Analysis Of Chinese Medical Constitution And Effect Of Sinomenine Preparation On The Expression Of HLA-DR4,PADI4,STAT4 In Peripheral Blood In Rheumatoid Arthritis Patients

Rheumatoid arthritis (rheumatoid arthritis, RA) is a systematic autoimmune disease which is characteristic of persistently progressive synovitis and extra-articular lesions. Articular cartilage and bone can be involved, resulting in joint deformity and ankylosis, even loss of joint function.The prevalence is about 0.4% of the population in our county.The pathogenesis of RA is still a dynamic area of researeh. The etiology of RA remains illusive and is most likely multifactorial. Heredity,environment and immunological regulation have palyed significant roles together. HLA-DR4,a member of HLAⅡmolecules, is known to be related to RA. The main function of HLAmolecules is to present antigen to T cells. HLAantigenic determinants will be changed when confronted with external stimuli or mutation. Through shared epitope and dosage effect of gene, HLA molecules become the target of immunological activation and then T cell was activated. A series of immune molecules are released such as cytokines which can induce RA and eventually lead to synovium, cartilage and bone destruction. Patients with HLA-DR4 are most likely to develop erosive lesions and poor prognosis and efficacy. It was considerable observed that a very high proportion of patients with RA have antibodies to citrulline-containing proteins. Peptidylarginine deiminase type4 (PADI4) post-translationally converts peptidylarginine to citrulline, a process called citrullination. As a result, uimmune tolerance is broken and autoimmune reactions occur. Since 2003, numerous studies have shown that PADI4 gene polymorphism has a strong correlation with RA. In addition, activated T cell plays an important role in RA pathogenesis. Transcription factors paly a key rolr in the process of CD4 +T cells differentiation STAT4 is considered as a key regulatory factor for Thl cell differentiation, also partly relevant to Th17 differentiation. A number of studies have demonstrated that STAT4 is one of RA susceptible genes since 2006. It is important to study the expression of these susceptible genes for understanding the pathogenisis and progression of RAIn Traditional Chinese medieine(TCM). the condition that is congruent with arthritis is called "Bi syndrome." Bi syndrome manifests as pain, soreness, or numbness of muscles, tendons and joints which is the result of the body being "invaded" by the external environmental factors such as Wind, Cold, Heat, and/or Dampness. The individual symptoms depend on which external pathogenic factor is strongest. Long-term Bi syndrome can lead to the formation of Phlegn and the deformation of bones and joints. In TCM pain and reduced range of motion indicates the presence of blockage; condition such as RA, therefore are always treated by means of removing Wind-Dampness and smoothing meridian. Sinomenine is an alkaloid extracted from the Chinese medicinal plant,Sinomenine acutum, which has been utilized to treat RA in China for over 2000 years. Sinomenine has been shown to mediate a wide range of pharmacological actions which include anti-inflammatory and anti-rheumatic effects. Qingfengtong capsules(Sinomenine preparation) has an effect of removing wind and dispersing dampness, clearing meridians and collaterals, expelling pathogenic cold from channel.Previous Pharmacological and clinical study on acute and chronic inflammation has shown a good anti-inflammatory and analgesic effects of Qingfengtong capsules. Study of Qingfengtong Capsules for the treatment of rheumatoid arthritis and its effect on the expression of HLA-DR4,STAT,PADI4 genes is helpful for the further study of the role and mechanism of sinomenine.Chinese medical constitution is individually morphological structure, physiological function and psychological state of an integrated, relatively stable characteristics, It is constrained and influenced by changes in the process of disease and is one of the basic factors with close correlation to the development and prognosis of disease.Because there are regional differences in RA morbidity and individual differences in clinical manifestations, it is necessary to make further research on the relationship between RA related gene expression and constitution despite of few research in this respect.Quantitative detection of the expression of genes at the same time can not be achieved by traditional methods. QGP (QuantiGene Plex) technology combines b-DNA (Branched DNA) signal amplification and the Luminex xMAP liquid-chip platform.The advantage is available directly from whole blood samples to detect mRNA expression, no RNA purification, no reverse transcription, no PCR amplification, and it can also high-throughput quantitatively detect 3 to 36 genes from less samples.Objectives1. To detect the expression level of HLA-DR4,PADI4,STAT4 in peripheral blood of RA patients and to explore the relationship between the expression of genes and disease activity.2. To evaluate the efficacy and safety of Qingfengtong Capsules in active rheumatoid arthritis (RA) patients at 12 weeks,and to study expression changes of HLA-DR4,PADI4,STAT4 genes after the treatment.3. To analyse distribution of Chinese Constitution in RA patients and HLA-DR4,PADI4,STAT4 gene expression of common types.Methods1. Research subjects38 RA patients were obtained from department of Traditional Chinese Rheumatology of Nan-fang hospital from July 2009 to December 2009., including 8 males and 30 females; for RA patients cohorts, including active 24 RA cases,4 males and 20 females; including remissive 14 RA cases,4 males and 10 females. Healthy controls include 4 males and 8 females, none of cases with other autoimmune diseases or severe heart, kidney and liver diseases.2. Research methods2.1 Study of the expression of HLA-DR4,PADI4,STAT4 genes in the peripheral blood of RA patients. The QGP method was used to measure the expression level of HLA-DR4,PADI4,STAT4 and PPIB, HPRT1 genes2.2 Clinical Evaluation of Qingfengtong Capsules(Sinomenine preparation) combined with MTX for treatment of RA and the effect on expression levels of HLA-DR4,STAT4,PADI4mRNA2.2.1 18 active cases were treated with Qingfengtong capsules united MTX for 12 weeks. The primary endpoints includethe proportion of patients achieving ACR20, ACR50, DAS28 score,duration of morning stiffness, joint swelling and joint tenderness count, VAS score, health assessment Questionnaire, the level of ESR,CRP.,Anti-CCP and RF. Regularly examine blood, urine routine test, liver and kidney function and record the occurrence of adverse events2.2.2 The QGP method was used to measure the expression level of HLA-DR4,PADI4. STAT4 mRNA before and after treatment.2.3 Classification of RA patients by using the standard of "Chinese Constitutions classification and determine "3 Statistical MethodsAll data were analysed using statistical software SPSS 13.0. Measurement data were expressed as means±SD. Independent sample t test was used for difference between two groups and paired test or Wilcoxon test for difference before and after treatment. Differences between different dose groups were analyzed by one-Way ANOVA followed by LSD test or Dunnetttest. Correlation analysis using Spearman or Person method. A two-sided P-value less than 0.05 was considered statistically significant.Results1. The QGP method was used to detect the expression level of HLA-DR4,PADI4,STAT4 mRNA1.1 The QGP detect the expression of HLA-DR4,PADI4,STAT4 mRNA,that AVG median fluorescence intensity of assay background (AVG MFI background) were respectively 6.0,9.0,5.5; the limit of detect (LOD) were 16.4,18.6,15.1; the intraplate assay coefficient of variation(CV) were5.4%,7.7%,6.8%; interplate assay coefficient of variation(CV) were 7.3%,10.1%,9.8%.1.2 The expressions of HLA-DR4, PADI4,STAT4 in RA patients were significantly higher than those of healthy controls (P<0.05 or P<0.01). The level of HLA-DR4 mRNA was no difference between active RA and Remissive RA. The expressions of PADI4 and STAT4 of active RA were significantly higher than remissive group (P<0.05).There were no correlation between the expression of HLA-DR4 of RA patients and DAS28, the level of anti-CCP antibody and RF (P>0.05). The level of PADI4 mRNA of patients was positively associated with DAS28 and the level of Anti-CCP antibody (r=0.521,P=0.01; r=0.558, P=0.000), while the expression of STAT4 of RA patients was positively associated with DAS28 and the level of RF(r=0.503,P=0.001; r=0.493,P=0.02).2. Clinical Evaluation of Qingfengtong Capsules(Sinomenine preparation) combined with MTX for Treatment of RA and its effect on expression levels of HLA-DR4,STAT4,PADI4mRNA2.1 At 12 weeks ACR20 was 72.2%, ACR50 was 22.2%;other indicators shows a similar level and trend of improvement (P<0.05). After 12 weeks of treatment clinical symptoms and laboratory parameters were significantly improved compared with baseline (P<0.05 or P<0.01)2.2 The expression level of HLA-DR4 mRNA was no difference before and after treatment (P＞0.05), while the expression levels of PADI4 and STAT4 after treatment were significantly lower than before treatment (P<0.05).3 Analysis of Chinese medical constitution in RA patients and HLA-DR4, PADI4, STAT4 expression in different types of Chinese medical constitution3.1 The most common type of Chinese medical constitutions were yang deficiency and blood stasis, and Chinese medical constitutions of damp heat, phlegm dampnes, yin deficiency and mild were followed.3.2 The level of HLA-DR4, STAT4 mRNA was no difference in various Chinese medical constitutions groups in RA patients (P＞0.05).The level of PADI4 mRNA of blood stasis constitution was higher than other constitutions (P＜0.05)Conclusion1. The advantages of QGP method to detect peripheral blood expression of HLA-DR4, PADI4, STAT4mRNA in RA patients are simple, reproducible, sensitive, saving samples.2. HLA-DR,PADI4 and STAT4 are overexpressed in peripheral blood of RA patients. The expressions of PADI4 andSTAT4 were closely related to disease activity,while HLA-DR4 expression was not The expressions of PADI4,STAT4 were related with disease progression and severity.The detection of PADI4 and STAT4 genes expression should be helpful for the judgement on disease activity of RA.3.Inflammation of active RA can significantly be relieved by Qingfengtong Capsules(Sinomenine preparation) combined with MTX therapeutics. After 12 weeks of treatment there were no serious adverse events and complications. The expression of PADI4 and STAT4 mRNA were decreased after treatment. That suggests Zhengqingfengtong capsules combined MTX can downregulate the expression of PADI4, STAT4 genes.4.The most common Chinese medical constitution in RA patients was yang deficiency and blood stasis. Morever, the level of PADI4 mRNA in this group was higher, prompting the expression of PADI4 may correlate with Chinese medical constitution types.