Acutum Alcohol Eluate In Treating Rheumatoid Arthritis And Explore Its Mechanism Of Action

BackgroundRheumatoid arthritis (Rheumatoid Arthritis, RA)is one of the slow synovial hyperplasia systemic autoimmune disease. RA belongs to the Arthralgia Syndrome in Chinese medicine, RA has a long history in Chinese medicine, Sinomenium acutum is the first choice drug therapy, and can relief the symptom of rheumatism, wipe out the meridians, diuresis, treatment rheumatism, joint swelling, itching, and other symptoms of paralysis. Sinomenine (sinomenine, SIN) is a monomer alkaloid extracted from Acutum, has the effect of analgesic, anti-inflammatory, effective clinical treatment of RA. Sinomenine has the function to alleviate the inflammation and inhibit bone destruction. In recent years, there are so many researches about the sinomenine, but Acutum alcohol eluate to RA therapeutic effectts are poorly researched.ObjectiveIn this study, inhibition of synovial hyperplasia and bone destruction in order to adjust the core of the Acutum 95% alcohol eluate for treatment of RA in vivo, in vitro studies, the use of renewal theory and techniques, understand the relevant mechanisms, explore Acutum advantages alcohol extract therapy of rheumatoid guide rational drug use.To compare the treatment effect of Caulis Sinomenii alcohol eluent and Sinomenine monomer for rheumatoid arthritis we observed the effect of acutum macroporous resin 95% alcohol eluate to the expression of vivo inflammatory in rat collagen-induced arthritis (CIA), at the same time, we researched the possible principles with the FLS and PBMC.Method1 Seprated of the effective parts1.1 Sinomenium acutum be crushed,8 times the dose of 95% ethanol were twice extracted, Every time 2 hours,the concentrates are combined, rotary evaporation to a thick extract form, addition of purified water used to dilute, formed Sinomenium acutum suspensions.1.2 By AB-8 macroporous resin to afford different concentrations of ethanol elution.Different concentrations elution ethanol content measured by HPLC in Sinomenine matter. 2 in vivo2.1 The collagen-induced arthritis (CIA) rat model was established, which were divided into five groups randomly as follow:(1) model group (MODEL, n=9);(2) high dose of Acutum alcohol eluate group (high does QFT, H-QFT, n=9);(3) low dose of Acutum alcohol eluate group (low does QFT, L-QFT, n=9);(4) sinomenine group (SIN, n=9);(5) blank control group (CONTROL, n=10)2.2 High dose of Acutum 95% ethanol eluate 120mg/kg.d by gavage, low dose of Acutum alcohol eluate 60mg/kg.d by gavage, Sinomenine group intragastric administration of an equivalent amount of Sinomenine suspensions, blank control group and modelgroup, gavage volume of saline.2.3 Every three days on rat arthritis index (AT) score, while observing and recording the state of rats.2.4 Analysis OPQ RANKL, IL-17secretion in rats’serum.3 In vitro3.1 FLS were isolated from the synovium of rheumatoid arthritis patients.3.2 Different administered groups’ proliferation of RA synovial fibroblast were detected by MMT method.3.3 Extracted total RNA from synovial fibroblast,Synovial fibroblasts’ RNA reverse transcription cDNA by RT-RCR.3.4 REAL TIME-PCR detected the expression of OPG and RANKL gene of different treatment groups, and osteoclast differentiation signaling molecules regulating the expression of NFATc1.Result1 Seprated of the effective parts1.1 Established of methodologies:λ=262nm, Column temperature:30℃, The mobile phase: Water (adjusted with phosphoric acid and triethylamine to PH 7.8)-acetonitrile.1.2 Determination of content:peak time and content of Acutum 95% ethanol eluate with similar SIN.2 in vivoThe first 21 days of the rat paw edema modeling reached the first peak. In the first 35 days the extent of rat paw edema model group reached the highest peak,36 successful model rats, according to the arthritis index (AI), were divided into H-QFT group, L-QFT group, model group,9 rats each group.2.1 Rats’weight of H-QFT group, L-QFT group and SIN group were under good control.2.2 Rats’Joint swelling of H-QFT group, L-QFT group and SIN group were significantly inhibited than those of MODEL group (P< 0.05).2.3 IL-17 level of H-QFT group were significantly lower than MODEL group (P< 0.05).2.4 the level of RANKL in H-QFT group had significantly decreased (P<0.05), while the level of OPG and O/R had significantly increased (P<0.05).3 in vitro3.1 Use type Ⅱ collagenase to extract rheumatoid arthritis (RA) patients’synovial fibroblasts(FLS).3-4 generation of FLS cells were used in the experiment, showed hastypical morphology.3.2.MTT assay administered group influence on cell proliferation, H-QFT could significantly inhibit the proliferation of synovial cells compared with the control group (P<0.05).3.3 RANKL, OPG expression of administered group to PBMC on 12h and 48h by RT-RCR, H-QFT could significantly reduce the expression of RANKL and up-regulate OPG expression.Conclusion:1. separation of effective components:8 times 95% ethanol extraction 2h, sinomenine content is highest.2. the vivo experiments showed that, compared with other treatment groups, H-QFT group could significantly reduce the symptom of joint swelling of CIA rats, reduce the level of IL-17 to further develop anti-inflammatory effects, inhibit synovial hyperplasia, cartilage protection from damage; decrease the RANKL expression, increase the OPG expression and OPG/RANKL ratio, prevent the bone destruction, to relieve symptoms of rheumatoid arthritis.3. the vitro experiment showed that, H-QFT could significantly inhibit proliferation of RA synovial cells, And also could depress the expression of the mRNA in the RANKL. NFATcl is the promoter factor for the development of osteoclast. We measured the number of their expression. After adding drugs, the number of the expression of NFATcl coule be inhibited. As a result, the developing of OC will be stopped and bines can be protected.