| 1. BackgroundRheumatoid arthritis (RA) is one kind of autoimmune disease which takes thejoint synovial membrane inflammation as it's pathology center. RA belongs to theChinese medicine rheumatism card, calendar festival category, and it is therepresentative disease in rheumatism card.. On clinical we still lack of the permanentcontroltional method and preventive measure to resist the disease,and the traditionaltreatment mainly aims at the inflammation and sequela. The research has demonstrat-ed that the synovial membrane becomes the ciliary cell massive proliferations andforms under the pannus con'osion cartilage and the cartilage bone cerebralortex,which causes the ossein to destroy, further causes joint abnormal. In this case of ill-ness process the most obvious characteristic is the synovial membrane in the synovialmembrane lining becomes the ciliary cell proliferation. Therefore, to treat RA, it isthe strategy key point that to seeks the way which corrodes from the most activecontroltional synovial membrane proliferation and the cartilage in the last few years. There are some compound traditional Chinese medicines for the treatment ofrheumatoid arthritis(RA) at current domestic and foreign, including the nonsteroidalantiinflammatory drugs(NSAIDs, as buprofen), disease—modifying drugs(DMARDS, also the name slow acting antirheumatic drugs, as MTX, and LEF[leflunomide]), corticosteroids, biologic therapies and gene therapy. One of themedicine, LEF, has been shown to retard joint destruction in RA and delay thecondition to progress obviously, definited for condition improvement medicine.As drugs for RA, The traditional Chinese medicines, Not only have the goodantiinflammation analgesic effect, moreover have distinct improvement to patient'ssurvival quality. It has few poisonous side effect, moreover, it can adjust theorganism immunity function and the joint synovial membrane ultrastructure as wellas cell function. Some effective compound prescription and simple medicine(traditional Chinese medicines) have been used as drugs for RA in clinicaltrials. According to the long-term clinical experience, We have drawn up a Comp-ound prescription for RA, named Shanshuibaihu Decoction (SSBHD), which hasbeen discovered that itcan produce clinical improvement in patients obviously.SSBHD is formed from classic prescription of the traditional Chinese medicine,named Baihu Decoction (BHD), specially supposesed for Re-Bi (mainly includingtwo kind of type: Re-Bi of the RA's and Re-Bi of the OA's). The medicines mainlyinclude Shuiniujiao, Hanshuishi, Bajiezi, Huzhang and Jixueteng,ect.).It's maineffect lies in eliminating "Re and Shi", improving the blood microcirculation,as well asalleviating pain.. Our research has revealed that, SSBHD is capable of controllingpartial inflammation effectively, eliminating partial swelling, alleviating ache.Simultaneously it is helpful in reducing experimental target like CCP, IgG-RF. Thiseffect of SSBHD appears to be related to its ability to produce control of immunityactivity of the RA in the body. In the animal experimentation study,we have uesed SSBHD alone to treat one group of CIA model big mouses when they were in theacute period of the disease RA, but to another group (control group) with the drug,LEF only. Finally the experiment indicated that, The SSBHD and LEF both canreduce the red,heat,swelling and the ache of the big mouse hind legs and jointsobviously.Thus further confirms that The SSBHD has antirheumatic activity inthe experimentation.This experimental paper is in publication.The SSBHD's mechanism of action is unknown at present. As a new medicinetreating for RA in clinical, It is a hot spot of the current domestic Chinese medicinetreatment and research for rheumatism card. Therefore, to study the compoundtraditional Chinese formula of the SSBND, we attempt to study the effect of drug-contained serum of SSBHD on proliferation inhibition of cultured synovial fibroblastcells in vitro.2,Objective and significance of the studyRefer to the foregoing words, In this case of RA process, the most obviouscharacteristic is that the synovial membrane in the synovial membrane liningbecomes the ciliary cell proliferation. In order to discover the mechanism ofantirheumatic action of SSBHD further clearly, we design this experimentation, inwhich we want to observe the effect of drug-contained serum of SSBHD onproliferation inhibition of cultured synovial cells from knee-joint in vitro. Furtherlywe want to prove preliminary that the SSBHD is able to inhibit the proliferation ofsynovial fibroblast cells or not. If it can be,, it may been proven that the way inhibitingthe proliferation of synovial cells plays a central role in the mechanism of the efficacyin RA for the SSBHD.3 Methods and projects3.1. The specimen source of synovial organizes were derived from cutting off inoperation for the surgery inpatient, whom had the knee osteoarthritic disease being in the Department of Spine and Orthopedics, Southern Hospital, with the number274447 (women, 68-year-old.) The. clinical manifestation of the patient had beenturn to accord with Re-Bi (hot numbness syndrome) diagnosing standard of thetraditional Chinese medical, as well as turn to according with knee OA diagnosingstandard recommended by the American consulting of rheumatic disease in vitro.3.2. The production and dividing into groups for the drug-contained serums ofSSBHD, LEF and NS(Saline group).15 Wistar female rats were divided into three groups randomly, 5 per set a group,including the group of SSBHD (A cgroup),the group of LEF (B cgroup t) andthe group of NS (C group) according to weight.The produce of the liquids ofSSBHD: Frieing the SSBHDin water twice, fetching the liquids twice andcommixturing them, carrying out concentration, ultimate thickness is 1 g/ml holdenin one crude drug. The produce of LEF's: Dissolving the LEF-piece in the normalsaline water, coming to make the ultimate concentration of the medicine liquidcontaining LEF with 0.18 g/ml. Then, Continuely, irrigating the rat stomach for7days with liquids of SSBHDfor A group twice a day (2 ml/one time),with liquids ofLEF for B group once a day (2 ml/one time), and with NS (the normal saline water)for C group once a day (2 ml/one time). At the seventh day, after fetching bloodrespectively from the rats' abdomens aorta and then taking centrifugation for them,the drug-contained serums come out,and then, preserving standby under -20℃..3.3. Biocytoculture of synoviocytes: tissue culture in vitroThe synoviocytes were isolated from mirror surgery of the osteoarthritic diseasepatients and then were handled in the ultraclean stage sterilitily. After inoculated, thesynoviocytes organization pieces were put into the culture box (at the condition of 37℃and 5%的CO2). After the synovial cells growing well enough, we digested theminto cells liquid and counted the number of synovial cells by direct counting method,then, we continued cultivating the cells to the 3-5 generations cells.At the 3generations,we carried out B type activity identifies of synoviocytes for the culturedcells by the means of immuohistochchemistry..In the whole process we observedthe morphology of the cultured cells.3.4. The inhibitory experiment of the proliferation of synovial fibroblast cells:analyzed through the viable cell count by direct counting method and MTTassay. (under designing random admeasurement).3.4.1 Direct counting method:The 3 generations synoviocytes were adjusted into 4×10~5 cell/ml,infused the 24 wellclusters(0.5ml/well), colleced supernatant of the synoviocytes after 24h culture.Itwas allotted four groups to adding diffrerent pecimens,including three groups for thedrug-contained serums of SSBHD (A group), LEF(B group) and NS(C group),and the fourth group of completelness culture liquid (D group) which was not addedany drug-contained serum. Inaddition, the ultimate concentration of the everydrug-contained serum group (A,B and C group) was corresponded respectively to5%,10%,20% of its primitive serum. and the ultimate in the same level ofevery concentration there were 3 wells(n=3) in which cell with just 1 ml liquidaltogether. After cultured to predetermined time in the culture box (under condition of37℃and 5%的CO2), all the cultured synoviocytes were digested and collected tobe counted.3.4.2 MTT assayThe 4 generations synoviocytes were adjusted into 5×10~6 cell/ml, inoculated into the96 well cluster(0.1 ml/well), colleced supernatant of the synoviocytes after 24h culture.It was allotted four groups at different serum dose with the drug-contained serums ofSSBHD (A group),LEF(B group) and NS(C group), being set respectively ultimateconcentration for 10%, 20%, 30%, 40%, 50%. and with the fourth group of completelness culture liquid (D group) which was not added any drug-containedserum. Inaddition, in the same level of every concentration there were 3 wells(n=3)in which cell with just 0.2 ml liquid altogether.. In order to make a tune-zero forhole MTT Analysis,in every cluster, a blank well was not interposeed any cellsliquid but interposeed a culture liquid only. And then, the 96 well cluster withsynoviocytes were cultured in the culture box (under condition of 37℃and5%CO2). At the predetermined time, adding 20μL of MTT solution liquid (5 mg/ml)into every well and carrying out the culture continuousIy for 4 hours,and then, givingup the culture serum liquid of every well, infusing 150μL of DMSO liquid tomixture them for 5-10 minutes.,Finnally, assaying the OD of every well liquid byMultiskan MK3 under 492nm,and drawing out the growth lines of them.3.5. Measurement of the nitric oxide in the supernatant of the synoviocytes:We Choose the supernatants of the synoviocytes cultured by the drug-containedserum in concentration of 20% and cultured by the Complete culture Solution only.Taking them out from the refrigerators(having been set under -20℃), afterunfreezeing them, the operation steps of the measurement was in progress accordingto specifications of the nitric oxide reagent box(ade by Company Nanjing establishes).Then we measured every sample by enzyme-unite-instrument in 340 nmwavelength, and according to the formula provided in specifications,we calculatedthe nitric oxide contents.3.6. Statistical analysis: analysis of variance of factorial design was used toanalyse the result of the three drug-contained serum groups. SNK test to compare theresult between two groups., the inspection lever is 0.05.4. Results4.1 The cells dashed forward from organization piece after fostering 72hs.They were prompt increasing after the 6th day., and 12th day or more, the cells growed up to full70％of the culture bottle bottom. The passage generations cells growed upcomparatively quickly.When having foll the culture bottle bottom, the cells weredigested into cell liquid and continued culture.Observing the morphology of thefostering cells under inverted microscope .It was found that the sharp of cells was likea shuttle, and the cells had the appearance form and characteristic of fibre cell. Theresult of the Vimentin masculine by the way SABC immunohistochemistrydemonstrated that the characteristic dyeing of the cells was B type activity identifiesof synoviocytes.4.2 Direct counting method: The viable cell count in NS group(C group) has nosignificant difference compares with the normal control group (D group) at the samedrug-contained serum and time,but we can not to conclue that there no significantstatistical difference between them because the sample number in the experimentwere designed only 4 in each unit.Inaddition, cultured in D group and C group at thefirst 7days ,the viable cell counts had significant increase following the increase ofculture days which can present the time effect dependent relationship (P＜0.001).Aftercultured 3 days later by the 5％dose of drug-contained serum, The viable cell countin the group of SSBHD(A cgroup) and LEF (B cgroup t) had marked reduction butsignificant increase in D group and C group, and there were remarkable statisticaldifference (P＜0.001) compared with C group or D group. Furdermore,such statisticaldifference was found after only one day culture when the concentration of the everydrug-contained serum increased to being 10％and 20％, which can present the timeand dose effect dependent relationship.4.3 MTT Analysis Cultured in D group and C group at the first 5days ,theviable cell counts had significant increase following the increase of culture dayswhich can present the time effect dependent relationship (P＜0.001).Compared with the control normal group(D group) or the control serum group (C group) at the sameculture condition, the count to cell proliferation of viable cells for A group and Bgroup both had significant reduction, which demonstrated that both thedrug-contained serum of A group and B group can suppressed the proliferation ofcultured synovial cells significantly. At the same concentration of drug-containedserum in A group(the drug-contained serum of SSBHD), before the first 5 daysculture, the decrease of viable cell count between any two uninterrupted days hadmarked reduction (P＜0.001), which indicated that the inhibitory action toproliferation of cultured synovial cells strengthened with days cultivating increasingby the drug-contained serum of SSBHD. Inaddition, at the same day but at differentconcentration in A group, the decrease of viable cell count had significant reductionwith the concentration increases (P＜0.001).Those situations and results werehappened in the B group(the drug-contained serum of LEF) too,which can present thetime and dose effect dependent relationship. Furdermore, The viable cell countdecrease in A group and B group has no significant difference between them at thesame concentration serum and time ,but we can not to conclude that there nosignificant statistical difference because the sample number in the experiment weredesigned only 3 in each unit.4.4 In the Measurement of the nitric oxide in the supernatant of the Synoviocytescultured in the concentration being 20％of the drug-contained serum, We foundthat there were large quantities of NO producted in the normal control group (D group)and the serum control group(C group). On contrary, there was little production ofNO by the synoviocytes after culture in the group of A or B, and the decrease ofthe production had significant reduction compared with the (D group) or C group(P＜0.001).5. Conclusion 5.1 Both the drug-contained serum of NS (in C group) and the completelnessculture liquid without any drug-contained serum(in D group), can increase the activityof proliferation of knee arthritic synoviocytes cultured in vitro.5.2 The proliferation of knee arthritic synoviocytes can be suppressed significant bydrug-contained serum of SSBHD or LEF, which can notablely reduce theproduction of the synoviocytes cultured in vitro.5.3 In the process of the significant inhibition effect on proliferation of kneearthritic synoviocytes cultured by drug-contained serums of SSBHDor LEF, at thesame time, the decrease of the production NO were notable, which not only hadshown the fact that inhibiting the synoviocytes proliferation and inhibiting the activityof synoviocytes to product NO were both in the action process of culture bydrug-contained serums of both SSBHDand LEF can, but also indicated that the twoinhibition effects maybe play an important role and relationship in the process.5.4. It can indicate from the studies that the inhibition effect on synoviocytesproliferation of knee synovitis arthritic maybe approach the mechanism thatShanshuibaihu Tang bear the treatment effect on arthritis synovitis such asrheumatoid arthritis and osteoarthritic,inaddition , decreasing the number and theactivity of NO producted by the synoviocytes maybe play an important role in the thetreatment on synovitis arthritic.
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