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OBJECTIVE@#To observe the effects of moxibustion on the ultrastructure of synovial cells of knee joint and serum cytokines in adjuvant arthritis (AA) rats, and to explore the potential mechanism of moxibustion in treatment of rheumatoid arthritis.@*METHODS@#Forty-five Wistar male rats were randomly divided into a normal group, a model group and a moxibustion group, with 15 rats in each group. In the model group and the moxibustion group, the AA model was replicated under wind, cold and humid environment and by injection with complete freund's adjuvant. In the moxibustion group, moxibustion at "Zusanli" (ST 36) and "Shenshu" (BL 23) was used, 20 min each time, once daily, for consecutive 21 days. In the normal group and the model group, no intervention was processed. The scores of the knee joint swelling degree (JSD) and arthritis index (AI) were compared among groups. The ultrastructure of synovial cells of knee joint were observed under transmission electron microscope (TEM). The levels of serum cytokines such as tumor necrosis factor-α (TNF-α), interieukin (IL)-1β, IL-6 and IL-10 were detected using ELISA method.@*RESULTS@#Compared with the normal group, JSD and AI scores, the levels of TNF-α, IL-1β and IL-6 were increased (P<0.01), while IL-10 was reduced (P<0.01) in the model group after intervention. JSD and AI scores, and the levels of TNF-α, IL-1β and IL-6 were lower (P<0.05, P<0.01), while the level of IL-10 was higher (P<0.01) in the moxibustion group compared with the model group. Compared with the normal group, the ultrastructure of synovial cell was obviously damaged in the model group, and the damage was attenuated in the moxibustion group compared with the model group.@*CONCLUSION@#Moxibustion can reduce the symptoms of arthritis in AA rats, which may be related to the improvement of the ultrastructure of synovial cells and the regulation of cytokines.
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Male , Rats , Animals , Cytokines , Interleukin-10 , Arthritis, Experimental , Tumor Necrosis Factor-alpha , Interleukin-6 , Moxibustion , Rats, Wistar , Knee JointABSTRACT
AIM: To investigate the molecular mechanism of lncRNA HOTAIR regulating miR-206 on the proliferation and apoptosis of rheumatoid arthritis synovial cells. METHODS: The synovial tissue from 30 cases of rheumatoid arthritis were collected. Rheumatoid arthritis synovial cells MH7A were cultured. The experiment was divided into si-NC group, si-HOTAIR group, miR-NC group, miR-206 mimic group, si-HOTAIR+NC inhibitor group, si-HOTAIR+miR-206 inhibitor group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression levels of HOTAIR and miR-206 in cells. CCK-8 method to detect cell proliferation; flow cytometry to detect cell apoptosis; Western blot to detect cell protein expression of CyclinD1, p21, Bax and Bcl-2; dual luciferase reporter assay to detect HOTAIR and miR-206 targets To combination relationship. RESULTS: Compared with the healthy control group, the expression level of HOTAIR in patients with rheumatoid arthritis was significantly up-regulated, and the expression level of miR-206 was significantly down-regulated (P<0.05). Compared with the si-NC group, the HOTAIR expression level in the si-HOTAIR group was significantly down-regulated, the cell survival rate were significantly down-regulated, and the apoptosis rate were significantly up-regulated (P<0.05). Compared with the miR-NC group, the expression level of miR-206 in the miR-206 mimic group was significantly up-regulated, the cell survival rate were significantly down-regulated, and the apoptosis rate were significantly up-regulated (P<0.05). Compared with the si-HOTAIR + NC inhibitor group, the cell survival rate in the si-HOTAIR+ miR-206 inhibitor group were significantly up-regulated, and the apoptosis rate were significantly decrease (P<0.05). CONCLUSION: Inhibiting the expression of HOTAIR and up-regulating the expression of miR-206 can reduce the proliferation of rheumatoid arthritis synovial cells and promote apoptosis.
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Aim To investigate the possible mechanism of paeonol inhibiting the inflammatory response of fibroblast synovial cells (RA-FLSS) in rheumatoid arthritis. Methods CCK-8 assay was used to detect Paeonol's inhibitory level on the abnormal proliferation of arthritis human fibroblast synovial cells (RA-FLSs). The levels of endoplasmic reticulum stress-related proteins MANF and ATF6 were detected by Western blot. Cell localization of transcription factor p65 and Mesencephalic Astrocyte Derived Neurotrophic Factor (MANF) was detected by immunofluorescence. RT-qPCR detected the changes of p65 target genes. Results Paeonol could significantly inhibit the abnormal proliferation of RA-FLSS cells. Paeonol activates ATF6 and increases the expression of MANF. Paeonol promoted the nuclear transfer of MANF protein and inhibited the transcriptional activity of p65. Conclusion Paeonol promotes the expression of MANF and nuclear transfer through the endoplasmic reticulum stress pathway and affects the progression of RA by inhibiting the transcriptional activity of p65.
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Aim To explore the effect of resveratrol (Res) on fibroblast synovial cells (FLS) inadjuvant arthritis (AA) rats treated with low concentration of H2O2, and the role of mitochondrial oxidative stress proteins deacetylase 3 (SIRT3) and manganese superoxide dismutase (MnSOD). Methods Twenty SD rats were randomly divided into normal group and model group. The AA model was induced by subcutaneous injection of the complete adjuvant in toes of model group. On 28th day after modeling, the rats were killed and the changes in serum oxidative stress index in two groups were detected. The expression of oxidative stress protein of SIRT3 and MnSOD in synovial tissues of knee joint of AA rats were detected by immunohistochemistry stainning. The effect of different concentrations of H2O2or Res treatment on FLS proliferation was observed by CCK-8 method. The effect of Res on apoptosis and the expression proteins SIRT3 and MnSOD of FLS treated with low H2O2concentration were detected by Hoechst 33258 and Western blot. Results Compared with normal group, the serum of AA rats showed oxidative stress. The expression of SIRT3 and MnSOD increased in synovial tissues of AA model rats. Low concentration of H2O2promoted FLS proliferation, and Res could dose-dependently inhibit proliferation treated with low concentration of H2O2, and increased FLS apoptosis. Moreover, with the increase of Res dose, the expression of SIRT3 and MnSOD protein decreased in FLS treated with low concentration H2O2. Conclusions AA rats are in a state of oxidative stress, low concentration of H2O2can promote FLS growth in AA ras, and Res treatment can inhibit FLS proliferation, promoting FLS apoptosis. The mechanism may be related to the reduction of anti-oxidative stress effect.
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Objective::To study the effects of Ermiaosan on migration, adhesion and invasion of human fibroblast-liked synovial cells(FLS) and explore its mechanism. Method::Using the human FLS as the research object, the nontoxic concentration of FLS.FLS was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay for the follow-up experiment. The transwell migration, adhesion and transwell invasion test were used to detect the migration and adhesion of the different concentration of Ermiaosan on FLS, respectively. The expression of interleukin (IL)-1 beta of FLS supernatant was detected by enzyme linked immunosorbent assay (ELISA). Protein in FLS was extracted and protein expression levels of phosphorylated Janus kinase 1 (p-JAK1), p-signal transducer and activator of transcription (STAT1) and p-STAT6 were detected by Western blot. Result::Compared with control group, tumor necrosis factor(TNF)-α (20 μg·L-1) increased the proliferation, migration, adhesion, invasion and the secretion of IL-1β of FLS (P<0.01). Ermiaosan(0.2, 0.4, 0.8 mg·L-1) had no significant effect on the proliferation of FLS induced by TNF-α for 24 h. Within 24 h, the migration, adhesion, invasion, invasion, and secretion of IL-1β of FLS cells induced by TNF-α were also decreased significantly(P<0.05, P<0.01). Compared with the blank group, TNF-α could induce abnormal elevation of p-JAK1, p-STAT1 and p-STAT6 in FLS (P<0.01), while Ermiaosan of 0.2, 0.4, 0.8 g·L-1 could significantly reduce the expression levels of p-JAK1, p-STAT1 and p-STAT6 (P<0.05, P<0.01). Conclusion::Ermiaosan can inhibit the migration, adhesion and invasion of FLS, and its mechanism may be related to the inhibition of the secretion of IL-1β, the mechanism may be related to JAK/STAT pathway.
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Objective To clarify the mechanisms that the response of fibroblast-like synovial (FLS) cellsto methotrexate (MTX) in rheumatoid arthritis (RA) and to provide theory basis for the drug treatment of RA.Methods Synovial fibroblasts were isolated from synovial tissue specimens obtained from patients with RA andexposed to MTX.Cell viability was measured using a MTT assay and cell apoptosis was valued by flow cytometry.Western blotting analysis of LC3 and immunocytochemistry were used to analyze the induction of autophagy in RA-FLS after treating with MTX.Transfection of siRNA was used to interfere the expression of Beclin1 to down-regulate the autophagy,cell apoptosis was valued by flow cytometry and western blot analysis was used to test the PARPp85 with or without the presence of MTX.Statistical product and service solutions (SPSS) 18.0 statistical software was used for statistical analysis of all experimental data.Independent sample t test was used according to data distribution status,homogeneity of variance,and normal distribution.GraphPad Prism 5.0 was used to draw statistical graphs.Results MTX induced apoptosis was increased in RA-FLS.MTX stimulated the autophagy response in RA-FLS by inducing autophagosome formation.In RA-FLS,transfection with Beclin1 siRNA inhibited autophagy and increased the susceptibility to MTX,which induced cell death.Conclusion Autophagy of RA-FLS contributes to the resistance to apoptosis induced by methotrexate.
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Objective To investigate the effect of Miao medicine Jinwu Jiangu decoction containing serum freeze-dried powder on levels of IL-17,ACT1 and TRAF6 in human rheumatoid arthritis fibroblast like synoviocytes (RA-HFLS).Methods Rabbits were randomly divided into blank control group (recieving normal saline of the same volume),Jinwu Jiangu decoction high-dose,medium-dose and low-dose group (intragastrically administrated with Jinwu Jiangu decoction at doses of 14.4,4.8 and 2.4 g·kg-1,respectively),tripterygium glycosides group and prednisone group (treated with human equivalent dosage).RA-HFLS primary cell model was established in the experiment.ELISA method was used to detect effect of lyophilized powder on IL-17 secretion.Expression of ACT1,TRAF6 mRNA was detected by RT-PCR.Results Compared with the blank control gorup,IL-17 in the supernatant of each medication administration group was significantly decreased (all P0.05).Conclusion Freeze-dried powder of Jinwu Jiangu decoction can decrease the secretion of IL-17 and down-regulate expression of ACT1,TRAF6 with RA-HFLS.
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Objective To study the relationship between E2F2 and the pathological features of RA and the mechanism of E2F2 expression.Methods The interference efficiency was detected by RT-PCR after RA FLs transfected with E2F2 siRNA.The cell proliferation was analyzed by MTS and the expression of numerous genes was tested by ELISA.To analyze the influence of E2F2 in the immune response by RT-PCR,RA FLs were separately treated with TNF-t and PDTC.The way of regulating E2F2 experssion was tested by Chip.Results Compared with negative control groups,the level of E2F2 mRNA were decreased in the cells transfected with E2F2 siRNA (P ＜ 0.01).Silencing E2F2 suppresses the proliferation of RA FLs (P ＜ 0.05).TNF-α regulates the expression of E2F2 in RA FLs via the NF-κB pathway.Conclusion Up-regulation of E2F2 is associated with the process of RA and TNF-α regulates the expression of E2F2 in RA FLs via the NF-κB pathway.Thus E2F2 might be a potential target for use in the development of new therapeutic approches of RA.
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K/BxN serum can induce arthritis in normal mice because of abundant autoantibodies that trigger an innate inflammatory response in joints. To determine whether IL-17 is involved in the pathogenesis of serum-induced arthritis, we injected wild-type and IL-17(−/−) mice with K/BxN serum and evaluated them for signs of arthritis. Unlike wild-type mice, IL-17(−/−) mice did not show any signs of arthritis. IL-17 was produced predominantly by CD3⁻ CD4⁻γδTCR⁻ NK1.1⁻ Sca1(int) Thy1(hi) cells residing in the inflamed synovial tissue. When synovial cells extracted from normal joints were stimulated with IL-23 or autoantibody-containing immune complexes, a substantial fraction of Sca1(int) Thy1(hi) cells produced IL-17. Thus, we have identified a novel population of IL-17-producing innate synovial cells that play a crucial role in the development of K/BxN serum-induced arthritis.
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Animals , Mice , Antigen-Antibody Complex , Arthritis , Autoantibodies , Interleukin-17 , Interleukin-23 , Interleukins , JointsABSTRACT
OBJECTIVE:To investigate the effects of triptolide(TP)on the proliferation of fibroblast-like synoviocytes(FLS) from patients with rheumatoid arthritis(RA)in vitro. METHODS:5 RA patients received knee arthroplasty or synovectomy to ob-tain synovial tissue. FLS was isolated,cultured and identified,and then incubated in the presence of TP [0 (blank control),50, 100 and 200 nmol/ml] for 24,48 and 72 h. The effects of TP on FLS was evaluated by MTT,and then proliferation inhibitory rate was calculated;flow cytometry was used to detect the apoptosis and cell cycle of FLS. RESULTS:The inhibitory rates of TP(50, 100 and 200 nmol/ml)on the proliferation of FLS were 17.46%-52.56%,which was positively correlated with drug concentration. Compared with blank control group,100 and 200 nmol/ml TP could increase the percentage of cells in G0/G1 phase and decrease the percentage of cells in S phase,with statistical significance(P<0.05);200 nmol/ml TP could induce cell apoptosis. CONCLU-SIONS:TP could inhibit the proliferation and also could induce the apoptosis of FLS in RA patients in vitro,which may be one of its mechanism for treating RA.
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Background & objectives: To study effects of drugs against rheumatoid arthritis (RA) synoviocytes or fibroblast like synoviocytes (FLS) are used. To overcome the drawbacks of using FLS, this study was conducted to show the validity of SW982 synovial cell line in RA study. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Annexin V propidium iodide (PI) staining, mitochondrial membrane potential assay, Triton X-114 Phase partitioning, and immunolot for apoptosis signaling in SW982 human synovial cell line were performed. Results: Fluvastatin induced apoptosis in a dose- and time-dependent manner in TNFα -stimulated SW982 human synovial cells. A geranylgeranylpyrophosphate (GGPP) inhibitor, but not a farnesylpyrophosphate (FPP) inhibitor, induced apoptosis, and fluvastatin-induced apoptosis was associated with the translocation of isoprenylated RhoA and Rac1 proteins from the cell membrane to the cytosol. Fluvastatin-induced downstream apoptotic signals were associated with inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Accordingly, 89 kDa apoptotic cleavage fragment of poly (ADP-ribose) polymerase (PARP) was detected. Interpretation & conclusions: Collectively, our data indicate that fluvastatin induces apoptotic cell death in TNFα-stimulated SW982 human synovial cells through the inactivation of the geranylgerenylated membrane fraction of RhoA and Rac1 proteins and the subsequent inhibition of the PI3K/Akt signaling pathway. This finding shows the validity of SW982 cell line for RA study.
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Objective To design and identify small interfering RNA (siRNA) targeting tumor necrosis factor‐α (TNF‐α) ex‐pression ,siRNAs were electroporated into synovial cells of Human to screen those which can effectively suppress TNF‐α expres‐sion .Methods Three TNF‐α specific double stranded siRNA were designed targeting different regions of TNF‐α mRNA(compared with negative control group) .RT‐PCR and Elisa were applied to detect TNF‐α ,mRNA expression and the secretion of TNF‐α in cell supernatant ,respectively .Results Two of the three customized TNF‐α siRNA could inhibit the expression of TNF‐α mRNA in synovial cells of Human (P< 0 .01) .At the same time ,the secretion of TNF‐α decreased in cell supernatant ,the difference was sig‐nificant statistically compared with the control group(P< 0 .01) .Conclusion TNF‐α siRNA can be successfully designed and syn‐thesized ,which can specifically and effectively suppress TNF‐α mRNA expression .
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AIM:To examine the effects of thromboxane A 2 receptor ( TXA2 R) , the downstream product of cy-clooxygenase-2 (COX-2), on the proliferative ability and COX-2 expression in rheumatoid arthritis (RA) synovial cells. METHODS:The effects of TXA2 R antagonist SQ29548 and agonist U46619 on the proliferation of RA synovial cell line MH7A were detected by MTS cell proliferation assay , and their effects on COX-2 mRNA expression in MH7A cells were al-so examined by real-time PCR.In addition, the possible effect of U46619 on the proliferation of MH7A cells, when COX-2 was knocked down by siRNA , was determined by BrdU cell proliferation assay .RESULTS:SQ29548 inhibited the cell proliferation and the mRNA level of COX-2 while U46619 enhanced them.Moreover, U46619 reconstitute the proliferative ability of MH7A cells to some extent that inhibited by COX-2 siRNA.CONCLUSION: In RA synovial cells, TXA2R is able to control COX-2 expression, while it also mediates the effects of COX-2, suggesting that TXA2R might be an ideal candidate for RA treatment .
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BACKGROUND: The aim of this in vitro study was to determine the effect of zoledronate, which is frequently used to treat osteoporosis, on osteoarthritis by analyzing zoledronate-induced expression of vascular endothelial growth factor-A (VEGF-A) in chondrocytes and synovial cells. METHODS: After chondrocytes and synovial cells were separated and cultured, zoledronate was added, and VEGF-A and pigment epithelium-derived factor (PEDF) expression were quantified by real-time polymerase chain reaction and Western blotting. RESULTS: There was no significant difference in the expression of VEGF-A mRNA in chondrocytes between the zoledronate group and the control group on the 8th day of culture. The expression of both VEGF-A and PEDF mRNA in synovial cells was significantly decreased in the zoledronate group (P<0.05). CONCLUSIONS: Zoledronate decreases the expression of VEGF-A in synovial cells and may affect the development and progression of osteoarthritis.
Subject(s)
Blotting, Western , Chondrocytes , Osteoarthritis , Osteoporosis , Real-Time Polymerase Chain Reaction , RNA, Messenger , Vascular Endothelial Growth Factor AABSTRACT
Objective:To study the effects of Anti-DR5 mAb on inducing synovial cells of adjuvant arthritis (AA) rats and its mechanisms.Methods:AA was induced by CFA in rats.The synovial cells of rats were separated and cultured in vitro system.The apoptosis induced by anti-DR5 mAb on synovial cells was measured by MTT analysis,DNA fragmentation and flow cytometry.Caspase-3 and Bcl-2 expression in synovial cells were detected by Western blot.The involvement of the apoptotic pathway was further proved by a caspase inhibition assay.Results:MTT result showed that Anti-DR5 mAb could inhibit synovial cells growth.And flow cytometry suggested that the cell death mode was apoptosis.The protein level of caspase-3 in synovial cells treated with anti-DR5 mAb was raised,while Bcl-2 level declined.When the caspase inhibitor was added to the synovial cells treated with anti-DR5 mAb,it was showed in a dose-dependence inhibition effect,indicating that anti-DR5 mAb inducing apoptosis might be through the caspase pathway.Conclusion:Anti-DR5 mAb could induce synovial cell apoptosis through caspase pathway.And this effect may be related to the protein level of Caspase-3 and Bcl-2.
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Objective To observe the effect of sinomenine(SINO) on immune function of adjuvant arthritis(AA) rats.Methods SD rats were randomized into normal group,model group,SINO groups(treated with gastric gavage of SINO at the doses of 60,120 and 240 mg/kg respectively),and glucosides of Tripterygium Wilfordii(30 mg/kg) group.Except the normal group,the rats in other groups received subcutaneous injection of the complete Freund's adjuvant 0.1mL into the left hind foot to induce AA.The medication began from 12 days after the modeling and lasted 12 days.The pedal swelling and joint function scores were observed in different time.Radioimmunoassay was used to detect the contents of interleukin-1?(IL-1?) and tumor necrosis factor ?(TNF-?) in synovial cells.Expression of IL-1? and TNF-? mRNA was examined by reverse transcription-polymerase chain reaction(RT-PCR) assay.Results SINO at different concentrations decreased the pedal swelling and arthritis scores to various degrees,inhibited the production of IL-1? and TNF-? in the synovial cells,reduced the expression of IL-1? and TNF-? mRNA,and recovered the normal histological features of synovial cells in AA rats.Conclusion The therapeutic mechanism of SINO for rheumatoid arthritis may be related with the inhibition of secretion of inflammatory mediators in synovial cells,and with the recovery of histological features of synovial cells.
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Objective To determine the expression of advanced glycation end products(AGE) binding proteins on human joint synovial cells for elucidating the pathobiological effects of ?2m modified with AGE(AGE-?2m) on joint resident cells. Methods Type A and type B synovial cells were isolated and cultured in vitro. The expression of AGE receptor 1 (ACE-R1 ), AGE receptor 2 (AGE-R2), AGE receptor 3 (AGE-R3) and 35 KD receptor for AGE(RAGE) on synoviocytes were detected by immunofluorescent staining using specific antibodies and flow cytometric analyses. mRNA of AGE receptors was examined by RT-PCR techniques.-Results RAGE and AGE-R3, but not AGE-R1 and AGE-R2, were constitutively expressed on the membrane surface of both type A and type B synovial cells. These two types of synovial cell also expressed mRNA of RAGE and AGE-R3. Conclusion Human joint synovial cells express specific AGE binding proteins, RAGE and AGE-R3, suggesting that these cells may be involved in AGE metabolism and might be the target of the biological effects of AGEs in dialysis-related amyloidosis.
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Homeostasis of multicellular organism is controlled by proliferation and differentiation of cells as well as by cell death. The defects in programmed cell death contribute to the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematous. RA is considered to be a proliferating disorder of synovial tissue which is accompanied by inflammatory cell infiltration and bone erosion. The aim of the study was to find whether potent inducers of apoptosis could be induced apoptosis in RA synovial cells. We examined the effects of drugs, such as dexamethasone, methotrexate, hydrogen peroxide, and ceramide on induction of apoptosis in cultured RA synovial cells. Used drugs did not induced apoptosis in RA synovial cells. Finally Fas antigen-mediated apoptosis of RA synovial cells was investigated by the addition of anti-Fas antibody. To examine the ICE (interleukin-1p-convertase; caspase-1) expression in synovial cells, RT-PCR of caspase-1 gene was performed. In synovial cells of RA, Fas induces that caspase-1 activation cause apoptosis.
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Humans , Apoptosis , Arthritis, Rheumatoid , Cell Death , Dexamethasone , Homeostasis , Hydrogen Peroxide , Ice , MethotrexateABSTRACT
To elucidate the effects of pro inflammatory mediators on mRNA expression of AGE receptors in type B synovial cells, type B synovial cells from normal subjects were isolated and cultured in vitro with human serum albumin modified with advanced glycation end products (AGE HSA), tumor necrosis factor ?(TNF ?)and interleukin 1?(IL 1?). The expression of RAGE mRNA and AGE R3 mRNA was examined by RT PCR techniques. TNF ?, AGE HSA and IL 1? up regulated the expression of AGE R3 mRNA in type B synovial cells in a dose and time dependent manner. In contrast, TNF ? and AGE HSA down regulated the expression of RAGE mRNA in a dose and time dependent manner. IL 1? had no effect on RAGE mRNA expression. The regulatory responses induced by AGE HSA were blocked by a neutralizing polyclonal anti human TNF ? antibody, suggesting that the effects of AGE HSA were mediated by TNF ?. The proinflammatory mediators may regulate the gene expression of AGE receptors in type B synovial cells, and the regulatory role of these receptors is different in response to the proinflammatory mediators.