| BackgroundRheumatoid 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.Objectives1.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.Methods1. Research subjects40 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 methods2.1 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 activityReal-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-1618 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 typeBased 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.Results1.Real time-PCR method used to detect the relative expression levels of miR-146a and miR-161.1 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-162.1 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 type3.1 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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