| BackgroundRheumatoid arthritis(RA)is a kind of autoimmune diseases with the major clinical manifestation of chronic progressive symmetric arthritis,its pathogenesis is not clear.The main manifestations of RA are synovitis,cell infiltration and pannus formation,bone destruction is its most common complications,with cartilage and bone involvement that lead to osteoporosis,and even joint structure damage caused by deformity and dysfunction,thereby increasing the RA morbidity,the harm is very serious.Therefore,even preventing bone destruction has become the main strategy for the treatment of RA.A great number of studies have shown that chronic inflammation of RA is the main factor to induce the bone destruction,In clinical practice,even RA patients with no symptoms,in the case of complete remission of disease activity,in some patients,there was still joint destruction,which is the so-called "inflammation and bone destruction"phenomenon.It is also difficult to inhibit the progress of bone destruction while the use of antirheumatic drugs to control the disease activity of RA,and although there are reports of bone damage can be delayed by biological agents,but it is difficult to completely block bone destruction progress.Therefore,the pathogenesis and treatment of RA bone destruction need to be further studied in order to inhibit the its occurrence and development.Hepcidin(Hepc)is a kind of small molecule polypeptide plays an important role in iron metabolism,its expression is influenced by many factors,such as the level of serum iron,erythropoietin,hypoxia and oxidative stress,and plays a key role in the regulation of iron metabolism balance of the body.Recent studies have found that the regulation of Hepc is also affected by inflammation,inflammation can stimulate the generation of Hepc,and the level of erum Hepc in RA patients is abnormally high,The results of the experiments in vitro showed that Hepc could promote the differentiation of osteoclast,and upregulate the expression of TRAP,CTK and MMP-9,which suggested that Hepc might be involved in the occurrence of RA bone destruction.In Traditional Chinese Medicine,RA belongs to the "Arthromyodynia"category,the main pathogenesis of bone destruction is the external evil invasion,simultaneous attack of wind and dampness,liver and kidney deficiency,tendons dystrophy,pathogenic factors invade the context,and obstruction of phlegm and blood stasis damage muscle and bone,resulting in joint deformation,bone destruction.Duanteng Yimu Decoction is based on the main pathogenesis of RA,reflects the basic treatment principles of "tonifying the liver and kidney,expelling wind and dampness,dispersing blood stasis and dredging collateral",can effectively control RA disease activity?However,the effect of RA on bone metabolism has not been observed.This research is supported by the natural science foundation of Guangdong Province(No.2014A030313403),in clinical research,we observed the bone metabolism and the expression of HEPC in RA patients,and their relevance;to evaluate the effect of Duanteng Yimu Decoction on serum bone metabolic markers in patients with RA;the CIA rat model was established to study the influence of Duanteng Yimu Decoction on RA bone destruction and the mechanism of action would be discussed.Part ? Clinical researchObjective:To observe the clinical efficacy and safety of Duanteng Yimu Decoction on RA bone destruction,through monitoring the level of the of Hepc,to analyze the correlation between Hepc and bone metabolism,to explore the mechanism of the treatment of Duanteng Yimu Decoction on RA bone destruction.Method:A total of 74 RA patients and 20 healthy volunteers were enrolled,74 cases of RA patients were randomly divided into control group of 37 cases,experimental group of 37 cases.Control group received Methotrexate Tablets treatment,the experimental group was given off Duanteng Yimu decoction.To detect and compare the RA patients and healthy volunteers serum Hepcidin(Hepc),type ? collagen carboxy terminal peptide(?-CTX),tartrate resistant acid phosphatase 5b(TRAP-5b),bone alkaline phosphatase(BALP)levels after treatment in two groups.The serum Hepc and bone metabolic indexes of two groups of patients with RA were reviewed after treatment,and the correlation between them would be analyzed.The effect of DAS28 was observed,at the same time,respectively before treatment and at 4,8,12 weeks,the disease activity index in two groups of TJC,SJC,PGA,MDGA,ESR,CRP,SDAI score of disease activity index and safety indicators were recorded,and to observe the occurrence of adverse reactions and the combination of medication.The treatment lasted for 12 weeks.Results:The levels of serum Hepc,?-CTX,TRAP-5b in patients with RA were higher than those in healthy group,the serum BALP level was lower,the difference was statistically significant(P<0.05).After treatment,of the patients in two groups,scores of TJC,SJC,PGA,MDGA were improved compared with before treatment,all four indexes in the control group were improved at the beginning of the eighth week(P<0.01),in twelfth week were more improved(P<0.01).The experimental group has improved from the fourth week after treatment(P<0.01),the 8th,12th week has been further improved(P<0.01).Comparison of TJC,PGA,MDGA between two groups,at 4,8,12 weeks in experimental group were significantly better than the control group(P<0.01);comparison of SJC between two groups,4th,8th week in experimental group were significantly better than the control group(P<0.01),but at twelfth weeks,there was no significant difference(P>0.05).Two groups of disease activity index were both significantly improved than before treatment(P<0.01),after four weeks of treatment,CRP,ESR,SDAI in the treatment group were beginning to improve(P<0.01 or P<0.05),at the 8th,12th week has been further improved(P<0.01);the SDAI score of control group was beginning to improve at 4th week,CRP,ESR index began improve at eighth week(P<0.01 or P<0.05),compared between two groups of CRP,treatment group was significantly better than the control group at eighth week(P<0.01),but at twelfth week,the two groups had no statistically significant difference(P>0.05);after 12 weeks of treatment,ESR,SDAI score of the treatment group were better than the control group(P<0.05).After 12 weeks of treatment,Hepc,?-CTX,TRAP-5b in two groups of patients all decreased(P<0.01),and the treatment group was better than the control group(P<0.01 or P<0.05),for BALP in two groups before and after treatment,there was no statistically significant difference(P>0.05).Before treatment,there was a significant positive correlation between serum Hepc level and level of ?-CTX,TRAP-5b,BALP,ESR,CRP and SDAI score(P<0· 01,P<0.05);after treatment,there was still a significant positive correlation between serum Hepc level and level of ?-CTX,TRAP-5b,BALP,ESR,CRP and SDAI score in RA(P<0.01),but no significant correlation with the BALP(P>0.05).Safety analysis showed that the level of WBC,PLT decreased,RBC,HGB in two groups of patients increased,the WBC in both group and PLT,RBC,HGB levels in control group were no significant differences(P>0.05),in the experimental group,RBC,HGB increased,PLT decreased,the difference was statistically significant(P<0.05).There was 1 case in the control group had get liver damage,and in the experimental group,4 cases of female patients with menstruation.Conclusion:1.Levels of serum Hepc elevates in RA patients,and is]significantly correlated with bone resorption index.2.While controlling RA disease activity,Duanteng Yimu Decoction can effectively improve the level of bone metabolism,inhibit bone destruction,the mechanism may be achieved by inhibiting the expression of Hepc in serum.Part ? Animal experimentObjective:To investigate the expression of Hepc in CIA rats and its relationship with bone metabolism,evaluate the therapeutic effect of Duanteng Yimu Decoction on RA bone destruction,and explore its mechanism of action.Methods:65 male Wistar rats were selected,and 60 rats were randomly divided into modele group,the other 5 rats were used as the control group.After the success of modeling,the rats were randomly divided into 6 groups:control group,model group,AG490 group,MIX group,Duanteng Yimu decoction low dose,medium dose,high dose.All groups were administered continuously for 6 weeks,to observe the general situation during modeling and medication,the body weight and a rthritis index(AI)were measured weekly.24 hours after the last administration,blood was selected from the abdominal aorta for detection of serum Hepc,TRAP-5b,CTX-1.The left femur were packaged with saline soaked gauze,used for micro-CT detection.Right femurs were fixed in 4%paraformaldehyde and stained for HE.Bilateral ankle joint synovial tissue were isolated to examine the changes of mRNA and protein level of p-JAK2 STAT3 and p-STAT3 of JAK2/STAT pathway by RT-PCR and Western blot.The experimental data was analyzed by stata11.0 statistical software.Results:1.The success rate of Wistar rats CIA model is 56.7%.Arthritis of the ankle and foot appearanced in the first 14-18 days,and reached the peak at about 4-5 weeks after the first immunization,then the swelling reduced,a few showed joint ankylosis,restricted activity,similar to the human RA pathology process,which proved that the model is successful.2.Compared with Control group,rats weight of Model group decreased,AI score increased,bone absorption index and serum Hepc level increased,and there was a positive correlation between them(correlation between Hepc and TRACP-5b:r=0.6483,P<0.01;correlation between Hepc and CTX-1:r=0.7149,P<0.01).Indicated that this model meets the needs of research model of this study,could be used in the research of pharmacodynamics and mechanisms further.3.After intervention of Duanteng Yimu Decoction,weight of rats increased(P<0.05),arthritis index decreased(P<0.05),bone histomorphometry increased(P<0.05),bone resorption index decreased(P<0.05),in a dose-dependent manner,the efficacy of the DTH group is better than in MTX group.4.After Duanteng Yimu Decoction treatment,serum Hepc decreased(P<0.05),in a dose-dependent manner.5.In the pathogenesis of CIA rats,the JAK/STAT signaling pathway was activated,the expression levels of JAK2,STAT3 and p-JAK2,p-STAT3 mRNA were increased,and the expression of p-JAK2 protein was up-regulated remarkably.6.After given intervention of AG490,the expression of JAK2 gene and protein could be inhibited(P<0.05),the expression of mRNA and phosphorylation of STAT3 molecule,which is downstream of JAK2,was significantly inhibited(P<0.05),serum Hepc level were significantly decreased(P<0.05),pathological staining of femur and bone hi stomorphometry parameters were significantly improved(P<0.05),bone absorption index decreased(P<0.05).7.Duanteng Yimu decoction can inhibit the JAK/STAT signaling pathway,down-regulated the expression of mRNA of JAK2 and STAT3,and the level of p-JAK2 and p-STAT3(P<0.05).Conclusion:1.During the course of RA inflammation,the level of bone resorption index and Hepc was increased,and there was a significant correlation between them.2.Duanteng Yimu decoction can effectively improve the condition of RA and inhibit bone destruction of RA,and the effect is dose-dependent.3.During the onset of rheumatoid arthritis,the JAK/STAT signaling pathway is activated,and can upregulate the expression of Hepc,and then promote the differentiation and function of osteoclast directly involved in the bone destruction of RA.4.Duanteng Yimu decoction may play a protective role in bone through down regulated expression of Hepc,by inhibiting the expression of related signaling molecules in the JAK/STAT pathway. |
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