0.517, P=0.001) and disease activity score in 28 joints(DAS28) (r=0.575, P=0.000),but not correlated with RF (r=0.239, P=0.137)
2. Sinomenine preparation (Qingfengtong Capsules) combinined with methotrexate for the treatment of rheumatoid arthritis and its effect on the expression of miR-146a and miR-16
2.1 The expression level of miR-146a was lower than before treatment (P=0.040)
2.2 The expression level of miR-16 was lower than before treatment (P=0.021)
3. The relationship between the expression levels of miRNAs and TCM syndrome type
3.1 The expression levels of miR-146a in each groups show significant difference (F=19.911, P=0.000).The expression levels of miR-146a in syndrome of wind-dampness combined with blood stasis are higher than that in syndrome of deficiency of liver and healthy control (P=0.000). The expression levels of miR-146a show no significant difference between syndrome of deficiency of liver and healthy control (P=0.852)
3.2 The expression levels of miR-16 in each groups show significant difference (F=9.229, P=0.000).The expression levels of miR-16 in syndrome of wind-dampness combined with blood stasis are higher than that in syndrome of deficiency of liver and healthy control (P=0.036, P=0.000). The expression levels of miR-146a show no significant difference between syndrome of deficiency of liver and healthy control (P=0.827)
3.3ESR and CRP in syndrome of wind-dampness combined with blood stasis RA patients are higher than that in syndrome of deficiency of liver and kidney (P=0.001, P=0.020)
3.4The level of RF in syndrome of wind-dampness combined with blood stasis RA patients are lower than that in syndrome of deficiency of liver and kidney (P=0.014).But the anti-CCP level have no significant difference between two TCM syndrome types (P=0.145)
Conclusion
1.The expression levels of miR-146a and miR-16 in the PBMCs of RA patients are upregulated which can indicate they may involve in the mechanism of RA.
2.The expression levels of miR-146a and miR-16 were positively correlated with swollen joints counts,tender joints counts,ESR,CRP and disease activity score in 28 joints(DAS28),but not correlated with RF.The elevated expression levels of miR-146a and miR-16 are correlated with RA disease activity,which suggest that they are very helpful to detemine the disease activity.
3. Sinomenine preparation (Qingfengtong Capsules) combined with methotrexate can downregulate the expression of miR-146a and miR-16,it suggests that the expression level of miRNAs can reflect therapeutic effect.
4.ESR,CRP,RF,anti-CCP,miR-146a and miR-16 are correlated with TCM syndrome types,and these datas can provide a reference for the objectively research about traditional Chinese medicine syndrome type.
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