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1.Objectives and significanceRheumatoid arthritis（RA） is a systemic autoimmune diseases characterized by chronic inflammation,abnormal immune responses and synovial membrane,It has a high disability rate and there is no effective specific treatment.Epidemiological investigation shows that there is about 0.5～1%Patients in the whole world.The adult prevalence rate of RA in China was about 0.4%.Recent studies suggest that RA is mainly characterized by the typical pathological inflammatory synovial hyperplasia, mononuclear cell infiltration and neovascularization.It’s growth characteristics similar to the limitations of invasive growth of tumors,rheumatoid arthritis can not therefore be considered as a benign.The disease,which seriously affected the quality of life of patients and in patients with shortened life expectancy,may be a local invasive tumors,the "second cancer".On the clinical,the treatment of rheumatoid arthritis is to alleviate pain,reduce inflammation,protect muscle and joint function.The pathogenesis of RA is still not entirely clear.Generally speaking,RA is a disease triggered on the base of genetic predisposition by external factors.T-cell lineage is considered as a milestone in the way to the recognition of RA.Many studies have confirmed that T cell-mediated immune response abnormalities is the main pathogenesis of RA.A large number of T lymphocyte infiltration in RA were found diffuse or nearby the blood vessels.CD4+/CD8+ of T cells play an important role in the pathogenesis of RA,especially in the excitation and continuity of RA. Besides,Cytokine network plays a key role in the damage of RA.Among the total,Tumor necrosis factor-α（TNF-α） mainly involved in the inflammation control; Interleukin-1（IL-1） may accelerate cartilage damage;（Vascular endothelial growth factor,VEGF） can promote the proliferation of synovial blood vessels,meanwhile, IL-1 and TNF-αare able to increase the secretion of VEGF.These cytokines stimulate each other in the pathogenesis of RA,promote the development of inflammation and accelerate the synovial lesions,and promote pannus formation,cause corrosion and damage of the Organization around the joints.While suppressing inflammatory cytokines Interleukin-10（IL-10） can inhibit the produce of metalloproteinases and the release of IL-1 and TNF-α,against the damage in patients with RA.But in generally speaking,the interaction between cytokines in RA is far from clear.The indeterminateness of path mechanisms results in multitargets and blindness in the treatment of RA.Though the classic immune depressive therapy can improve the disease condition to some extent,even clinical cure can be attained,a long-term use of immunosuppressive agents may lead to adverse reactions,which have been frequently reported and studied.Recent years researches on biologic agents’ aimed at each target of treatment in cytokine network have made great progress..However, because of the high cost,potential health risks and too many contraindications,these agents can not widely use.On the contrary,it had been clearly explained that how and why arthromyodynia happened in the ancient traditional Chinese medicine（TCM） files.In recent years,the traditional theory of wind-cold-damp stasis was greatly improved and extended. Accordingly,TCM plays an more and more important role in the treatment of RA.As a result,the issues on how to fully explore the advantages and potential superiorities of TCM,and how to strengthen the research of the effective TCM formula,are the key points in the treatment of RA.SHI RE BE tablet functions as eliminating dampness,clearing heat,freeing the network vessels and relieving pains.The formula involves in Rhizoma Atracylodis, Caulis lonicerae,Earthworm Lumbricus,Forsythia suspensa Vahl,Amur Corktree Bark, Coix Seed,etc.We herein using mice models to study the anti-inflammatory and analgesic effects of SHI RE BE tablet;We also copied the model of adjuvant arthritis （AA） in SD rats,aimed to observe the effects of SHI RE BE tablet on pathological changes of AA,and explore its related pharmacological mechanism.2.Methods2.1 Effects of anti-inflammatory and analgesic2.1.1 Hot-plate test40 mice,female,were put on hot plate during（55±0.5）℃,and recorded the time when the mice licking the foot,the time is called pain threshold.Screening the mice that pain threshold is during 10 and 30 seconds,were randomly divided into 5 groups, including Blank control group,Ibuprofen group,SHI RE BE tablet low-dose, high-dose groups.Mice are intragastric administration for 4days,and record the pain threshold at 60min after the last day.2.1.2 Writhing experiment40 mice,female,were randomly divided into 4 groups,including Model control group,Ibuprofen group,SHI RE BE tablet low-dose,high-dose groups.Mice are intragastric administration for 4 days,30 minutes after the last one,each mouse is intraperitoneal injection by 0.6%acetic acid,and the number of writhing is recorded in 20 minutes.2.1.3 Ear swelling experiment40 mice,female,were randomly divided into 4 groups,including Model control group,Prednisone group,SHI RE BE tablet low-dose,high-dose groups.Mice are intragastric administration for 4 days,30 minutes after the last one,15μl xylene is spread evenly on right ear,after 15 minutes,mice are to put to death,the ears are cut, draw the materials from the same place of two ears by hole puncher,then weighing in electronic balance and calculate it.2.1.4 Permeability test of capillary vessel 40 mice,female,were randomly divided into 4groups,including Model control group,Prednisone group,SHI RE BE tablet low-dose,high-dose groups.Mice are intragastric administration for 4 days,30 minutes after the last one,caudal vein injection by 0.5%Evans Blue solution,and immediately intraperitoneal injection by 0.6%acetic acid,20 minutes later,the mice are to put to death,open the abdominal cavity,wash with 5ml Sodium Chloride,and suction the solution,centrifuge and determine the absorbance of the supernatant on extreme ultraviolet spectroscopy in 595nm.2.2 The effects of SHI RE BE tablet on the Mtb-induced arthritis（AA） in rats 50 SD rats were randomly divided into 5 groups,including Blank control group, model group,LEF group,SHI RE BE tablet low-dose,high-dose groups.In addition to the rats of the blank control group,other rats were immunized s.c.at the base of the tail with Mtb in mineral oil.On the day after the initial immunity,rats are intragastric administration for 28 days.During the treatment the arthritis score and the paw volume of the rats were observed.X-ray was filmed to observe the foot joint lesions in rats after 28 days.Then all the knee joints and ankle joints of hind limbs were sampled.Immunohistochemical observation and scoring was preformed.2.3 The pharmacological mechanisms of SHI RE BE tablet2.3.1 The effects of SHI RE BE tablet on the T lymphocyte subsets in periphery blood Take 100μl anticoagulated blood after 28 days,add the APC-CD4、PE-CD8a in order and incubation,then add the hemolysin,wash the cell by PBS,suspension the cell and detected by flow cytometer.2.3.2 The effects of SHI RE BE tablet on the abundance of TNF-αAfter 28 days,non-anticoagulant blood samples were prepared followed the instructions and the abundance of TNF-αwere detected by ELIA.2.3.3 The effects of SHI RE BE tablet on the levels of IL-1、IL-10 and VEGF After 28 days,enzyme linked immunosorbent assay was performed to detect the levels of IL-1、IL-10 and VEGF in joint tissue homogenate of SD rats,and the mean concentration of the five groups was compared and analyzed.3.Results3.1 Effects of anti-inflammatory and analgesicIn the hot-plate test,Compared with the control group,the pain threshold are extend in SHI RE BE tablet low-dose,high-dose groups（P＜0.05）.In writhing experiment,compared with the model group,the times of writhing in 20 minutes are less in SHI RE BE tablet low-dose,high-dose groups（P＜0.01）,it provide that SHI RE BE tablet has significant analgesic effects.In xylene-induced ear edema experiment,the ear swelling in SHI RE BE tablet groups and Prednisone group is significantly lighter than the model group.And in permeability test of capillary vessel, the formula could significantly restrain the permeability of capillary vessel.SHI RE BE tablet contributes to control the acute inflammation of adjuvant arthritis model in rats.3.2 The pathological changes of AA ratsAfter the immunity,there is a significantly difference of the paw volume and the clinical score in different time and different groups,both the changes is the accordant. The mean of the paw edema and the clinical score in the model group shows significantly high.However,the one of LEF group and SHI RE BE tablet group shows down from the 13th and 16th.Observation of pathological changes and X-ray showed that bone destruction and synovial damage in LEF group and SHI RE BE tablet group are significantly improved.It can prove that SHI RE BE tablet has a role in controlling the acute inflammation.3.3 The related mechanism of anti-inflammatory effects3.3.1 Changes of T-lymphocyte subsets in peripheral bloodThe content of CD8+T cell and the CD4+/CD8+ are significantly different in the model group and other medication administration groups,but the content of CD4+T cell is almost the same.Compared with the model group,the content of CD8+T cell is higher（P＜0.05） and the CD4+/CD8+ is obviously lower（P＜0.05） in the treatment groups.3.3.2 The influense to pro-inflammatory factorsThe abundance of serum TNF-α、IL-1 and VEGF are significantly different in model group and other medication administration groups.Compared with the blank group,these pro-inflammatory factors are obviously increased（P＜0.05）.Compared with the model group,the concentration of TNF-α、IL-1 and VEGF are lower（P＜0.05）.3.3.3 The influense to anti-inflammatory factorsThe level of IL-10 is significantly different in model group and other medication administration groups.Compared with the model group,the level of IL-10 is obviously higher（P＜0.05）.4.Conclusion4.1 SHI RE BE tablet has significant anti-inflammatory and analgesic effects on acute inflammation.It can extend the pain threshold,contributes to control the ear swelling and the permeability of capillary vessel.All of these can make clear that SHI RE BE tablet has significant anti-inflammatory and analgesic effects.4.2 SHI RE BE tablet contributes to control the acute inflammation of AA rats.SHI RE BE tablet can suppress the pathological changes in the procession stage of AA, including synovial hyperplasia and fibrosis,inflammatory cell infiltration,the destruction of cartilages and bones.4.3 The probable pharmacological mechanisms of SHI RE BE tablet are as following: SHI RE BE tablet can regulate the turbulence of periphery abnormal T lymphocyte subsets of AA rats;SHI RE BE tablet can regulate the balance of Cytokine Network.
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