Effect of modified citrus pectin on synovial fibroblasts / 国际生物医学工程杂志

Objective:To study the effects of modified citrus pectin (MCP) on the viability and gene expressions of synovial fibroblasts (SF) as well as SF treated by galectin-3 (Gal-3).Methods:Rabbit SF was isolated and cultured in vitro. Then SF was treated with different concentrations of MCP (0, 250, 500, and 750 mg/L). In addition, SF was further treated with the same different concentrations of MCP after treatment with 10 μg/ml Gal-3 for 24 h. The viability of SF was detected by CCK-8 on the first, third, and fifth day after treatment. The mRNA expression of transforming growth factor-β1 (TGF-β1), type I collagen (COL1A2), and Gal-3 in SF was detected by real-time quantitative PCR. The synthesis of type I collagen in SF was investigated by immunofluorescence staining. Results:MCP, especially at a concentration of 500 mg/L can inhibit the proliferation of SF significantly (all P < 0.05) on the first, third, and fifth day after treatment. Compared with the control group, MCP at different concentrations induced different gene expression profiles. In particular, MCP at high concentrations can upregulate the expression of TGF-β1, COL1A2 and Gal-3 in SF. However, MCP shows no significant effect on the synthesis of type I collagen in SF. MCP can down-regulate the expression of TGF-β1, COL1A2, and significantly reduce the synthesis of type I collagen in SF after Gal-3 treatment. Particularly, the effect of MCP at a concentration of 500 mg/L on inhibiting the expression of TGF-β1, COL1A2, and Gal-3 in SF is significant. Conclusions:MCP can inhibit the excessive proliferation of SF and regulate gene expression in SF.

Effect of Synovial Fibroblasts in Rheumatoid Arthritis and Intervention of Chinese Medicine： A Review / 中国实验方剂学杂志

Rheumatoid arthritis （RA） is a common autoimmune disease. Excessive hyperplasia of synovial tissues and osteoclastic bone absorption are two main causes of bone and joint destruction in RA. Synovial fibroblasts in RA （RA-FLS） are important cells in the synovial tissues of RA. The changes in their growth characteristics and inhibition of apoptosis lead to the proliferation of synovial tissues， stimulate inflammatory reactions， damage joint structure， and result in joint dysfunction. Therefore， regulating abnormal proliferation and promoting apoptosis of RA-FLS can interfere with the pathogenesis of RA. At present， there are many studies on the effect of Chinese medicine and its monomer components on the excessive proliferation and apoptosis of RA-FLS. The present study reviewed the effect of RA-FLS in RA and the intervention of Chinese medicinal monomers and compounds by regulating RA-FLS. The results showed that monomer components mainly included terpenoids， flavonoids， alkaloids， and anthraquinones. Despite different types， they can effectively intervene in RA through different approaches. For instance， they can prevent bone and cartilage injury by inhibiting the secretion of inflammatory cytokines and inhibiting the generation of chondrocytes and osteoclasts. They can achieve apoptosis by up-regulating the pro-apoptotic genes B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and cysteinyl aspartate-specific protease （Caspase） in the Fas/FasL， nuclear factor-κB （NF-κB）， Janus kinase （JAK）/signal transducer and activator of transcription protein （STAT）， and p38 mitogen-activated protein kinase （p38 MAPK） signaling pathways， and down-regulating the anti-apoptotic genes Bcl-xl and Bcl-2. They can also inhibit the proliferation of RA-FLS by inhibiting the protein expression of microtubule-associated protein 1 light chain 3Ⅱ（LC3Ⅱ）/LC3Ⅰ and Beclin-1. Although their molecular mechanisms of action and targets are different， they all exert corresponding roles. The above research results provide a scientific basis for elucidating the multi-component and multi-target characteristics of Chinese medicine in the treatment of RA.

Effects of daphresin on synovial fibroblasts in rheumatoid arthritis based on PERK/ATF4/CHOP signaling pathway / 中国药理学通报

Aim To investigate the effects of DAP on human rheumatoid arthritis synovial fibroblasts(RA-FLS)and its relationship with endoplasmic reticulum stress(ERS)PERK/ATF4/CHOP signaling pathway.Methods RA-FLS cells were cultured and identified by immunofluorescence assay for Vimentin and CD68.CCK-8 detected(0,5,10,20,30,40,50,60,70)mg·L-1 DAP on the proliferation activity of RA-FLS cells.According to the proliferation activity,the experiment was divided into blank group(Blank),low dose group(L-DAP),medium dose group(M-DAP),high dose group(H-DAP)and high dose+specific inhibitor 4-PBA group(H-DAP+4-PBA).The levels of TNF-α and IL-6 were detected by ELISA.Cell apoptosis was detected by flow cytometry.The invasion and migration of cells were detected by Transwell and scratches assays,and the expressions of endoplasmic reticulum stress-related proteins GRP78,PERK,P-PERK,ATF4,CHOP,Caspase-12,C-Caspase-12 and Bcl-2 were assessed by Western blot.Results CCK-8 results showed that compared with 0 mg·L-1 DAP group,the proliferation activity of each group in 20-70 mg·L-1 DAP group was significantly different(P<0.05),and the proliferation rate corresponding to 0,20,40 and 60 mg·L-1 DAP group was significantly different(P<0.05).This concentration was used as the basis for blank,low-dose,medium-dose,high-dose groups and high-dose+4-PBA experimental grouping.DAP could inhibit the release of inflammatory cytokines IL-6 and TNF-α from RA-FLS cells in concentration,reduce the invasion ability of cells,promote apoptosis,upregulate the expression of PERK,P-PERK,ATF4,GRP78,CHOP,Caspase-12, C-caspase-12 proteins and decrease the expression level of anti-apoptotic protein Bcl-2.Another set of experiments demonstrated that high dose DAP+4-PBA could up-regulate the expression of IL-6 and TNF-α,as well as the invasion of cells,and inhibit the expression of apoptosis and ERs-related proteins.Conclusions Daphresin regulates the secretion of inflammatory factors by activating the PERK/ATF4/CHOP signaling pathway,inhibits the proliferation and invasion of RA-FLS cells,and induces apoptosis,which is expected to be a potential therapeutic pathway for RA.

Xinfeng Capsules promotes apoptosis of synovial fibroblasts and attenuates inflammation in rheumatoid arthritis by regulating lncRNA MAPKAPK5-AS1 / 中国中药杂志

To explore the regulatory effects of Xinfeng Capsules(XFC) on the apoptosis of synovial fibroblasts(FLS) and inflammation in rheumatoid arthritis(RA) via lncRNA MAPKAPK5-AS1(MK5-AS1). Thirty healthy people and 30 patients with RA due to spleen deficiency and dampness exuberance were collected for extracting the peripheral blood mononuclear cells(PBMCs) before and after XFC treatment, which were used to observe the correlation between MK5-AS1 and clinical indicators as well as MK5-AS1 expression before and after XFC treatment. Following the establishment of RA-FLS cell line and the preparation of XFC-containing serum, MK5-AS1-overexpression plasmid was constructed and transfected into RA-FLS for investigating the efficacy of XFC-containing serum in regulating inflammation and apoptosis of RA-FLS via MK5-AS1. The expression of MK5-AS1 in PBMCs of patients with RA due to spleen deficiency and dampness exuberance was decreased(P<0.001). The ROC curve analysis revealed the AUC of 83.9%. Correlation analysis showed that MK5-AS1 was negatively correlated with ESR, CRP, RF, CCP, and spleen deficiency and dampness exuberance syndrome score. The expression of MK5-AS1 increased significantly after XFC treatment(P<0.001). As demonstrated by association analysis, XFC decreased MK5-AS1, ESR, CRP, RF, and spleen deficiency and dampness exuberance syndrome score, with the degree of support all greater than 83%, confidence greater than 80%, and lift greater than 1. The results of RT-qPCR showed that the MK5-AS1 RNA expression significantly decreased after TNF-α stimulation(P<0.01), which, however, increased significantly after the intervention with XFC-containing serum(P<0.05). Such expression rose again after the transfection of pcDNA3.1-MK5-AS1(P<0.01). ELISA results showed that TNF-α stimulation elevated the expression of pro-inflammatory factor IL-17 but lowered the expression of anti-inflammatory factor IL-4(P<0.01). After intervention with XFC-containing serum, the expression of IL-17 decreased while that of IL-4 increased(P<0.01). The transfection of pcDNA3.1-MK5-AS1 contributed to the reduction in IL-17 expression but the elevation in IL-4 expression(P<0.01). The immunofluorescence(IF) findings demonstrated that the expression of pro-apoptotic protein Bax was down-regulated, whereas that of the anti-apoptotic protein Bcl-2 was up-regulated after TNF-α stimulation(P<0.01). After the intervention with XFC-containing serum, the Bax expression was increased, while Bcl-2 expression was decreased(P<0.01), which were remarkably collaborated by the transfection of pcDNA3.1-MK5-AS1(P<0.05). The expression of MK5-AS1 is significantly decreased in both RA-PBMCs and RA-FLS, implying that XFC inhibits inflammatory reaction and promotes the apoptosis in RA by regulating the expression of MK5-AS1.

Inhibitory effect of baicalin on autophagy of synovial RSC-364 cells of rats induced by lipopolysaccharide / 吉林大学学报(医学版)

Objective: To investigate the effect of baicalin on the autophagy of the synovial RSC-364 cells of the rats induced by lipopolysaccharide (LPS) through Pl3k/Akt/mTOR signal pathway, and to clarify its mechanism. Methods: The rat synovial RSC-364 cells in logarithmic growth phase were divided into control group, model group. dexamethasone (DXMS) group. 10/imol • L 1 baicalin group. 20/imol • L 1 baicalin group and 40/imol • L baicalin group. The RSC-364 cells in control group were only supplemented with culture medium, and the RSC-364 cells in the other groups were stimulated with 1 mg • L 1 LPS for 12 h to make the inflammatory cell models. The survival rates of RSC-364 cells were detected by MTT assay, and the expression levels of P13k. Akt, mTOR. Beclinl. and LC3-II mRNA in the RSC-364 cells in various groups were detected by RT-PCR method; Western blotting method was used to detect the expression levels of Beclinl. Atg5. Atg7. Atgl2. microtubule-associated protein-light chain 3-II (LC3-II ). and P62 proteins in the RSC-364 cells in various groups. Results: Compared with control group, the survival rate of RSC-364 cells in model group was significantly increased ( P-'-CO. 01). the expression levels of P13k. Akt. mTOR mRNA and P62 protein in the RSC-364 cells in model group were significantly decreased (P<0. 05 or P<0. 01). and the expression levels of Atg5. Atg7. Atgl2. LC3-II . Beclinl mRNA and proteins were significantly increased ( P<0. 01) ; compared with model group, the survival rates of RSC-364 cells in different concentrations of baicalin groups were significantly decreased (P<0. 05 or P<0. 01). the expression levels of P13k. Akt. mTOR mRNA and P62 protein in the RSC-364 cells in different concentrations of baicalin groups were significantly increased (P-'-CO. 05 or P<0. 01). and the expression levels of Atg5. Atg7. Atgl2. LC3-II and Beclinl mRNA and proteins were decreased ( P<0. 05 or P<0. 01). Conclusion: Baicalin may inhibit the LPS-induced autophagy of the RSC-364 cells by activating the Pl3k/Akt/mTOR signaling pathway.

The reaserch about miR-17 targeting signal transducer and activator of transcription 3 to regulate the proliferation of rheumatoid arthritis synovial fibroblasts and the release of inflammatory factors from rheumatoid arthritis synovial fibroblasts in the pathogenesis of rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate whether miR-17 plays a role in re-gulation of signal transducer and activator of transcription 3 (STAT3) expression,affecting the proliferation of rheumatoid arthritis synovial fibroblasts (RASFs) and the release of inflammatory factors from RASFs.Methods The synovial tissues of rheumatoid arthritis (RA) patients were collected.As a comparison,synovial tissues from osteoarthritis (OA) patients were collected as control,the expression of miR-17,STAT3 were detected.The results were analyzed by Mann-Whitney U test.To analyze the effects of interleukin (IL)-17A treatment on the expression of micro RNA-17 and STAT3,cell proliferation and secretion of IL-6 and IL-8 in RASFs cells.The effects of cell proliferation and secretion of IL-6 and IL-8 were analyzed by cell transfection of micro RNA-17 mimic and siRNA-STAT3.The results about RASFs cells were analyzed by t-testof two independent samples.Results Compared with OA patients,the expression of miR-17 in synovial tissues of RA patients decreased significantly (Mann-Whitney U=6,P＜0.01),while the expression of STAT3 increased obviously (Mann-Whitney U=32,P＜0.01).The expressions of IL-17A,IL-6 and IL-8 in synovial fluid of patients with OA were (53±12),(43±9) and (33±5),respectively,significantly lower than those of patients with RA [(170±30),(222±37) and (156±34),t=18.83,24.28,19.23,P＜0.01].IL-17A treatment significantly lowered the expression of miR-17 [(1.00 ±0.12) vs (0.37±0.04),t=8.63,P＜0.01],while up-regulated the expression of STAT3 [(1.00±0.14) vs (1.92 ±0.23),t =5.92,P＜0.01),promoted the proliferation of RASFs cells,and promoted the release of inflammatory factors IL-6 and IL-8.The results of the double luciferase reporter gene showed that there was a targeting regulation relationship between miR-17 and STAT3.Transfection of miR-17 mimic or siRNA-STAT3 could significantly reduce the expression of STAT3 and p-STAT3 in RASFs cells,along with the inhabitation of cell proliferation [miR-NC vs miR-17 mimic (26.9±2.8) vs (41.5±3.1),t=6.06,P＜0.01;siRNA-NC vs siRNA-STAT3 (23.5±2.4) vs (43.2±3.2),t=8.58,P＜0.01] and reduction of the secretion of inflammatory factors [miR-NC vs miR-17 mimic (110±13) vs (66±9),t=4.88,P＜0.01;siRNA-NC vs siRNA-STAT3 为(117±12) vs (70±6),t=6.10,P＜0.01] and IL-8 [miR-NC vs miR-17 mimic (127±10) vs (72±7),t=8.10,P＜0.01;siRNA-NC vs siRNA-STAT3 (123±11) vs (52±6),t=10.19,P＜0.01].Conclusion Decreased expression of miR-17 may play an important role in enhancing STAT3 expres-sion and promoting the pathogenesis of RA.Overexpression of miR-17 could inhibit STAT3 expression,atten-uate RASFs cell proliferation and inflammatory factor release.

The role and mechanism of S100 calcium binding protein B in osteoarthritis cartilage damage repair / 中国修复重建外科杂志

Objective: To investigate the role and mechanism of S100 calcium binding protein B (S100B) in osteoarthritis (OA) cartilage damage repair. Methods: Twenty New Zealand rabbits were randomly divided into control group and model group, with 10 rabbits in each group. Rabbits in the model group were injured by the right knee joint immobilization method to make the artilage injury model, while the control group did not deal with any injury. After 4 weeks, the levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in synovial fluid were detected by ELISA method; the mRNA and protein expressions of S100B, fibroblast growth factor 2 (FGF-2), and FGF receptor 1 (FGFR1) in cartilage tissue were examined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot assay. Human synovial fibroblasts (SF) were isolated and cultured in vitro. The effects of S100B overexpression and knockdown on the levels of IL-1β and TNF-α (ELISA method) and the expressions of FGF-2 and FGFR1 gene (qRT-PCR) and protein (Western blot) were observed. Moreover, the effects of FGFR1 knockdown in above S100 overexpression system on the levels of IL-1β and TNF-α (ELISA method) and the expressions of FGF-2 and FGFR1 gene (qRT-PCR) and protein (Western blot) were observed. Results: ELISA detection showed that the expressions of IL-1β and TNF-α in the synovial fluid of the model group were significantly higher than those of the control group ( P<0.05); qRT-PCR and Western blot detection showed that the mRNA and protein expressions of S100B, FGF-2, and FGFR1 in cartilage tissue were significantly higher than those of the control group ( P<0.05). Overexpression and knockdown S100 could respectively significantly increase and decrease lipopolysaccharides (LPS) induced IL-1β and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1 ( P<0.05); whereas FGFR1 knockdown could significantly decrease LPS induced IL-1β and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1 ( P<0.05). Conclusion: S100B protein can regulate the inflammatory response of SF and may affect the repair of cartilage damage in OA, and the mechanism may be related to the activation of FGF-2/FGFR1 signaling pathway.

Baicalin alleviated rheumatoid arthritis synovitis of SD rats through TLR2-NF-κB pathway / 中国药理学通报

Aim To evaluate the effects of different doses of baicalin on rheumatoid arthritis (RA) in SD rats and explore the possible mechanism.Methods Firstly,the model of RA in SD rats was prepared and the hind foot swelling was measured;HE staining was used to observe the pathological changes of the knee joint synovial tissue;RT-qPCR was adopted to determine the mRNA expressions of TLR2 and MyD88 in synovial tissue;Western blot was used to determine the protein expressions of TLR2,MyD88 and NF-κB p65 aher intragastric administration of different doses of baicalin solution.Results Compared with the model,baicalin (60 and 30 mg · kg-1) could inhibit the proliferation of fibroblasts and inflammatory damages in synovial tissue,significantly cut down mRNA expressions of TLR2 and MyD88 (P ＜ 0.05) and markedly reduce protein expressions of TLR2,MyD88 and NF-κB p65 (P ＜ 0.01).Conclusion Baicalin has good effects on RA,which may be realized by inhibiting the activation of TLR2-NF-κB signaling pathway.

Comparison of Three Methods for Culturing Rheumatoid Arthritis Synovial Fibroblasts / 昆明医科大学学报

Objective To compare the three kinds of methods for in vitro primary culturing of rheumatoid arthritis synovial fibroblast-like cells (RASFs), in order to get fast and effective culture methods. Methods Synovial tissue from RA synovial arthroscopic resection were treated by collagenase digestion method, modified tissue culture method, double enzyme digestion method respectively. By using an inverted phase contrast microscope, cell morphology and growth characteristics were observed and identified with vimentin staining. Trypan blue was used to count the number of living cells after culturing for 14d. Results The three primary methods could successfully isolate and culture RASFs, and RASFs met the morphological characteristics of vimentin-positive cells>95%, namely, the proportion of RASFs cell confluence was 70% after 16-20days by the collagenase digestion method,whose cell confluence proportion reached 95%after 4 weeks;and the cell confluence proportion was above 70%after 10-14days by modified tissue culture method,and the cell confluence proportion reached 85%after 4 weeks by the double enzyme digestion method. The comparison of the viable cells number cultured same number of synovial tissue by the three methods show the viable cells number cultured by the modified tissue culture method were (1.60±0.08) ×106, those by the collagenase digestion method were (1.41±0.08) ×106, those by the double enzyme digestion method were (1.19 ±0.05) ×106, which were with significant difference among them (P<0.05) .The comparison of incubation time of RASFs primary cells showed it took (267.50±16.58) mins by the collagenase digestion method, (183.75 ±11.08) mins by the double enzyme digestion method, and 149.10 ±13.71mins by the modified tissue culture method, with significant differences (P<0.05) .Conclusion Modified tissue culture for RASFs is an efficient and fast culture method, the number and purity of RASFs can meet the requirements for biology experiments.

Interleukin-22 promotes Th17 cells differentiation through up-regulating IL-6 production by rheuma-toid arthritis synovial fibroblasts / 中华微生物学和免疫学杂志

Objective To investigate the effects of interleukine-22 ( IL-22 ) on the expression of interleukin-6 (IL-6) by rheumatoid arthritis synovial fibroblasts (RASF), and to analyze their association with IL-17+CD4+T (Th17) cells differentiation.Methods RASF were isolated from six patients with rheu-matoid arthritis ( RA) and cultured in vitro.The expression of IL-6 at mRNA and protein levels by RASF were detected by qRT-PCR analysis and ELISA after treatment with different concentrations of IL -22 for dif-ferent periods of time.Anti-IL-22R1 blocking antibody and inhibitor assay were used to analyze the specific receptor and its downstream signaling pathways associated with IL-6 production.IL-22 pre-treated RASF and CD4+T cells were co-cultured for 3 days in the presence or absence of anti-IL-22R1 or anti-IL-6 to measure the percentage of Th 17 cells by flow cytometry .Results The expression of IL-6 by RASF was increased up-on IL-22 stimulation in a dose and time dependent manner (P<0.05), and that was closely related to IL-22R1 and its downstream signaling pathways of p38 and JAK2 (P<0.05).Co-culturing CD4+T cells with RASF and Transwell system indicated that the percentage of Th 17 cells was increased in IL-22 pre-treated group as compared with that in IL-22 untreated group , but it could be down-regulated by either blocking IL-22R1 or IL-6.Conclusion IL-22 promoted the expression of IL-6 by RASF and further enhanced Th 17 dif-ferentiation.Neutralizing IL-22 in synovium of patients with RA might be an effective therapeutic strategy for RA treatment.

Effects of microRNA-16 on proliferation, invasion and cytokine secretion of synovial fibroblasts from rheumatoid arthritis patients / 中国病理生理杂志

AIM:To investigate the effect of microRNA-16 ( miR-16) on the proliferation, invasion and cyto-kine secretion of rheumatoid arthritis ( RA) synovial fibroblasts ( RASFs) from the RA patients.METHODS: miR-16 mimic and miR-16 inhibitor were synthesized, and then Transfected into RASFs isolated from RA patients with lipo-fectamine.MTT assay, Transwell chamber and flow cytometry were used to determine the effect of miR-16 on proliferation, invasion and apoptosis of RASFs.The expression of matrix metalloproteinase 3/13 ( MMP3/13) and interleukin 1β( IL-1β) was measured by RT-PCR and Western blotting.RESULTS: The proliferation and invasion of RASFs were signifi-cantly inhibited by miR-16 mimic.The result of flow cytometry demonstrated that miR-16 had no effect on apoptosis of RASFs.Furthermore, miR-16 down-regulated the expression of MMP3/13 and IL-1β.CONCLUSION:miR-16 plays an important role in the development of RA and may inhibit the proliferation and invasion of RASFs through down-regulating the expression of MMP3/13 and IL-1β.

Hypoxia/reoxygenation Regulates Expression of Tumor Suppressor PTEN in Rheumatoid Synovial Fibroblasts / 대한류마티스학회지

OBJECTIVE: The lack of phosphatase and tensin homolgue deleted on chromosome ten (PTEN) expression was described in rheumatoid synovial tissues and synovial fibroblasts, but exact cause of that in RA is not well known. Hypoxic conditions are thought to exist in inflamed arthritic synovium and therefore, this study was designed to investigate the effects of hypoxia/ reoxygenation on the expression of PTEN in synovial fibroblasts of rheumatoid arthritis. METHODS: Synovial fibroblasts were isolated from synovial tissues of patients suffering from RA and hypoxic culture was performed by incubating cells in 5% CO2 incubator held at 3% oxygen by the addition of nitrogen gas for 24 hours. Then synovial fibroblasts were cultured for 10 min under normoxic condition for reoxygenation. To know the expression of PTEN and phosphorylated Akt (p-Akt) in synovial fibroblasts, Western blotting analysis was performed. The expression of PIP3 kinase and PTEN was analyzed by immunocytochemical staining. RESULTS: There were less PTEN expression in rheumatoid synovial fibroblasts than that of healthy controls. Hypoxic/reoxygenation stimuli induced down-regulation of PTEN expression in the rheumatoid synovial fibroblasts. In contrast, the expression of PIP3 and p-Akt was increased after stimulation with hypoxia/reoxygenation. CONCLUSION: These studies suggest that hypoxia/reoxygenation could cause the reduced expression of PTEN in rheumatoid synovial fibroblasts and thus it might thereby contribute to the invasive behaviour of rheumatoid synovial fibroblasts by maintaining their aggressive phenotype in RA.

Effects of Antioxidant on the Hypoxia-induced Expression of ICAM-1 in Cultured Human Synovial Fibroblasts

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and joint destruction. The synovial fibroblasts express cell adhesion molecules and have a role in adhesive interation with inflammatory cells in synovial tissue. It has been suggested that hypoxic conditioins are thought to exist in arthritic joints, and several studies indicate that reactive oxygen species (ROS) produced in hypoxic condition can initiate events that lead to pro-adhesive changes via increased expression of adhesion molecules. So, this study wsa designed to examine whether antioxidant can inhibit hypoxia-induced expression of ICAM-1 in cultured human synovial fibroblasts. METHODS: Synovial fibroblasts were isolated from synovial tissue in patients with RA and cultured at hypoxic condition. Antioxidant, PDTC (pyrrolidine dithiocarbamate) were pre-treated for an hour before the hypoxic culture and synovial fibroblasts were harvested at 0, 6, 12, 24, 48 hours time points. Cell surface ICAM-1 expression in synovial fibroblasts was examined by the flow cytometric analysis. To analyse the expression of ICAM-1 mRNA, reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. The levels of cytokines in culture supernatants were measured by ELISA, and activation of NF-kB was analysed by electrophoretic mobility shift assay. The adhesive reaction between synovial fibroblasts and lymphocytes was assayed by measurement of fluorescent intensity of BCECF-AM in lymphocytes. RESULTS: Hypoxic stimuli up-regulated the ICAM-1 expression as well as the adhesive interaction of human synvial fibroblasts to lymphocytes in a time-dependent manner, and PDTC inhibited hpyoxia-induced ICAM-1 expression and cell-cell interaction. PDTC also inhibited the hypoxia-induced activation of intracellular transcription factor, NF-kB. PDTC decreased the amount of hypoxia-induced production of IL-1beta and TNF-alpha. CONCLUSION: These studies demonstrate that PDTC inhibit the hypoxia-induced expression of the adhesion molecule, ICAM-1 and activation of NF-kB in cultured human synovial fibroblasts.

Hypoxia Increases the Expression of ICAM-1 in Cultured Human Synovial Fibroblasts / 대한류마티스학회지

OBJECTIVE: Hypoxic conditions are thought to be exist in inflamed arthritic synovium.Several in vitro and in vivo studies indicate that hypoxia can initiate events that lead to pro-adhesive changes.Therefore,this study was designed to examine the effects of hypoxia on the expression of ICAM-1 by cultured human synovial fibroblasts. METHODS: Synovial fibroblasts were isolated from patients with RA and cultured at hypoxic condition.To quantify the expression of ICAM-1 mRNA in synovial fibroblasts,RT-PCR was performed.The levels of cytokines in culture supernatants were measured by ELISA.The activation of NF-

Differential proteome of human synovial fibroblasts from rheumatoid arthritis patients / 第三军医大学学报

ObjectiveTo understand the possible role of some proteins expressed by human synovial fibroblasts(SFs)in the pathogenesis of rheumatoid arthritis.MethodsThe expression difference of synovial fibroblast proteins between rheumatoid arthritis(RA)patients and healthy controls was analyzed by 2-DE.The differential expression spots were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry(MALDI-TOF-MS),followed by bioinformatics analysis,some of which were validated by Western blot.ResultsUsing 12% SDS-PAGE following pH 4-7 IPG strips in IEF,averagely 837 and 852 protein spots were detected in RA patients and normal subjects,respectively.Gel image analysis revealed that there were 49 differential protein spots.By peptide mass fingerprinting strategy,we identified 40 protein spots derived from gels of SFs in RA patients among differential spots and 23 valid proteins were obtained.Western blot analysis showed that expressions of Enolase ?,Annexin I,Cathepsin D,SOD2,Peroxiredoxin 2 were significantly higher in SFs from RA patients than those from normal subjects,which was consistent with proteome analysis.ConclusionThe differential proteins might be involved in inflammation of synovitis in RA.