The Clinical And Experiment Research Of Duanteng Yimu Decoction Treatment Of Rheumatoid Arthritis

Objective:This subject include three parts. The first part is Literature Review, mainly introduced three aspects:illustrated the relation between pathogenesis of rheumatoid arthritis(RA) and Rheumatoid fibroblast-like synoiocytes (RA-FLS)activation, research summary about oral TCM therapy for RA, previous correlation study about Duanteng Yimu Decoction(DYD). The second part is a12-weeks trial to assess the efficacy and safety of DYD and Leflunomide for RA patients in active period. The third part is experiments to observe effects of DVD medicinal serum on RA-FLS and TLR4/NF-κ B signal transduction pathway, all above work is to provide to provide experimental and clinical basis for Using DYD to treat RA.Methods1. Clinical part:Accord RA classification criteria from American College of Rheumatology (ACR) Promulgated in1987for diagnosis,70RA cases were divided into test and control group according to the time sequence of seeing doctor.,35cases of test group, to DYD, a does one day,150ml Bid,35cases of control group,to LEF,20mg Qd.Observed clinical indexes of4time points (0w,2w,6w,12w) such as tender joint count,swollen joint count, morning stiffness duration,patient global assessment, overall pain VAS from patients and researcher, HAQ. Lab indexes of4time points (0w,2w,6w,12w) include RF, CRP, ESR. And serum TNF-α of2time points (0w,12w)is also measured and recorded. DAS28change, numbers of cases reached ACR20, ACR50were caculated according to above informations, index of security included routine blood test, renal (BUN,Cr), Liver function (ALT,AST).2. Experimental parts:Synovial specimens obtained from the joint of patients were minced into small pieces of RA-FLS.The morphological features of RA-FLS subcultured for3-5generations were observed under inverted microscope.Our study was a two-stage procedure:evaluation of DYD effects on the the prolifration of RA-FLS at first, and then analysis of its effects on TLR4mRNA, MyD88mRNA,. NF-κ B of cells total protein, TNF-a from culture supernatants. In evaluation of DYD effects on RA-FLS, RA-FLS cells were divided into three groups:the control, LEFgroup(10%serum containing LEF) and DYD group(10%serum containing DYD) and then evaluated by MTT at three time points (24,48,72h). In evaluation of DYD effects on TLR4mRNA, MyD88mRNA. RA-FLS cells were divided into four groups:the control, model control(LPS20μg/ml), LEFgroup(LPS20μg/ml+10%serum containing LEF) and DYD groups(LPS20u g/ml+10%serum containingDYD), after24h, TLR4mRNA and MyD88mRNA of cells RNA from each groups were evaluated by RT-PCR, NF-κ B of cells total protein from each groups were evaluated by Elisa, TNF-α from culture supernatants of different groups were tested by Elisa.Result1. Clinical part:(1)At last,31cases in control and33cases in test finished whole trail, cases shedded because of bad compliance.Accord to standard of DAS28, the total effective rate of test group is51.42%, this rate in the control is57.1%, no significant difference in total effective rate was found between the two groups (P>0.05). After treatments, DAS28, number of cases reach ACR20, ACR50in the two groups were improved obviously than12weeks before.(P<0.01).In test group, the rate reach ACR20is57.14%, the rate reach ACR50is20.00%, In the control group, the rate reach ACR20is62.86%, the rate reach ACR50is22.86%。 There is not significance between the rate reach ACR20, ACR50from the two groups (P>0.05).After12weeks, symptoms of patients from both groups were improved in tender joint number, swollen joint number, morning stiffness duration, patient global assessment, overall pain VAS from patients and researcher, HAQ.(P<0.01). At the end of2rd week, test group has obviously improved in the aforementioned aspects except morni ng stiffness (P<0.05), and compare to the control, the test group improved more significant in swollen and tender joint number, patient global assessment, overall pain VAS from patients and researcher, HAQ (P<0.05), until he end of6th week, in the aforementioned6aspects had been improved obviously in the control.(P<0.05),In the end, compare to the test group, overall pain VAS from patients and researcher, HAQ in the control group had been improved more obviously (P<0.05), no significant difference was found in any other Symptoms observation index.(P>0.05)(2)Lab indexes:after treating for6wand12w, ESR,CRP and RF reduced significantly than baseline data in both groups (P<0.01), in the test group, CRP reduced significantly than baseline at the end of2rd week,(P <0.05) in the control, all the three indexes reduced obviously after6week (P<0.05).At the end of6and12week, TNF-α reduced obviously compare to baseline in both groups (P<0.01), but no difference between the two groups at the same time point (P>0.05)(4) safety indexes:after12weeks, PLT in test group reduced compared to the baseline (P<0.05), no obvious difference was found in other indexes at any time point in the two groups (P>0.05)(5) adverse reaction, lpatient got skin rash in the test group, but soon passed,3women had got few menstrual than before, but all of them adhere to the end. In the control,10patients lost their hair, there were2cases got AST increased to double than before, but released with treatments.2. Experimental parts:At the time point of48h,72h, compare to the control group, LEF and DYD serum have obviously inhibitive effects on the prolifration of RA-FLS from patients (P<0.05) Inhibition rate in LEF is higher than DYD groups after72h.(P <0.05), Similar efficacy is found in reduce of the expression of TLR4mRNA after intervene of LPS (P<0.05), but the effect has not a significant difference between the LEF and DYD group (P>0.05), expression of MyD88mRNA has not a significant difference between all the groups (P>0.05). Test result of TNF-α from control and LPS groups were not different particularly (P>0.05).while TNF-α in DYD group is less than LPS group (P<0.05). NF-κ B in LPS group is more than the control (P<0.05), compare to the control group, NF-κ B reduced more significantly in the two serum groups (P<0.05), LEF group is less than control group (P<0.05), no significant difference were found between LEF and DYD groups (P>0.05).Conclusion1. after12weeks, DYD and LEF effectively improved clinical RA patients' symptoms, joint function and quality of life, reduced ESR, CRP, and RF, TNF-α in serum, but action of DYD occurs more faster than LEF. DYD reduced PLT partly may be explained as the leonurus in DYD has a effect of antiplatelet.Though3female patients in test group had got irregular menstruation, it remained to be seen in further clinical trail because climacterium may also result the same symptoms. While increasing levels of transaminases in LEF group were in agreement with references,No Serious adverse reaction happened in both groups meant the two drugs were safe withinl2weeks of treatment.2. Serum contained DYD or LEF can inhibit RA-FLS proliferation in regular doses dose, and the does can reduce the expression of TLR4mRNA, NF-κB of cells total protein, but has no effects on expression of MyD88mRNA.The result suggested the therapeutic mechanism of DYD for RA may included two aspests, first DYD could restrain proliferation of RA-FLS, second DYD may downregulated the expression of components from TLR4/NF-κ B pathway, but it is not clear wether MyD88dependent pathway is activated be no association with MyD88.