Attenuation of the Activation of NLRP3 Inflammasome in Fibroblast Like Synoviocytes of Rheumatoid Arthritis by Baicalin through Regulating the Let-7i-3p/PI3K/Akt/NF-κB Signaling Axis

[Objectives]To study the effect and mechanism of baicalin on the activation of NLRP3 inflammasome in human fibroblast like synoviocytes of rheumatoid arthritis(HFLS-RA).[Methods]To confirm that baicalin alleviated the activation of NLRP3 inflammasome in HFLS-RA,the expression of NLRP3 before and after baicalin treatment was observed by immunofluorescence.Western blot was used to detect the protein expression of p-PI3K,p-Akt,NF-κB p65,NLRP3,ASC and caspase-1 after baicalin treatment for 48 h,and the contents of IL-1 and IL-18 in the supernatents were detected by ELISA.In order to explore the mechanism of baicalin alleviating the activation of NLRP3 inflammasome,the corresponding relationship between let-7i-3p and PIK3CA was verified by double luciferin and Westen blot analysis.The expression of let-7i-3p and PI3K before and after baicalin intervention was detected by RT-qPCR.let-7i-3p interference was used to verify whether baicalin mitigated the activation of enhanced NLRP3 inflammasome.[Results]Baicalin(50 and 100 mg/L)significantly reduced the activation of NLRP3 inflammasome,inhibited the protein expressions of p-PI3K,p-Akt,NF-κB p65,NLRP3,ASC and caspase-1,and the secretion of IL-1 and IL-18.let-7i-3p and PIK3CA had a targeted correspondence,and baicalin up-regulated the expression of let-7i-3p and down-regulated the expression of PIK3CA.Baicalin attenuated the activation of NLRP3 inflammasome enhanced by let-7i-3p interference.[Conclusions]Baicalin can up-regulate let-7i-3p expression,inhibit PI3K/Akt/NF-κB signal transduction,and thus reduce the activation of NLRP3 inflammasome in HFLS-RA.

Triptolide Inhibits Expression of Inflammatory Cytokines and Proliferation of Fibroblast-like Synoviocytes Induced by IL-6/sIL-6R-Mediated JAK2/STAT3 Signaling Pathway

Triptolide,a component of the Chinese herb Tripterygium wilfordii Hook F,has been proved to be effective in the treatment of rheumatoid arthritis(RA).However,its underlying mechanisms on RA have not yet been well established.We observed the inhibitory effect of triptolide on the expression of inflammatory cytokines and proliferation of fibroblast-like synoviocytes(FLS)induced by the complex of interleukin-6(IL-6)and the soluble form of the IL-6 receptor(sIL-6R).Furthermore,to clarify the underlying mechanisms,we treated FLS with the Janus-activated kinase 2(JAK2)inhibitor/signal transducer and activator of transcription 3(STAT3)activation blocker AZD1480.In this study,immunohistochemical staining was used to identify vimentin(+)and CD68(−)in FLS.The FLS proliferation was measured by cell proliferation assay,and the cell cycles were analyzed by flow cytometry.Furthermore,ELISA was used to detect the expression of the inflammatory factors in culture solution.The expression levels of p-JAK2,JAK2,p-STAT3 and STAT3 were investigated through Western blotting analysis.The results showed that IL-6/sIL-6R significantly increased the cell proliferation and expression of inflammatory cytokines,including IL-6,interleukin-1β(IL-1β)and vascular endothelial growth factor(VEGF).Triptolide or AZD1480 inhibited the cell proliferation and inflammatory cytokine expression in IL-6/sIL-6R-stimulated FLS by suppressing JAK2/STAT3.The study suggested that the physiological effects of triptolide on RA were due to its contribution to the inhibition of the inflammatory cytokine expression and FLS proliferation by suppressing the JAK2/STAT3 signaling pathway.It may provide an innovative insight into the effect of triptolide in preventing RA pathogenesis.

Therapeutic Actions of the Chinese Herbal Formulae with Cold and Heat Properties and Their Effects on Ultrastructures of Synoviocytes in Rats of the Collagen-Induced Arthritis

The therapeutic actions of Qing Luo Yin (QLY清络饮) with heat property and Wen Luo Yin (WLY温络饮) with cold property on pain, swelling of the ankle, arthritis index and ultrastructures of synoviocytes were compared in rats of type II collagen-induced arthritis (CIA), with tripterygium glycosidorum (TG) used as control. The results indicated that both QLY and WLY could reduce pain, swelling of the ankle and the arthritis index of CIA, and QLY had better effects in reducing the swelling of the ankle and controlling the secondary pathological lesions as compared with WLY. Investigation on the ultrastructures of synoviocytes indicated that both QLY and WLY could reduce the number of Golgi apparatus, rough surface endoplasmic reticulum, dense bodies, matrix filaments and vacuoles so as to suppress the excessive secretion of synoviocytes in rats of CIA.

Effect of Autologus Platelet-Rich Plasma on IL-6, MMP-3 and MCP-1 Expression in Synoviocytes from Osteoarthritic Patients Knees

Nowadays, some studies reported promising results of platelet-rich plasma (PRP) for the treatment of osteoarthritis (OA). However, the effects of PRP on prevention of osteoarthritis in knee joints have been debated. The present study investigated the effects of PRP on osteoarthritis related inflammatory cytokines expressed in fibroblast-like synoviocytes (FLS) from osteoarthritic knees. The synovial tissues were harvested from eight osteoarthritic patients who had undertaken total knee arthroplasties (TKAs) and cultured in DMEM containing 10% FBS. Platelet-rich plasma releasate (PRPr) was made by clotting or activation of PRP by citrate. The levels of PDGF-AA and VEGF in PRPr and whole blood were measured with ELISA method. The FLS were isolated and cultured from osteoarthritic knees. The IL-1β stimulated FLS were cultivated with three different conditions (none, 1% and 10% of PRP). To determine the expression of IL-6, MMP-3, and MCP-1, we used reverse transcriptase polymerase chain reaction (RT-PCR). The concentrations of platelet count in PRP were about 7 to 9 times higher than those of whole blood. The levels of PDGF-AA in PRPr were approximately 3 to 4 times higher than those of whole blood. The levels of VEGF in PRPr were also significantly 7 to 18 times higher than those of whole blood. Without induction of the FLS with IL-1β, 1% or 10% PRPr did not reduce expressions of inflammatory proteins (MMP-3, MCP-1), except for IL-6. However, with induction of the FLS with IL-1β, both concentrations (1% and 10%) of PRPr reduced significantly all inflammatory protein expressions (IL-6, MMP-3, MCP-1). PRPr diminished inflammatory IL-1β-mediated effects on human osteoarthritic fibroblast-like synoviocytes. These results suggest that platelet-rich plasma can be a good therapeutic option for the treatment of osteoarthritis.

Paeoniflorin-6′-O-benzene sulfonate ameliorates progression of adjuvant-induced arthritis by inhibiting interaction between Ahr and GRK2 of fibroblast-like synoviocytes

OBJECTIVE Aryl hydrocarbon receptor(Ahr)is thought to be a crucial factor that regulates immune responses,which may be involved in the pathogenesis of autoimmune inflammation including rheumatoid arthritis(RA).The results of our group in recent years have shown that CP-25,a novel ester derivative of paeoniflorin,has a good effect on improving RA animal models.However,whether the anti-arthritis effect of CP-25 is related to Ahr remains unclear.METHODS CP-25 treatment ameliorated adjuvant-induced arthritis(AA),a mouse model of RA,by inhibiting Ahr-related activities in fibroblasts like synoviocytes(FLS).AA rats were treated with CP-25 or paroxetine from day 17 to 33 after immunization.RESULTS CP-25 alleviated arthritis symptoms and the pathological changes,decreased the expression of Ahr in the synovium and FLS of AA rats.Besides,treatment with CP-25 reduced the proliferation and migration of MH7A caused by Ahr activation.In addition,we also demonstrated that CP-25 down-regulated the co-expression and co-localization of Ahr and G protein-coupled receptor kinase 2(GRK2)in MH7A.CONCLUSION The data presented here demonstrated that CP-25 suppressed FLS dysfunction in rats with AA,which were associated with reduced Ahr activation and the interaction between Ahr and GRK2.

Protective effects of Dioscin on TNF-α-induced collagen-induced arthritis rat fibroblast-like synoviocytes involves in regulating the LTB4/BLT pathway

Background and Objective:LTB4 has been shown to be involved in rheumatoid arthritis(RA)pathogenesis.The effect of Dioscin(Dio)on the LTB4 pathway of RA have not been reported yet.This study aimed at further exploring whether Dioscin’s effects on TNF-αinduced collagen-induced arthritis(CIA)rat fibroblast-like synoviocytes(FLS)connected with the LTB4 and its receptor pathway.Materials&Methods:In this experiment,control group,TNF-αgroup,and different concentrations of Dioscin groups were established.Cell viability was evaluated using MTT assay.The levels of LTB4 in the samples of above groups were measured using ELISA.The mRNA expression levels of LTA4H,BLT1,and BLT2 were detected by quantitative real time PCR,while the expression level of LTA4H proteins were detected using western blot.The distribution of LTA4H was assessed by immunofluorescence assay.Results:the LTB4 level of TNF-αgroup in sample supernatant was higher than both control group and Dioscin groups with decreased LTB4 levels(p<0.05).Compared with the control group,the expression of LTA4H was significantly increased in TNF-αgroup(p<0.05),whereas LTA4H expressions were significantly decreased in all Dioscin groups when compared to TNF-αgroup(p<0.05).The mRNA expressions of BLT1 and BLT2 were markedly higher in TNF-αgroup than those in control group while Dioscin treatment significantly inhibited the increased expressions of BLT1 and BLT2 induced by TNF-α(p<0.05).Conclusions:These results firstly demonstrate that the protective effect of Dioscin on TNF-αinduced FLS may involve in its reducing LTB4 production by down-regulating LTA4H expression,and may inhibit its downstream pathway by decreasing LTB4 receptors levels.This findings suggest that dioscin produces a potential therapeutic effects for RA via its influencing LTA4H/LTB4/BLT pathway.

Nuciferine alleviates collagen-induced arthritic in rats by inhibiting the proliferation and invasion of human arthritis-derived fibroblast-like synoviocytes and rectifying Th17/Treg imbalance

Rheumatoid arthritis(RA)is a chronic autoimmune disorder marked by persistent synovial inflammation and joint degradation,posing challenges in the development of effective treatments.Nuciferine,an alkaloid found in lotus leaf,has shown promising anti-inflammatory and anti-tumor effects,yet its efficacy in RA treatment remains unexplored.This study investigated the antiproliferative effects of nuciferine on the MH7A cell line,a human RA-derived fibroblast-like synoviocyte,revealing its ability to inhibit cell proliferation,promote apoptosis,induce apoptosis,and cause G1/S phase arrest.Additionally,nuciferine significantly reduced the migration and invasion capabilities of MH7A cells.The therapeutic potential of nuciferine was further evaluated in a collagen-induced arthritis(CIA)rat model,where it markedly alleviated joint swelling,synovial hyperplasia,cartilage injury,and inflammatory infiltration.Nuciferine also improved collagen-induced bone erosion,decreased pro-inflammatory cytokines and serum immunoglobulins(IgG,IgG1,IgG2a),and restored the balance between T helper(Th)17 and regulatory T cells in the spleen of CIA rats.These results indicate that nuciferine may offer therapeutic advantages for RA by decreasing the proliferation and invasiveness of FLS cells and correcting the Th17/Treg cell imbalance in CIA rats.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes(FLSs)are the key drivers in the pathogenesis of rheumatoid arthritis(RA)and joint destruction.Abundant Yes-associated protein(YAP),which is a powerful transcription co-activator for proliferative genes,was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms.Using Gene Expression Omnibus database analysis,it was found that Salvador homolog-1(SAV1),the pivotal negative regulator of the Hippo-YAP pathway,was slightly downregulated in RA synovium.However,SAV1 protein expression is extremely reduced.Subsequently,it was revealed that SAV1 is phosphorylated,ubiquitinated,and degraded by interacting with an important serine-threonine kinase,G protein-coupled receptor(GPCR)kinase 2(GRK2),which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2(PGE2)in RA.This process further contributes to the decreased phosphorylation,nuclear translocation,and transcriptional potency of YAP,and leads to aberrant FLSs proliferation.Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration.Similarly,paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations.Collectively,these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and Rho A GTPases and activation of the PI3K/Akt and JNK signaling pathways

Tetrandrine （Tet）, the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synovioeytes （RA-FLS） and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet （0.3, 1 μmol L-1） was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC （against PI3K, Akt, JNK, ERK, p38 MAPK and NF-kB-p65, respectively） were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac 1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Racl, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.

Therapeutic effect of neohesperidin on TNF-α-stimulated human rheumatoid arthritis fibroblast-like synoviocytes

During the pathogensis of rheumatoid arthritis(RA),activated RA fibroblast-like synoviocytes(RA-FLSs)combines similar proliferative features as tumor and inflammatory features as osteoarthritis,which eventually leads to joint erosion.Therefore,it is imperative to research and develop new compounds,which can effectively inhibit abnormal activation of RA-FLSs and retard RA progression.Neohesperidin(Neo)is a major active component of flavonoid compounds with anti-inflammation and anti-oxidant properties.In this study,the anti-inflammation,anti-migration,anti-invasion,anti-oxidant and apoptosis-induced effects of Neo on RAFLSs were explored to investigate the underlying mechanism.The results suggested that Neo decreased the levels of interleukin IL-1β,IL-6,IL-8,TNF-α,MMP-3,MMP-9 and MMP-13 in FLSs.Moreover,Neo blocked the activation of the MAPK signaling pathway.Furthermore,treatment with Neo induced the apoptosis of FLSs,and inhibited the migration of FLSs.It was also found that Neo reduced the accumulation of reactive oxygen species(ROS)induced by TNF-α.Taken together,our results highlighted that Neo may act as a potential and promising therapeutic drug for the management of RA.

Inhibition of smoothened decreases proliferation of Synoviocytes in rheumatoid arthritis

Fibroblast-like synoviocytes （FLSs） contribute to synovial hyperplasia in rheumatoid arthritis （RA）. Smoothened （Smo） is a key component of sonic hedgehog （Shh） signaling and contributes to tumor cell proliferation. The objective of this study was to investigate the role of Smo in RA synoviocyte proliferation. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist （GDC-0449） and small interfering RNA （siRNA） targeting the Smo gene in FLSs. Cell proliferation was quantified by using kit-8 assay and cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell cycle-related genes and proteins were detected by real-time PCR and western blot. FLSs treated with GDC-0449 or Smo-siRNA showed significantly decreased proliferation compared to controls （P〈0.05）. Incubation with GDC-0449 or transfection with Smo-siRNA resulted in a significant increase of G1 phase cells compared to controls （P 〈 0.05）. Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells （P 〈 0.05）. Protein expression of cyclin D1 was also downregulated after Smo gene knockdown （P 〈0.05）.The results suggest that Shh signaling plays an important role in RA-FLSs proliferation in a Smo-dependent manner and may contribute to synovial hyperplasia. Targeting Shh signaling may help control joint damage in patients with RA.

Madecassoside impedes invasion of rheumatoid fibroblast-like synoviocyte from adjuvant arthritis rats via inhibition of NF-κB-mediated matrix metalloproteinase-13 expression

Fibroblast-like synoviocytes(FLS) play a pivotal role in Rheumatoid arthritis(RA) pathogenesis through aggressive migration and invasion. Madecassoside(Madec), a triterpenoid saponin present in Centella asiatica herbs, has a potent anti-inflammatory effect. In the present study, Madec exerted an obvious therapeutic effect in reversing the histological lesions in adjuvant-induced arthritis(AIA) rats. To recognize the anti-rheumatoid potentials of Madec, we further investigated whether Madec interfered with FLS invasion and metalloproteinase(MMP) expression. In cultures of primary FLS isolated from the AIA rats, Madec(10 and 30 μmol·L–1) was proven to considerably inhibit migration and invasion of FLS induced by interleukin 1β(IL-1β), but exhibiting no obvious effect on cell proliferation. Madec repressed IL-1β-triggered FLS invasion by prohibiting the expression of MMP-13. Additionally, Madec suppressed MMP-13 transcription via inhibiting the MMP-13 promoter-binding activity of NF-κB. Our results further showed that Madec down-regulated the translocation and phosphorylation of NF-κB as demonstrated by Western blotting and immunofluorescence assays. In conclusion, our results suggest that Madec exerts anti-RA activity via inhibiting the NF-κB/MMP-13 pathway.

Targeted inhibition of GRK2 kinase domain by CP-25 to reverse fibroblast-like synoviocytes dysfunction and improve collagen-induced arthritis in rats

Rheumatoid arthritis(RA)is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes(FLS).The up-regulated cellular membrane expression of G protein coupled receptor kinase 2(GRK2)of FLS plays a critical role in RA progression,the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor(EP4)signaling and FLS abnormal proliferation.Recently,although our group found that paeoniflorin-6’-O-benzene sulfonate(CP-25),a novel compound,could reverse FLS dysfunction via GRK2,little is known as to how GRK2 translocation activity is suppressed.Our findings revealed that GRK2 expression up-regulated and EP4 expression down-regulated in synovial tissues of RA patients and collagen-induced arthritis(CIA)rats,and prostaglandin E2(PGE2)level increased in arthritis.CP-25 could down-regulate GRK2 expression,up-regulate EP4 expression,and improve synovitis of CIA rats.CP-25 and GRK2 inhibitors(paroxetine or GSK180736 A)inhibited the abnormal proliferation of FLS in RA patients and CIA rats by down-regulating GRK2 translocation to EP4 receptor.The results of microscale thermophoresis(MST),cellular thermal shift assay,and inhibition of kinase activity assay indicated that CP-25 could directly target GRK2,increase the protein stability of GRK2 in cells,and inhibit GRK2 kinase activity.The docking of CP-25 and GRK2 suggested that the kinase domain of GRK2 might be an important active pocket for CP-25.G201,K220,K230,A321,and D335 in kinase domain of GRK2 might form hydrogen bonds with CP-25.Site-directed mutagenesis and co-immunoprecipitation assay further revealed that CP-25 down-regulated the interaction of GRK2 and EP4 via controlling the key amino acid residue of Ala321 of GRK2.Our data demonstrate that FLS proliferation is regulated by GRK2 translocation to EP4.Targeted inhibition of GRK2 kinase domain by CP-25 improves FLS function and represents an innovative drug for the treatment of RA by targeting GRK2.

uPAR promotes tumor-like biologic behaviors of fibroblast-like synoviocytes through PI3K/Akt signaling pathway in patients with rheumatoid arthritis

Urokinase-type plasminogen activator receptor(uPAR),is a multifunctional receptor on cell surface,widely present in endothelial cells,fibroblasts,and a variety of malignant cells.Current studies have suggested that uPAR overexpressed on synovial tissues or in synovial fluid or plasma in patients with rheumatoid arthritis(RA).However,there are limited researches regarding the role of uPAR on fibroblast-like synoviocytes of rheumatoid arthritis(RA-FLSs)and its underlying mechanisms.Here,our studies show that the expression of uPAR protein was significantly higher in fibroblast-like synoviocytes(FLSs)from RA than those from osteoarthritis or traumatic injury patients.uPAR gene silencing significantly inhibited RA-FLSs cell proliferation,restrained cell transformation from the G0/G1 phase to S phase,aggravated cell apoptosis,interfered with RA-FLSs cell migration and invasion,and reduced activation of the PI3K/Akt signaling pathway,which may be associated withβ1-integrin.Cell supernatants from uPAR gene-silenced RA-FLSs markedly inhibited the migration and tubule formation ability of the HUVECs(a human endothelial cell line).Therefore,we demonstrate that uPAR changes the biological characteristics of RA-FLSs,and affects neoangiogenesis of synovial tissues in patients with RA.All of these may be associated with theβ1-integrin/PI3K/Akt signaling pathway.These results imply that targeting uPAR and its downstream signal pathway may provide therapeutic effects in RA.

Efficacy of Zhonglun'a-decoction-containing serum on fibroblastlike synoviocyte apoptosis in rats with collagen-induced arthritis via inhibiting Janus kinase/signal transducer and activator of transcription signaling pathway

OBJECTIVE: To investigate the efficacy of Zhonglun'a-decoction-containing serum(ZHONGL-CS)on the in vitro apoptosis of fibroblast-like synoviocytes(FLS) from rats with collagen-induced arthritis(CIA) by investigating the Janus kinase/signal transducer and activator of transcription(JAK/STAT) signaling pathway.METHODS: A CIA rat model was established using bovine type Ⅱ collagen. FLS were isolated, cultured and identified. A cell counting kit-8 was used to detect cell activity. The half maximal inhibitory concentration(IC50) was calculated. Experimental subjects were divided into control, CIA,ZHONGL-CS, JAK2 inhibitor AG490, and ZHONGLCS with AG490 groups. The in vitro cell cycle and apoptosis rate were detected in FLS by flow cytometry. Bcl-2-associated X protein(Bax), B-cell lymphoma 2(Bcl-2), caspase-3, cyclin D1, phosphorylated JAK2, STAT1, and STAT3 protein expressions in FLS were examined by Western blotting. JAK2, STAT1 and STAT3 mRNA levels were examined by quantitative real-time polymerase chain reaction.RESULTS: Compared with the CIA group, FLS proliferation was inhibited, the FLS G0/G1 cell cycle was arrested, and the rate of FLS apoptosis was increased in the ZHONGL-CS group. In the ZHONGLCS group, the protein levels of Bcl-2 and cyclin D1 were reduced compared with the CIA group and the levels of Bax and caspase-3 in FLS were increased. In the ZHONGL-CS group, the expressions of JAK2, STAT1, and STAT3 mRNA and the levels of phosphorylated JAK2, STAT1, and STAT3 proteins were reduced.CONCLUSION: ZHONGL-CS may induce FLS apoptosis in CIA rats. Activation of the JAK/STAT signaling pathway was inhibited in FLS in vitro.

Activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting siRNA

Background:Fibroblast-like synoviocytes(FLSs),resident mesenchymal cells of synovial joints,play an important role in the pathogenesis of rheumatoid arthritis(RA).Dickkopf-1(DKK-1)has been proposed to be a master regulator of bone remodeling in inflammatory arthritis.Here,potential impairation on the activity of FLSs derived from RA to small interfering RNAs(siRNAs)targeting DKK-1 was investigated.Methods:siRNAs targeting DKK-1 were transfected into FLSs of patients with RA.Interleukin(IL)-1β,IL-6,IL-8,matrix metalloproteinase(MMP)2,MMP3,MMP9,transforming growth factor(TGF)-pi,TGF-β2 and monocyte chemoattractant protein(MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Invasion assay and 3H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation,respectively.Western blotting was performed to analyze the expression of nuclear factor(NF)-kB,interleukin-1 receptor-associared kinase(IRAK)1,extracellular regulated protein kinases(ERK)1,Jun N-terminal kinase(JNK)and p-catenin in FLSs.Results:DKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs(P<0.01).siRNAs induced a significant reduction of the levels of IL-6,IL-8,MMP2,MMP3 and MMP9 in FLSs compared to the control group(P<0.05).DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs(P<0.05).Important molecules of pro-inflammatory signaling in FLSs,including IRAKI and ERK1,were decreased by the inhibition of DKK-1 in FLSs.In contrast,β-catenin,a pivotal downstream molecule of the Wnt signaling pathway was increased.Conclusions:By inhibiting DKK-1,we were able to inhibit the proliferation,invasion and pro-inflammatory cytokine secretion of FLSs derived from RA,which was mediated by the ERK or the IRAK-1 signaling pathway.These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

Activation of human fibroblast-like synoviocytes by uric acid crystals in rheumatoid arthritis

Hyperuricemia-mediated uric acid crystal formation may cause joint inflammation and provoke the destruction of joints through the activation of inflammasome-mediated innate immune responses.However,the immunopathological effects and underlying intracellular regulatory mechanisms of uric acid crystal-mediated activation of fibroblast-like synoviocytes(FLS)in rheumatoid arthritis(RA)have not been elucidated.Therefore,we investigated the in vitro effects of monosodium urate crystals,alone or in combination with the inflammatory cytokines tumor-necrosis factor(TNF)-a or interleukin(IL)-1b,on the activation of human FLS from RA patients and normal control subjects and the underlying intracellular signaling mechanisms of treatment with these crystals.Monosodium urate crystals were able to significantly increase the release of the inflammatory cytokine IL-6,the chemokine CXCL8 and the matrix metalloproteinase(MMP)-1 from both normal and RA-FLS(all P,0.05).Moreover,the additive or synergistic effect on the release of IL-6,CXCL8 and MMP-1 from both normal and RA-FLS was observed following the combined treatment with monosodium urate crystals and TNF-a or IL-1b.Further experiments showed that the release of the measured inflammatory cytokine,chemokine and MMP-1 stimulated by monosodium urate crystals were differentially regulated by the intracellular activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways but not the p38 mitogen-activated protein kinase pathway.Our results therefore provide a new insight into the uric acid crystal-activated immunopathological mechanisms mediated by distinct intracellular signal transduction pathways leading to joint inflammation in RA.

Expression of C5aR ( CD88) of synoviocytes isolated from patients with rheumatoid arthritis and osteoarthritis

Objective To investigate the expression of anaphylatoxin receptor C5aR (CD88) in synoviocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).Methods The expression of C5aR was assessed in synoviocytes isolated from 27 RA and 12 OA patients using reverse transcription-polymerase chain reactions (RT-PCR), flow cytometry, and immunofluorescence analysis. The effects of C5a on the release of tumor necrosis factor a (TNFα) from synoviocytes were assayed using enzyme-linked immunosorbent assays (ELISA).Results C5aR mRNA was detected in 24 of 27 samples from RA patients, and 10 of 12 samples from OA patients. Flow cytometric analysis and immunofluorescence study demonstrated the cell surface expression of C5aR in a significant proportion of synoviocytes from both RA and OA patients, and the level of C5aR expression in synoviocytes was significantly correlated with joint swelling, erythrocyte sedimentation rate ( ESR) and C-reactive protein ( CRP) in RA patients. Finally, interaction of C5aR with its ligand C5a was shown to enhance lipopolysaccharide (LPS) -induced TNFa release from synoviocytes.Conclusions The expression of C5aR in synoviocytes from inflammatory joint diseases and also the induction of TNFa release in activated synovicytes by the interaction of C5a and C5aR suggest that the C5a/C5aR system may play an important role in joint inflammation process.

改良组织块法分离培养佐剂性关节炎兔成纤维样滑膜细胞

目的 通过改良组织块细胞培养法,建立一种简单、可行的佐剂性关节炎兔成纤维样滑膜细胞（FLS）体外培养方法并观察细胞生物学特性.方法 通过注射弗氏完全佐剂建立关节炎模型,无菌条件下取佐剂性关节炎兔膝关节滑膜组织,剔净后剪成碎块,用灭菌滤纸吸掉组织上附着的水分,碎片接种于培养皿,观察细胞生长状况及形态特征.取第3代对数期细胞,用噻唑蓝（MTT）法绘制其生长曲线,并用免疫荧光法检测波形蛋白的表达情况.结果 体外培养的佐剂性关节炎兔FLS形态为长梭形纤维样,符合FLS的生长特征,免疫荧光检测显强表达波形蛋白.结论 改良组织块法可以分离培养出兔佐剂性关节炎成纤样维滑膜细胞,方法简便,成功率高,满足实验要求.

Paeoniflorin-6＇-O-benzene sulfonate, a novel compound, protects against autoimmune arthritis by modulating inflammation and bone damage

Aim Paeoniflorin （Pae） is the principal bioactive component of total glucosides of peony （TGP）, which has been widely used in therapy for rheumatoid arthritis （RA）. Paeoniflorin-6＇-O-benzene sulfonate （code： CP-25） , a novel compound that is a newly ester derivatives of Pae, was evaluated in rats with adjuvant-induced ar- thritis （AA） to study its potential anti-arthritic activity. Methods AA rats were randomly divided into different groups and then treated with CP-25 （25, 50, 100 mg· kg^-1） and methotrexate （0. 5 mg · kg^-1）, from day 16 to day 32 after immunization. Arthritis severity was evaluated by clinical manifestation and histopathological examina- tion. The cells proliferation was determined by CCK-8 assay. Activities of IL-1β, IL-6, IL-17, IL-10, TGF-β1, TNF-oL, IIANKL and OPG were assessed by ELISA. The subsets of CD4 ＋T cells were assayed by flow cytometry. Results CP-25 treatment effectively reduced clinical severity scores and blinded histopathological scores compared with AA groups. CP-25-treated rats exhibited a decrease in the pro-inflammatory cytokines （IL-1β, IL-6, IL-17, and TNF-α） , coupled with an increase in the anti-inflammatory cytokines IL-10 and TGF-β1 in serum and macro- phages of AA rats. The flow cytometry analyses of CD4 ＋T cells dramatically demonstrated the immunomodulatory effects of CP-25 on abnormal immune dysfunction. Apart from the anti-inflammatory activity, treatment with CP-25 inhibited the fibroblast-like synoviocyte （FLS） activation and function. Furthermore, CP-25 treatment of AA rats restored the balance between RANKL and OPG in favor of its anti-osteoclastic effects. Conclusions Data presen- ted here demonstrated that administration of CP-25 significantly inhibited the progression of rat AA, with reductions both in arthritic inflammation and bone damage. The protective effects of CP-25 in AA highlight an attribute that is potential as an ideal new anti-arthritic agent for the treatment with human RA.