| Rheumatoid atthritis （RA） is a chronic systemic autoimmune disordercharacterized by arthrosynovitis. The disease can cause multi-joints swellingpain, late stage can cause joint deformities and lost of function. RA is one ofthe major diseases causing disability in our nation, with the prevalence beingabout0.4%.RA is promoted by three interrelated pathophysiology processes:hyperplasia of synovium, inflammation and autoimmune. Numerousinflammatory cytokines directly or indirectly involved in the articularinflammatory response, the destruction of the synovium and articular cartilage,during which TNF-α, IL-1β and IL-6cytokines play a major role.Nuclear factor NF-κB is the central transcription factor involved ininflammatory and immune responses. It can be activated by a variety ofcytokine, such as TGF-β, TNF-α, IL-1β, IL-6, etc., and can further promotemultiple cytokines transcribe in inflammatory responses. This feedbackmechanism can lead to inflammatory response, and make destruction ofrheumatoid atthritis be sustained.The basic pathological feature of RA is arthromeningitis, and it cantrigger erosion and damage of bone and articular cartilage, and systemic bonemetabolism changes. The studies suggest that the receptor activator of NF-κBligand （RANKL） is the initiation factor of precursor osteoclast differentiationand maturation, and that osteoprotegerin （OPG） is the natural antagonist ofRANKL receptor.IL-6can induce the production and release of cytokines such as TNF-α,IL-1, etc., and can enhance its destructive effect of inflammation, is considered to be the amplification factor for biological effect of cytokines ininflammatory response. IL-10can inhibit the secretion of pro-inflammatorycytokines such as TNF-α, IL-1β, IL-2, IL-6, etc., as well as the conglutinationand invasion of pro-inflammatory cells.The experiments of the earlier stage proved that the Saponins of theThladiantha Dubia Roots （STDR） has significant inhibition for pain reactionto several kinds of pain model, and has obvious inhibition for thecarrageenan-induced rat paw edema.Preliminary evidences indicated thatSTDR can obviously reduce PGE2, MDA and NO levels in animal serums andinflamed tissues, and it is proved by safety evaluation to have low toxicity andno obvious side effects. In this study, we established rat model of adjuvantarthritis, then observed the therapeutic effect of STDR for model rats, andexplored the action mechanism of STDR for rheumatoid arthritis. This studywill provide experimental evidence and theoretic basis for furtherdevelopment of Thladiantha dubia Bunge.Objective:To evaluate the effect of STDR in treatment of RA using the jointswelling degree and arthritic index as index.To explore the mechanisms ofSTDR in treating RA using the expressions of NF-κB p65、OPG、RANKL、IL-6、IL-10as index,and to provide experimental data for clinical applicationsand further studies.Methods:1Model preparation and treating10rats were randomly selected from80male SD rats as control group,and the remaining70were given freund’s adjuvant induced adjuvant arthritis.50rats with arthritic index being≥6were evenly divided into five groups:model group, high dosage STDR group, middle dosage STDR group, lowdosage STDR group and Tripterygium Glycosides1l group. Each group wasgiven the corresponding medicine, and control group was given distilled water,for consecutive21days． 2. Determination of the joint swelling degreeThickness of vola pedis was measured using vermier cliper befer18daysafter injection and5,9,13,17and21days after the administration. Jointswelling degree （%）=[thickness of vola pedis （cm）-thickness of vola pedisof befer （cm）]/thickness of vola pedis of befer （cm）.3arthritis indexThe arthritic index of rats was calculated by arthritis scloring method18days after injection and21days after the administration.4detect the expression of factors4.122th after the administration, all rats were anesthetized byintraperitoneal injection of3%pentobarbital. Venous blood was gathered fromabdominal aorta, tissue samples were takened.4.2Immunohistochemistry and Westren Blotting were used to detect theexpression of NF-κB p65protein in each group.4.2.1Immunohistochemistry: fixing tissues with4%paraformaldehyde,embedding with paraffin, slicing, dewaxing, antigen retrieval, adding sealingliquid （A）, dropping the primary antibody, dropping C, DAB coloration,dehydration of gradient ethanol, Xylene transparent, mounting.4.2.2Wertern Blot: extracting the total proteins of the tissues; measuringprotein concentration; SDS-PAGE electrophoresis; the expression protein istransferred to PVDF membrane; detecting target protein; development andfixation; film processing.4.3RT-PCR was used to detect the expression of NF-κB p65, IL-6, IL-10mRNA in each group.Extracting total RNA of the tissues; testing RNA concentration, purityand integrity; reverse transcription; PCR amplification; analysis of PCRreaction products.4.4ELISA was used to detect serum levels of OPG, RANKL, IL-6andIL-10in each group.Results:1. The effects of joint swelling degree and arthritic index of RA rats bySTDR Joint swelling degree （left hindlimb） was measured using befer18daysafter injection and5,9,13,17,21days after the administration. Joint swellingdegree of the STDR160mg·kg-1group was significantly lower than that of themodel group521days after the administration （P＜0.05or P＜0.01）, andwas obviously lower than that of the TG group （P＜0.05）. The STDR80mg·kg-1group indicated obvious lower joint swelling degree relative to themodel group921days after the administration （P＜0.05or P＜0.01）. Jointswelling degree of the STDR40mg·kg-1group was conspicuously lower thanthat of the model group1321days after the administration （P＜0.05or P＜0.01）.1.2The arthritic index of the STDR groups （6.00±0.40,5.10±0.18,4.20±032） were evidently lower than that of the model group （7.20±0.32） onthe21st day of treatment （P＜0.05or P＜0.01）.2. The effects of NF-κB p65protein expression in tissue of AA rats bySTDR2.1Detect by immunohistochemistry assay, NF-κB p65positive productswere brown or dark brown, no positive staining was detected in the normalcontrol group, and NF-κB p65immunohistochemical staining was extensivepositive in model groups. Expression of NF-κB p65in each STDR group wasobviously lower than that in the model group （P＜0.05or P＜0.01）.2.2The Western method uses the ratio of NF-κB p65to β-actin toevaluate the expression levels of NF-κB p65. Compared to the normal controlgroup （0.0177±0.001）, the relative expression levels of NF-κB p65protein inthe model group （0.676±0.016） were significantly higher （P＜0.05）. Distinctlower values of relative expression levels of NF-κB p65protein were observedin the three STDR groups （0.445±0.023,0.181±0.008,0.0754±0.002） relativeto the model group （P＜0.05or P＜0.01）.3. The effects of STDR on NF-κB p65, IL-6, IL-10mRNA expression ofRA rats tissues （the ratio of the optical density of target band to the opticaldensity of β-actin was used to evaluate the expression levels of its mRNA）.3.1Compared to the normal control group （0.096±0.003）, the relative expression levels of NF-κB p65mRNA in the model group （0.615±0.006）were apparently higher （P＜0.01）. The relative expression levels of NF-κBp65mRNA of the three STDR groups （0.405±0.009,0.164±0.002,0.063±0.003） were significantly lower than that of the model group （P＜0.01）in a dose-dependent manner.3.2The model group （0.706±0.010） presented obvious higher relativeexpression levels of IL-6mRNA （P＜0.01） compared to the normal controlgroup （0.144±0.005）. The relative expression levels of IL-6mRNA of thethree STDR groups （0.536±0.010,0.115±0.007,0.081±0.003） weresignificantly lower than that of the model group （P＜0.01） in adose-dependent manner.3.3Obvious lower （P＜0.01） relative expression levels of IL-10mRNAwere detected in the model group （00.153±0.004） compared to the normalcontrol group （0.295±0.007）. The three STDR groups （0.272±0.004,0.280±0.005,0.254±0.007） showed conspicuous higher relative expressionlevels of IL-10mRNA than the model group did （P＜0.01）.4. The content change of OPG, RANKL, IL-6, IL-10in serum of AA ratsby STDR4.1Detected by ELSIA, the levels of RANKL of the three STDR groups(98.74±10.17pmol·L-1,87.81±17.78pmol·L-1,77.75±14.61pmol·L-1) weresignificantly lower （P＜0.05or P＜0.01） than that of the model group(120.80±12.14pmol·L-1), while the levels of OPG (71.67±2.62pmol·L-1,77.50±0.93pmol·L-1,91.04±7.26pmol·L-1) were conspicuouslyhigher （P＜0.05or P＜0.01） relative to the model group (53.64±3.46pmol·L-1). Compared to the model group （0.43±0.06）, the three STDR groups（0.71±0.08,0.89±0.17,1.13±0.24） had higher OPG/RANKL （P＜0.05,P＜0.01）.4.2Detected by ELSIA, the levels of IL-6of model group (1.58±0.04pg·L-1) were apparently higher （P＜0.01） than that of the normal control group(0.75±0.02pg·L-1); the levels of IL-6of the three STDR groups (0.79±0.06pg·L-1,0.95±0.04pg·L-1,1.13±0.03pg·L-1) were distinctly lower than that of the model group （P＜0.01）; the levels of IL-10of model group(1.91±0.06ng·L-1) were significantly lower （P＜0.01） than the normal controlgroup (2.60±0.056ng·L-1), the levels of IL-10of the three STDR groups(2.92±0.236ng·L-1,3.06±0.056ng·L-1,3.08±0.056ng·L-1) were conspicuouslyhigher than the model group （P＜0.01）.Conclusion:STDR has therapertic effect on RA induced by freund’s adjuvant in rats.Its mechanism maybe concern regulated pathway of OPG/RANKL/NF-κB,decrease of NF-κB and IL-6expression, improvement of the expression levelsof OPG and IL-10, which balance network of inflammatory cytokines. |
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