RESUMO
Anti-TNF-α therapy fails in 30% of patients, where TNF-α may not be the key causative factor in these patients. We developed a bispecific single-domain antibody block TNF-α and VEGF (V5-3).The experiments showed that V5-3 effectively activated proliferation and migration of RA-FLS and HUVEC, tube-forming role of HUVEC, and expression of inflammatory factors in vitro. Besides, the experiments indicated that the anti-RA activity of V5-3 was superior to Anbainuo in vivo. Application of V5-3 reduced the expression of inflammatory factors, extent of synovial inflammation and angiogenesis and attenuated the severity of autoimmune arthritis in collagen-induced arthritis (CIA) mice. Mechanistically, V5-3 suppressed p65, AKT and VEGFR2 phosphorylation, as well as production of TNF-α and VEGF in joint tissues. These results demonstrated that V5-3 displayed a superior effect of anti-RA, may be a new therapy to overcome the limitations of anti-TNF-α monoclonal antibody.
Assuntos
Artrite Experimental , Artrite Reumatoide , Humanos , Camundongos , Animais , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores do Fator de Necrose Tumoral/farmacologia , Inflamação/metabolismo , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Fibroblastos , Membrana Sinovial , Fragmentos Fc das Imunoglobulinas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Receptores Tipo II do Fator de Necrose TumoralRESUMO
Long non-coding RNA (lncRNA) is involved in the regulation of rheumatoid arthritis (RA) and many other diseases. In this study, a new lncRNA, NR-133666, was identified to be highly expressed in the adjuvant-induced arthritis rat model using the Agilent lncRNA microarray assay. qRT-PCR verified that NR-133666 was upregulated in fibroblast-like synoviocyte of a collagen-induced arthritis (CIA) rat model. Fluorescence in situ hybridization analysis showed that NR-133666 is mainly expressed in the cytoplasm of collagen-induced arthritis FLS. MTT assay and EdU staining results showed that the proliferation of CIA FLS was inhibited after NR-133666 was knocked down, and the wound healing assay showed that the migration of CIA FLS was also suppressed. Dual luciferase detection was used to confirm the relationship among NR-133666, miR-133c and MAPK1. MAPK1 is the target gene of miR-133c, where NR-133666 acts as a sponge of miR-133c to reduce the inhibitory effect of miR-133c on MAPK1. Overexpression of NR-133666 and MAPK1 can promote the proliferation and migration of CIA FLS, and overexpression of miR-133c can reverse this phenomenon. Western blot indicated that it may be related to the ERK/MAPK signaling pathway. Collectively, we identified that lncRNA NR-133666 acted as a miR-133c sponge that can promote the proliferation and migration of CIA FLS through regulating the miR-133c/MAPK1 axis.
