Journal: Journal of Orthopaedic Surgery and Research

Article Title: Circ_0000479 promotes proliferation, invasion, migration and inflammation and inhibits apoptosis of rheumatoid arthritis fibroblast-like synoviocytes via miR-766/FKBP5 axis

doi: 10.1186/s13018-023-03700-0

Figure Lengend Snippet: Circ_0000479 was upregulated in MH7A cells. A Relative expression of circ_0000479 in MH7A cells and normal FLSs. B Relative expression of EPSTI1 in MH7A cells and normal FLSs. C The expression of EPSTI1 protein in MH7A cells and normal FLSs. D Relative RNA levels of circ_0000479 and EPSTI1 after RNase R treatment. E Analysis for RNA abundance of circ_0000479 and EPSTI1 after Actinomycin D treatment at indicated time points. F Detected by qRT-PCR, circ_0000479 was mainly enriched in the cytoplasm. **P < 0.01; ****P < 0.0001

Article Snippet: RA-FLSs (MH7A cells) and normal FLSs (primary cells) were purchased from Wuhan Procell Life Technology Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Circ_0000479 promotes proliferation, invasion, migration and inflammation and inhibits apoptosis of rheumatoid arthritis fibroblast-like synoviocytes via miR-766/FKBP5 axis

doi: 10.1186/s13018-023-03700-0

Figure Lengend Snippet: MiR-766 was the target of circ_0000479. A Detection of miRNA expression levels in MH7A cells by qRT-PCR. B Relative expression of miR-766 in MH7A cells and normal FLSs. C Relative expression of miR-766 in MH7A cells transfected with miR-NC or miR-766. D The binding sites between circ_0000479 and miR-766. E Relative luciferase activity of WT-circ_0000479 and MUT-circ_0000479 after transfection of miR-NC or miR-766. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: RA-FLSs (MH7A cells) and normal FLSs (primary cells) were purchased from Wuhan Procell Life Technology Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Luciferase, Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of isoflavone derivatives on the production of inflammatory cytokines by synovial cells

doi: 10.3892/etm.2021.10735

Figure Lengend Snippet: Effects of daidzein, genistein, and glycitein on IL-6 and IL-8 production by IL-1β-stimulated MH7A cells. Cells were treated without (-) or with (+) 1 or 5 µg/ml of (A and D) daidzein, (B and E) genistein or (C and F) glycitein for 3 h, and then stimulated without (-) or with (+) 50 pg/ml IL-1β for 18 h. Alternatively, cells were treated without (-) or with (+) 0.5% DMSO (a solvent of isoflavones) in the absence of isoflavones, and then stimulated with IL-1β. Thereafter, culture supernatants were recovered, and (A-C) IL-6 and (D-F) IL-8 levels were measured by ELISA. Data are expressed as a ratio to that in IL-1β-stimulated MH7A cells without isoflavone derivatives, and compared between without and with isoflavones. Results are shown as the means ± SD of five independent experiments. DMSO, dimethyl sulfoxide.

Article Snippet: The authors would like to thank Dr Keiji Miyazawa (Kissei Pharmaceutical Co., Ltd., Nagano, Japan) for the establishment of MH7A cells, and Dr Mamoru Igarashi, Dr Kaori Suzuki, Dr Taisuke Murakami and Dr Yumi Kumagai Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine, for their technical assistance and helpful discussions.

Techniques: Solvent, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of isoflavone derivatives on the production of inflammatory cytokines by synovial cells

doi: 10.3892/etm.2021.10735

Figure Lengend Snippet: Effects of daidzein on the phosphorylation of NF-κB, ERK1/2, and p38 MAPK. Cells were treated without (-) or with (+) 10 µg/ml daidzein for 16 h, and then stimulated without (-) or with (+) 200 pg/ml IL-1β for 15 min. Cells were harvested, the supernatants (containing 12 µg protein) obtained from cell lysates were resolved on 10% SDS-PAGE, and phosphorylated and total (A) NF-κB p65, (B) ERK1/2 and (C) p38 MAPK were evaluated by western blotting. GAPDH (an internal control) was also detected by western blotting. Images are representative of three separate experiments. The phosphorylation of NF-κB p65, ERK1/2, and p38 MAPK was normalized with total NF-κB p65, ERK1/2, and p38 MAPK, respectively, and expressed as a ratio to that in IL-1β-stimulated MH7A cells without daidzein. Data were compared between without and with daidzein. Results are expressed as the means ± SD of three independent experiments. *P<0.05. NF-κB, nuclear factor-κB; ERK, extracellular signal regulated kinases; p38 MAPK, p38 mitogen-activated protein kinase.

Article Snippet: The authors would like to thank Dr Keiji Miyazawa (Kissei Pharmaceutical Co., Ltd., Nagano, Japan) for the establishment of MH7A cells, and Dr Mamoru Igarashi, Dr Kaori Suzuki, Dr Taisuke Murakami and Dr Yumi Kumagai Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine, for their technical assistance and helpful discussions.

Techniques: Phospho-proteomics, SDS Page, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of isoflavone derivatives on the production of inflammatory cytokines by synovial cells

doi: 10.3892/etm.2021.10735

Figure Lengend Snippet: Effects of daidzein on the phosphorylation of NF-κB, ERK1/2, and p38 MAPK. Cells were treated without (-) or with (+) 10 µg/ml daidzein for 16 h and then stimulated without (-) or with (+) 200 pg/ml IL-1β for 30 min. Thereafter, cells were harvested, the supernatants (containing 12 µg protein) obtained from cell lysates were resolved on 10% SDS-PAGE, and phosphorylated and total (A) NF-κB p65, (B) ERK1/2 and (C) p38 MAPK were evaluated by western blotting. GAPDH (an internal control) was also detected by western blotting. Images are representative of three separate experiments. The phosphorylation of NF-κB p65, ERK1/2, and p38 MAPK was normalized to total NF-κB p65, ERK1/2, and p38 MAPK, respectively, and expressed as a ratio to that in IL-1β-stimulated MH7A cells without daidzein. Data are compared between without and with daidzein. Results are expressed as the means ± SD of three independent experiments. *P<0.05. NF-κB, nuclear factor-κB; ERK, extracellular signal regulated kinases; p38 MAPK, p38 mitogen-activated protein kinase.

Article Snippet: The authors would like to thank Dr Keiji Miyazawa (Kissei Pharmaceutical Co., Ltd., Nagano, Japan) for the establishment of MH7A cells, and Dr Mamoru Igarashi, Dr Kaori Suzuki, Dr Taisuke Murakami and Dr Yumi Kumagai Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine, for their technical assistance and helpful discussions.

Techniques: Phospho-proteomics, SDS Page, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of isoflavone derivatives on the production of inflammatory cytokines by synovial cells

doi: 10.3892/etm.2021.10735

Figure Lengend Snippet: Schematic model of IL-1β-stimulated signaling pathways of NF-κB p65, ERK1/2, and p38 MAPK, and inhibitory effects of daidzein on NF-κB/ERK1/2 signaling in synovial cells. TAK1 is activated by a stimulation with IL-1β, and activated TAK1 phosphorylates the IKK. Activated IKK phosphorylates IκB in the complex with NF-κB, and phosphorylated IκB is degraded by the ubiquitin-proteasome system, which allows the release and nuclear translocation of NF-κB, leading to the transcription of inflammatory cytokine genes. Moreover, activated IKK activates Tpl2, which, in turn, activates MKK1/2, leading to the activation of ERK1/2. Alternatively, TAK1 activates the p38 MAPK pathway via the activation of MKK3 and MKK6. p38 MAPK may also be activated by TAK1 via the IKK/Tpl2 pathway (dotted line). Since daidzein inhibited the phosphorylation of NF-κB p65 and ERK1/2, but not p38MAPK in IL-1β-stimulated MH7A cells, it appears to inhibit the TAK1/IKK-mediated activation of NF-κB p65 and ERK1/2, but not the TAK1/MKK3/MKK6-mediated activation of p38 MAPK. IKK, IκB kinase; IκB, inhibitor of κB. NF-κB, nuclear factor-κB; ERK, extracellular signal regulated kinases; p38 MAPK, p38 mitogen-activated protein kinase; TAK1, transforming growth factor-β-activated kinase 1; MAPKKK, MAP kinase kinase kinase; IκB, inhibitor of κB; IKK, IκB kinase; Tpl2, tumor progression locus 2; MKK and MAPKK, mitogen-activated protein kinase kinase.

Article Snippet: The authors would like to thank Dr Keiji Miyazawa (Kissei Pharmaceutical Co., Ltd., Nagano, Japan) for the establishment of MH7A cells, and Dr Mamoru Igarashi, Dr Kaori Suzuki, Dr Taisuke Murakami and Dr Yumi Kumagai Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine, for their technical assistance and helpful discussions.

Techniques: Protein-Protein interactions, Ubiquitin Proteomics, Translocation Assay, Activation Assay, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: A Modified Compound From Paeoniflorin, CP-25, Suppressed Immune Responses and Synovium Inflammation in Collagen-Induced Arthritis Mice

doi: 10.3389/fphar.2018.00563

Figure Lengend Snippet: CP-25 down-regulated excessive synoviocyte activation by decreasing GRK2-mediated β2-AR desensitization. MH7A (A,B) proliferation (1 nM: t = 1.067, P < 0.05; 0.1 μM: t = 3.584, P < 0.01; 1 μM: t = 5.768, P < 0.01; 10 μM: t = 5.685, P < 0.01), (C,E) migration (0.1 μM: t = 5.268, P < 0.01; 1 μM: t = 6.141, P < 0.01; 10 μM: t = 6.070, P < 0.01), and (D,F) invasion (0.1 μM: t = 2.514, P < 0.05; 1 μM: t = 5.477, P < 0.01; 10 μM: t = 5.691, P < 0.01). Data are expressed as the mean ± SD. ## P < 0.01 vs. control; ∗ P < 0.05, ∗∗ P < 0.01 vs. TNF + ISO. The results are representative of at least three independent experiments. (G–I) Expression of β2-AR ( t = –12.745, P < 0.01) and GRK2 ( t = 5.986, P < 0.01). The results are representative of at least three independent experiments and data are expressed as the mean ± SD. ∗∗ P < 0.01 vs. TNF.

Article Snippet: The human RA fibroblast-like synoviocyte (FLS) cell line, MH7A, was purchased from the China Center for Type Culture Collection.

Techniques: Activation Assay, Migration, Control, Expressing

Journal: AMB Express

Article Title: Inhibitory effects of H-Ras/Raf-1-binding affibody molecules on synovial cell function

doi: 10.1186/s13568-014-0082-3

Figure Lengend Snippet: Experimental protocol (A) and western blot analysis for confirmation of cytosolic synthesis of affibody molecules (B). (A) Affibody molecules were introduced into MH7A cells encoded by DNA or as proteins. After 24-h culture, the cells were stimulated with TNF-α (100 ng/mL) and cultured for an additional 24-48 h. (B) Western blot analysis of each soluble fraction from MH7A cells harboring plasmid-encoded affibody protein molecules was analyzed at 48 and 72 h after the introduction of the plasmid into MH7A cells. Actin was analyzed as a control.

Article Snippet: Percent inhibition was calculated as follows: ([(IL-6 or PGE2 levels in affibody protein-introduced MH7A cells) - (IL-6 or PGE2 levels in MH7A cells introduced with standard peptides)]/([IL-6 or PGE2 levels in MH7A cells introduced with standard peptides]) × 100.

Techniques: Western Blot, Cell Culture, Plasmid Preparation

Journal: AMB Express

Article Title: Inhibitory effects of H-Ras/Raf-1-binding affibody molecules on synovial cell function

doi: 10.1186/s13568-014-0082-3

Figure Lengend Snippet: IL-6 (A) and PGE2 (B) production by MH7A cells with plasmid-expressed affibodies. MH7A cells were transfected with plasmids encoding affibodies. After 24 h of culture, the cells were stimulated with TNF-α (100 ng/mL) and cultured for an additional 24-48 h. Data represent the percent inhibition of IL-6 or PGE2 production by MH7A cells. Percent inhibition was calculated as follows: ([(IL-6 or PGE2 levels in MH7A cells transfected with affibody DNA) - (IL-6 or PGE2 levels in MH7A cells transfected with pcDNA3.1)]/[IL-6 or PGE2 levels in MH7A cells transfected with pcDNA3.1]) × 100. Data represent the means × SD of three independent experiments. * p <0.05.

Article Snippet: Percent inhibition was calculated as follows: ([(IL-6 or PGE2 levels in affibody protein-introduced MH7A cells) - (IL-6 or PGE2 levels in MH7A cells introduced with standard peptides)]/([IL-6 or PGE2 levels in MH7A cells introduced with standard peptides]) × 100.

Techniques: Plasmid Preparation, Transfection, Cell Culture, Inhibition

Journal: AMB Express

Article Title: Inhibitory effects of H-Ras/Raf-1-binding affibody molecules on synovial cell function

doi: 10.1186/s13568-014-0082-3

Figure Lengend Snippet: Western blot analysis for the confirmation of ERK1/2 phosphorylation. MH7A cells were transfected with plasmids encoding affibodies. After 24 h of culture, the cells were stimulated with TNF-α (100 ng/mL) and cultured for an additional 24-48 h. Each soluble fraction of MH7A cells harboring plasmid-encoded affibody molecules was analyzed via western blot for the expression of p-ERK and actin at 16 and 24 h after TNF-α stimulation. Representative data are shown for p-ERK and actin expression by MH7A cells.

Article Snippet: Percent inhibition was calculated as follows: ([(IL-6 or PGE2 levels in affibody protein-introduced MH7A cells) - (IL-6 or PGE2 levels in MH7A cells introduced with standard peptides)]/([IL-6 or PGE2 levels in MH7A cells introduced with standard peptides]) × 100.

Techniques: Western Blot, Transfection, Cell Culture, Plasmid Preparation, Expressing

Journal: AMB Express

Article Title: Inhibitory effects of H-Ras/Raf-1-binding affibody molecules on synovial cell function

doi: 10.1186/s13568-014-0082-3

Figure Lengend Snippet: IL-6 (A) and PGE2 (B) production by affibody protein-introduced MH7A cells. MH7A cells were transfected with affibody proteins. After 24 h of culture, the cells were stimulated with TNF-α (100 ng/mL) and cultured for an additional 24-48 h. IL-6 and PGE2 levels were analyzed, and percent inhibition of IL-6 or PGE2 production was determined. Percent inhibition was calculated as follows: ([(IL-6 or PGE2 levels in affibody protein-introduced MH7A cells) - (IL-6 or PGE2 levels in MH7A cells introduced with standard peptides)]/([IL-6 or PGE2 levels in MH7A cells introduced with standard peptides]) × 100. Data represent the means × SD of three independent experiments. * p <0.05.

Article Snippet: Percent inhibition was calculated as follows: ([(IL-6 or PGE2 levels in affibody protein-introduced MH7A cells) - (IL-6 or PGE2 levels in MH7A cells introduced with standard peptides)]/([IL-6 or PGE2 levels in MH7A cells introduced with standard peptides]) × 100.

Techniques: Transfection, Cell Culture, Inhibition

Journal: AMB Express

Article Title: Inhibitory effects of H-Ras/Raf-1-binding affibody molecules on synovial cell function

doi: 10.1186/s13568-014-0082-3

Figure Lengend Snippet: Cell proliferation assay for MH7A cells transfected with plasmid-expressed affibody molecules (A) and MH7A cells introduced affibody molecules as protein (B). After 24 h of culture, the cells were stimulated with TNF-α (100 ng/mL) and cultured for an additional 24-48 h. Cell proliferation was analyzed with a CCK assay. Data represent the means × SD of three independent experiments. * p <0.05.

Article Snippet: Percent inhibition was calculated as follows: ([(IL-6 or PGE2 levels in affibody protein-introduced MH7A cells) - (IL-6 or PGE2 levels in MH7A cells introduced with standard peptides)]/([IL-6 or PGE2 levels in MH7A cells introduced with standard peptides]) × 100.

Techniques: Proliferation Assay, Transfection, Plasmid Preparation, Cell Culture