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This study started from the effect of baicalin (BC), the main active component of the labiaceae plant Scutellaria baicalensis, on collagen-induced arthritis (CIA) in rats, to explore the mechanism of glucose metabolism reprogramming in fibroblast like synoviocytes (FLSs), a key effector cell of synovial inflammation in rheumatoid arthritis (RA). First of all, CIA rats and tumor necrosis factor-α (TNF-α)-induced RASFs in vitro and in vivo models were established, the arthritis index (AI) score and histopathological changes of CIA rats after BC administration were observed, and the levels of inflammatory factors in serum and cell supernatant were quantified by ELISA, immunocytochemistry and Western blot were used to detect the expression of G-protein-coupled receptor 81 (GPR81) and pyruvate dehydrogenase kinase 1 (PDK1) proteins. In addition, the kit was used to measure the levels of key products and enzyme activities in glucose metabolism reprogramming. The results showed that BC (50, 100 and 200 mg·kg-1) could alleviate the symptoms of arthritis in CIA rats in a dose-dependent manner, inhibit synovial hyperplasia, alleviate the infiltration of inflammatory cells, down-regulate the levels of pro-inflammatory factors TNF-α and interleukin (IL)-1β, and up-regulate the levels of anti-inflammatory factor IL-10 in CIA rats. At the same time, the secretion levels of lactate, pyruvate, acetyl-CoA, citrate and the activity of lactate dehydrogenase B (LDH-B) were decreased, and the expressions of GRP81 and PDK1 were down-regulated, suggesting that BC mediated the reprogramming process of glucose metabolism. However, when GPR81 inhibitor 3-OBA inhibited lactate uptake, the activity of LDH-B was significantly increased, suggesting that BC inhibited the expression of PDK1, a key enzyme in the reprogramming metabolism from glycolysis to oxidative phosphorylation. All animal experiments in this study were conducted in accordance with the ethical standards of the Laboratory Animal Care Center of Anhui University of Chinese Medicine (approval number: AHUCM-rats-2021049). These studies revealed that baicalin mediated metabolic reprogramming of RASFs from glycolysis to oxidative phosphorylation by inhibiting PDK1 protein expression, and alleviated joint inflammation in CIA rats.
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Objective:To observe the effect of Fengshi Qutong capsule (FSQTC) on protein kinase B(Akt) and mitogen-activated protein kinase (MAPK) signaling pathways of rheumatoid arthritis (RA). Method:Collagen-induced arthritis (CIA) was induced in SD rats, and the synovial membranes of the knee joints were prepared after 19 days of oral administration of 0.25, 0.5, 1 g·kg-1 FSQTC. MH7A cells were induced by tumor necrosis factor-α (TNF-α, 20 μg·L-1) in vitro, and human umbilical vein endothelial cells (HUVEC) were induced by vascular endothelial growth factor (VEGF). FSQTC (0.02, 0.1, 0.5 μg·L-1) were added to MH7A/HUVEC cells, and then the cells were collected. Proteins of synovial tissue, MH7A and HUVEC cells were extracted, and then were detected the expresstion of p-Akt, p-p38 MAPK, p-extracellular signal-regulated kinase(ERK) and p-Jun n-terminal kinase(JNK) by Western blot. Result:The expression levels of p-Akt, p-p38 MAPK, p-ERK and p-JNK in the synovial membrane of CIA model were significantly increased compared with normal group (P-1·d-1 FSQTC significantly decreased their expression levels (PPα or VEGF were increased (P-1 FSQTC (PPConclusion:FSQTC can down-regulate the abnormal activation of Akt and MAPK signaling pathways in the synovial membrane of CIA rats, fibroblast synovial cells and vascular endothelial cells, which is related to the inhibition of synovial angiogenesis in the treatment of RA.
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Objective: To study the effects of Fengshi Qutong capsule (FSQTC) on proliferation, migration, adhesion, invasion and secretion of human synovial cells in rheumatoid arthritis (RA) induced by tumor necrosis factor-α (TNF-α) and explore its mechanism. Method: Human synovial cells (MH7A) in RA patients were induced in vitro by using TNF-α (20 μg·L-1). After treatment with different concentrations of FSQTC (0.02,0.1,0.5 μg·L-1), MTT colorimetric assay, transwell migration, adhesion and invasion tests were used to detect the proliferation, migration, adhesion and invasion of the MH7A, respectively. The expression levels of vascular endothelial growth factor (VEGF) and interleukin-1β (IL-1β) in MH7A supernatant were detected by enzyme linked immunosorbent assay (ELISA). Result: As compared with blank control group, TNF-α (20 μg·L-1) significantly increased the proliferation, migration, adhesion, invasion and secretion of IL-1β and VEGF of MH7A cells (P-1) had no significant effect on proliferation of TNF-α-induced MH7A cells after treatment for 24 hours. After 48 hours of treatment, proliferation of MH7A cells induced by TNF-α was decreased in a concentration-dependent manner (PPPPConclusion: FSQTC can inhibit the proliferation, migration, adhesion, invasion and secretion of IL-1β and VEGF in MH7A cells.
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Objective:It has been found that serum CXCL16 concentration in rheumatoid arthritis (RA) patients are significantly higher than those in osteoarthritis (OA) and normal subjects, and are positively correlated with disease activity and bone erosion.However, how is CXCL16 involved in the pathogenesis of RA is unclear.To evaluate the expression of CXCL16 and its receptor CXCR6 in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) patients, and to explore the role of CXCL16 in the proliferation of RA-FLS.Methods: FLS were isolated from knee synovial tissues obtained from 8 patients of RA, 7 osteoarthritis (OA) and 3 normal controls.The diagnosis of RA was in line with the 1987 American Rheumatology Association (ACR) RA classification criteria, osteoarthritis met the 1996 ACR revised knee osteoarthritis classification criteria.Control synovium were obtained from trauma caused knee joint injury in healthy individuals who required surgery.Human knee FLS were cultured by tissue explants adherent method.FLS between passages 3 and 5 were used in the experiment.Expression of CXCL16 and its receptor CXCR6 were performed in Western blot analysis.FLS proliferation follo-wing stimulation with TNF-α and different concentrations of CXCL16 was examined by cell counting kit-8 (CCK-8).Expression of phosphorylated AKT (pAKT) in RA-FLS stimulated by CXCL16 was quantified by Western blot.Different concentrations of recombinant human CXCL16 were added to the culture medium of RA-FLS.After 48 h culture, supernantants were collected, and TNF-α, IL-6, RANKL and MMP3 in culture supernatants of RA-FLS were determined by enzyme-linked immunosorbent assays (ELISA) operated following the kit instructions.Results: Expression of CXCL16 and CXCR6 in RA-FLS was significantly higher than that of OA and controls (P<0.05), but no significant difference was found between OA-FLS and control FLS.Proliferation of RA-FLS was markedly up-regulated after stimulation of CXCL16 (P <0.05).In the case of the CXCL16 stimulated OA-FLS and control FLS, the FLS proliferation remained basically unchanged.Expression of phosphorylated AKT in RA-FLS increased remarkably in condition of CXCL16 (50,100, 200 μg/L) stimulation.The levels of IL-6 and RANKL in culture supernatants of RA-FLS were obviously increased under CXCL16 (200 μg/L) stimulation, while TNF-α and MMP-3 levels in the culture supernatants remained unchanged after CXCL16 (200 μg/L) stimulation.Conclusion: This study shows that the expression of CXCL16 and its receptor was highly elevated in RA-FLS.Recombinant CXCL16 promoted RA-FLS proliferation and activation in vitro.All these indicate that CXCL16 play an important role in the pathogenesis of RA, anti-CXCL16 treatment may help to relieve inflammation and bone damage of RA patients.However, due to the limitations of this study, the role of CXCL16 and its receptors in RA-FLS remains to be elucidated by further research.
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This study was aimed to observe the influence of Discornin Tablets on activation nuclear transcription factor NF-κBp65 of rheumatoid arthritis (RA) cell model as well as the expression of MMP-9, VEGF and tumor necrosis factor-α (TNF-α). Interleukin-17 (IL-17) and TNF-α were used for stimulating RSC-364 cells. Discornin Tablets at different concentrations were used for intervention. The influence of Discornin Tablets in different concentrations on cell viability was detected by MTT method. Expressions of NF-κBp65 and its inhibitory protein (IκB-α) in each group were detected by western blot method. Changes in VEGF, MMP-9 and TNF-α protein levels in cell broth supernatant were checked by ELISA. The results showed that Discornin Tablets can promote the expression of κB inhibitory pro-tein, reduce the high expression of NF-κB protein level, and inhibit the cellular secretion of VEGF, MMP-9 and TNF-α. It was concluded that Discornin Tablets had negative regulation effect on nuclear transcription factor κB of RSC-364 cells. It can increase the expression of IκB-α, as well as reduce the secretion of inflammation factors and blood vessel newborn factors. It suggested that Discornin Tablets may have the potential regulation effect on RA.
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This study was aimed to observe the influence of water-solubility nipponica saponin on activation of TNF-α+IL-17-induced rat fibroblast-like synovial cell line RSC-364 cellular model nuclear transcription factor NF-κB pathway as well as TNF-α, IL-1, ICAM-1, MMP-2, MMP-3 secretion. IL-17+ TNF-α were used for stimulating RSC-364 to establish rheumatoid arthritis (RA) cellular model. Water-solubility nipponica saponin in different con-centrations was used for intervention. The influence of water-solubility nipponica saponin in different concentrations on cell viability was detected by semi-quantitative RT-PCR method. Changes in the level of TNF-α, IL-1, ICAM-1, MMP-2, and MMP-3 of culture supernatant were detected by ELISA. The results showed that the activation of NF-κB p65 in RSC-364 stimulated by TNF-α+ IL-17 can be inhibited by water-solubility nipponica saponin ac-cording to its concentration. It improved IκB-α expression, and inhibited TNF-α, IL-1, ICAM-1, MMP-2 and MMP-3 secretion. It was concluded that water-solubility nipponica saponin can inhibit the activation of NF-κB pathway, hinder the secretion and activation of multiple downstream genes, which may be its effect in inhibiting syn-ovial inflammation in RA.
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Objective To replicate experimental animal model of knee osteoarthritis and to investigate the method of culture and biological characteristics of rats synoviocytes of gonarthritis in vitro.MethodsAnimal models of knee osteoarthritis were made by the Modified Hulth method.4 weeks after the replicating experiment,synovial tissues were mechanically isolated and enzyme-digested and the growth of the synovial cells was investigated.Results The synovial tissues were obviously hyperplasia in the model made by the Modified Hulth method.The synovial cells were abundant after enzyme-digested cultivation and the cell activity was higher than 98%.Conclusion The study exhibits that the Modified Hulth method apparently promotes the hyperplasia of synovial tissues.The methods of isolation and cultivation of the synovial cells in vitro is proved to be simple and feasible.
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Objective To study effect of Tongbiling (TBL) on VEGF mRNA expression in RA synovlcytes cultured in vitro and explore its mechanism. Method Synovial cells from the knee joint in patient with RA were digested, divided and separately cultured after arthroscopy. Blank controls:DMEM culture containing 10% FBS in saline;Low-dose admission group of TBL:DMEM culture containing 10% FBS in 50 mg/L TBL;High-dose admission of TBL:DMEM culture containing 10% FBS in 200 mg/L TBL. RNA were distilled. Expression of VEGF mRNA were detected with RT-PCR. Result Compared with control group, expression of VEGF mRNA in low and high-dose admission groups of TBL both decreased (P
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AIM:To observe the effects of recombi- nant human endostatin(rh-end)on the expression of p53,fas and bcl-2 genes in synovial tissue in rats with adjuvant arthritis(AA).METHODS:The effects of rh- end on the expression of p53,fas and bcl-2 mRNA in synovial tissue in AA rats were examined quantitatively by SYBR GreenⅠreal-time fluorescent quantitative PCR (FQ-PCR).RESULTS:The expression offas and bcl-2 mRNA in synovial tissue in AA rats treated with rh-end was significantly increased,as compared with model con- trois(P
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Objective To investigate the proliferative characteristics of fibroblast-like synovial cells (FLS)in osteoarthritis in vitro and the mechanism of the immunosuppressive effect of total glucusides of paeony(TGP).Methods FLS of OA and non-inflamed synovium(NS)were cultured and identified in vitro in the presence or absence of TGP.After incubation,the survival fraction(SF)of FLS was evaluated by MTI' and the TNF-?,IFN-?and bFGF level in cultured FLS supernatant was measured by ELISA.The expression of FLS c-los mRNA and cell cycle of OA-FLS was observed by RT-PCR and flow eytometry respectively at the same time.Results No statistical significant differences were noted between the OA and NS FLS in pro- liferating double time.High doses of TGP suppressed FLS-SF more evidently in OA patients than in NS(P0.05).Conclusion High dose TGP can inhibit OA-FLS proliferation,modulate cy- tokine secretion and c-fos expression in OA.This suggests that TGP has immunosuppressive effect on OA syn- ovitis,probably by preventing the synovial hypertrophy in OA.
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Aim To investigate the effects and mechanisms of CCK-8 on IL-1? induced proliferation of RSC-364, a rat fibroblast-like synovial cell line. Methods MTT colorimetric assay and Western blot were used to measure cell proliferation and p38MAPK phosphorylation level to elucidate the mechanism of CCK-8 in IL-1? induced RSC-364 proliferation. Results CCK-8 significantly inhibited IL-1?-induced RSC-364 proliferation at 10 -12 , 10 -10 , 10 -8 , 10 -6 mol ? L -1 , and IL-1?-activated p38MAPK activity at 10 -10 , 10 -8 , 10 -6 mol?L -1 in a dose-dependent manner. The effect of CCK-8 was blocked by CR1409 (a CCKA-receptor antagonist) and CR2945 (a CCKB-receptor antagonist). Conclusion CCK-8 inhibits IL-1?-induced RSC-364 proliferation, probably by reducing p38MAPK activity through CCKA and CCKB receptors.
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Objective To study the effect of the extract of total flavonoids of chrysanthemum indicum(TFC) on adjuvant arthritis synovial cells. Methods 0.1ml of the complete Freund's adjuvant was subcutaneously injected into the right hind feet pads of the SD rats.24 days after immunity synovial cells in knee joint were treated with TFC and inhibition of proliferation was measured with MTT assay.DNA fragmentations were analyzed with DNA gel electrophoresis.Fluorescence staining of Hoechst 33258 to observe apoptotic body.Results The IC_50 of TFC on synovial cells was 112mg/L.DNA gel electrophoresis showed ladder-like strap.Apoptotic bodies were observed by Hoechst 33258.Conclusion TFC can inhibit proliferation and induce apoptosis in synovial cells,and exerts therapeutical effect on rheumstoid arthritis.
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The study was performed to detect the binding proteins for advanced glycation end products (AGEs) on human joint synovial cells (HSCs). Normal human synovial cells (type A and type B cells) were isolated and cultured in vitro. Binding assay was performed with radiolabeled human serum albumin modified by AGE (AGE HSA). Specific binding was defined as total binding minus binding in the presence of excess unlabeled AGE HSA. The result showed that: specific dose dependent binding of 125 I AGE HSA to immobilized HSCs was observed with R=4.90 0.75 10 4 /cell , Kd = 1.27 0.19 10 -6 M in type A HSCs , and R= 3.48 0.32 10 5 /cell, Kd= 1.38?0.16 10 -7 M in type B HSCs. TNF ?,IL 1? and AGE HSA upregulated the expression of AGE binding proteins on HSCs. Normal HSCs express specific AGE binding proteins. TNF ?, IL 1? and AGE HSA upregulate the expression of these proteins, suggesting that joint resident cells may be involved in the pathogenesis of dialysis related amyloidosis.
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Rheumatoid arthritis is characterized by a chronic inflammatory synovitis, eventually leading to destruction of bone and cartilage. Significant hyperplasia and infiltration of activated inflammatory cells play a major role in the destruction of joint. The proliferation of synovial cells could be derived from imbalance between apoptotic cell death and excessive proliferation of synovial cells. However, many reports regarding on the apoptosis or proliferation of synovial cells showed a little bit contradictory up to date. Induction of synovial cell apoptosis could be an interesting and attractive way of treatment by way of many signal transduction pathway, such as NFkB, P53, sentrin, FADD, etc. We discussed on the apoptosis and proliferation of synovial cells, and focused on the proposed mechanisms of resistance for apoptosis. Here, we reviewed literatures on the apoptosis and abnormal proliferation of synovial cells, and focused on the proposed mechanisms of resistance against apoptosis. In addition, we mentioned about the possibility of apoptosis induction as a modality of treatment against rheumatoid arthritis in future.
Subject(s)
Apoptosis , Arthritis, Rheumatoid , Cartilage , Cell Death , Hyperplasia , Joints , Signal Transduction , SUMO-1 Protein , SynovitisABSTRACT
Objective To investigate the proliferation and differentiation characteristics of fibroblast like synovial cells(FLS)in rheumatoid arthritis(RA)in vitro and the mechanism of the immunosuppressive effect of differentiation inducers, such as all trans retinoid acid(ATRA), ealcitriol [1,25(OH)_2D_3] and dexamethasone(DEX). Methods FLS of knee synovial tissues from RA patients were cultured and identified in vitro in the presence or absence of ATRA, 1,25(OH)_2D_3 and DEX respectively. Synoviocyte proliferation in RA were measured by MTT colorimetrie assay and the survival fraction(SF)of FLS was evaluated. Cell cycle of FLS was observed using fluorescence-activated cell sorting(FCS)method in RA patients. Results The identified synovial cells in patients with RA were FLS(Vimentin and Fibronectin expression was positive), and hadn't been transformed or differentiated to adipocytes and osteoblasts with the three inducers. The SF of all RA-FLS interfered by ATRA, 1,25(OH)_2D_3 and DEX was much lower than that without drugs vehicle group in RA-FLS(P
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No abstract available.
Subject(s)
Female , Humans , Arthritis, Rheumatoid , Bone Density , Estrogens , Etidronic Acid , NF-kappa B , Osteoporosis, PostmenopausalABSTRACT
To investigate effects of cytokines on rheumatoid synovial cells, proliferation and expression of cytokine and metalloproteinase genes were studied with the primary culture of rheumatoid synovial cells which was treated with TNF-alpha, GM-CSF, TGF-alpha, PDGF and IL-B. By [3H] thymidine incorporation assay, TGF-beta and PDGF increased proliferation of synovial cells by 1.5 and 2.5 folds respectively. Cytokine gene expression was assessed by RT-PCR. Rheumatoid synovial cells expressed constitutively TGF-beta and IL-B at a high level and IL-1B, GM-CSF, and MIP-1a at a relatively low level. TGF-beta, GM-CSF and PDGF increased IL-B expression. Expression of pro-inflammatory cytokines and chemokines was increased by GM-CSF and PDGF. Both GM-CSF and PDGF increased the expression of IL-1B, GM-CSF MIP-la and IL-8. In addition, GM-CSF enhanced expression of TNF-alpha. Stromelysin and collagenase are the major proteinases responsible for destruction ot joints in rheumatoid arthritis (RA). These genes were expressed constitutivefy in rheumatoid synovial cells. In summary, PDGF and GM-CSF may piay an important role by inducing or increasing expression of IL-1B, TGF-beta and PDGF by increasing proliferation of rheumatoid synovial cells.
Subject(s)
Tumor Necrosis Factor-alphaABSTRACT
Objective To test the hypothesis that attachment of synovial cell to &?2-microglobulin modified with advanced glycation end products (ACE-?2m) would affect cell adhesion, spreading and proliferation.Methods Normal human synovial cells (type B cells) were isolated and plated in culture dishes coated with AGE-?2m or with normal extracellular matrix proteins (EMP). Adhesion was analyzed by counting the isotope-labelled cells. Spreading was tested using a light microscope and proliferation determined by 3H-TdR incorporation and counting the number of cells. Results Synovial cells adhered less effectively to AGE-?2m, ?2m and AGE-collagen than to the normal EMP (collagen and fibronectin). Cells interacting with AGE-?2m, ?2m or AGE-collagen also demonstrated less extensive spreading throughout the examined time intervals (60-120 minutes after plating), and decreased 3H-TdR incorporation and cell numbers after 72 hours of plating when compared to cells interacting with normal EMP. Conchusion AGE-?2m in amyloid may alter synovial cell behavior in situ in ways which cods contribute to the development of dialysis-related amyloidodsis(DRA).
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An ultrastructral study was carried out to investigate distributional character of various synovial cells in different aress of synovial membrane of rabbit knee joints. Synovial membranes were surgically obtained from both knee joints of 5 rabbits, and were observed by both light and electron micrescopies. Type A synovial cells were distributed msinly on the luminal surface of the synovial membrane but type B cells were mostly in deep stromal areas. B cells were more than A cells in over-all numbers. Cellular density was found different sccording to the areas observed. Generally mid-central areas of the synovial membrane were less cellular than peripheral areas (medisl, lateral, upper and lower aress). Differences of cellular populations and cellular densities in various areas of rabbit synovial membranes were considered to be closely related to underlying structures and function of individual cell types.