Therapeutic Effect of a Recombinant betaig-h3 Fragment-RGD Peptide for Chronic Inflammatory Arthritis

OBJECTIVE: betaig-h3 is a 68kDa extracellular matrix protein which is overexpressed in synovial tissues of rheumatoid arthritis (RA). Previous results proved that betaig-h3 fragments are relevant to adhesion and migration of synovial fibroblast and angiogenesis through interaction with alphavbeta 3 integrin. We designed a recombinant betaig-h3 protein consisting of a fas-1 domain and RGD motif and evaluated the therapeutic efficacy in RA. METHODS: Inhibitory effect of adhesion and migration of NIH3T3 cell line was evaluated in 96 well microtiter and transwell plates coated with betaig-h3. Clinical arthritis index was evaluated after treating CIA mice with MFK12. Immunohistochemical staining in synovial tissues were performed. Expression of transcripts and proteins of inflammatory mediators were analyzed by semi-quantitative RT-PCR and immunoblotting. RESULTS: Recombinant protein consisted of 4th fas-1 domain truncated for H1 and H2 sequences and RGD peptide (MFK12), had M.W. of 10.4kDa. betaig-h3 mediated adhesion and migration of NIH3T3 cell line were significantly inhibited in a dose-dependent manner. Arthritis severity and incidence were efficiently reduced when CIA mice were treated with MFK12 at 30 mg/kg/day compared with the control. Immunohistochemical staining of joint tissues in MFK12 treated mice exhibited reduced angiogenesis. In treated mice, expression of transcripts regarding inflammatory mediators was markedly suppressed and immunoblotting of ICAM-1 and RANKL from whole extract of hind paws also showed a significant reduction. CONCLUSION: This study shows that MFK12 is effective in treating RA, although further study is warranted to improve the therapeutic efficacy.

beta ig-h3-Mediated Adhesion of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVE: beta ig-h3 is an extracellular matrix protein, which is overexpressed in synovial tissues of rheumatoid arthritis (RA) similar to adhesive glycoproteins. We sought to evaluate the compensatory role of beta ig-h3 with adhesive glycoproteins in mediating the adhesion of fibroblast- like synoviocytes (FLS) and to confirm the inhibitory effect of YH18 peptide of the 2nd fas-1 domain in beta ig-h3-mediated adhesion. METHODS: The adhesion of FLS isolated from synovial tissues of RA, was evaluated in 96 well microtiter plate coated with matrix proteins. Inhibitory effect of YH18 peptides from the 2nd and 4th fas-1 domains was estimated in beta ig-h3-mediated adhesion of FLS. RESULTS: The adhesion of FLS on beta ig-h3 was weaker than that of fibronectin and vitronectin. The beta ig-h3-mediated adhesion was enhanced by the stimulation with phorbol myristate acetate (PMA), but not by cytokines and growth factors. Combination of fibronectin with beta ig-h3 synergistically enhanced the adhesion of FLS, in contrast to the additive effect of vitronectin combined with beta ig-h3. YH18 peptide of the 2nd fas-1 domain did not block the beta ig-h3-mediated adhesion of FLS. CONCLUSION: Our results reveal that beta ig-h3 may regulate the adhesion of FLS through the interaction with adhesive glycoproteins and confirm that the essential motifs mediating adhesion on beta ig-h3 are different according to the type of cells.

Cinobufacini induces the apoptosis of U937 cells and its mechanism / 肿瘤

Objective: To investigate the effect of cinobufacini on apoptosis of U937 cells and its possible mechanism. Methods: Cell viability was measured by MTT assay. The morphological changes were observed by Wrightś staining and immunofluorescence staining. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptotic rate was evaluated by teminal deoxynucleotidyl transferase (TdT) labeling in situ. Expression of bel-2 protein was analyzed by flow cytometry. The levels of Fas and Fas-1 mRNA were measured by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results: Compared with the control group, treatment with cinobufacini at 0.225 to 1.8 μg/ mL for 1-3 d significantly inhibited growth of U937 cells in a time and dose dependent manner. The IC50 value was 1.36 μg/ mL at 24 h. The typical morphological changes of apoptosis and typical apoptotic DNA ladder were observed after incubation with cinobufacini at 0.9 μg/mL for 1 d. The apoptotic rate evaluated by TdT labeling in situ were 4.8%, 13.57%, and 24.33% after exposure to cinobufacini at 0.225, 0.45, and 0.9 μg/ mL for 1 d, respectively. The expression levels of bcl-2 protein and Fas-1 mRNA significantly decreased and the expression of Fas mRNA significantly increased in apoptotic cells. Conclusions: Cinobufacini inhibits growth and inducs apoptosis of U937 cells via inhibition of bcl-2 and Fas-1 genes and activation of Fas gene.