RESUMEN
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.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Humanos , Ratones , Animales , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inhibidores del Factor de Necrosis Tumoral/farmacología , Inflamación/metabolismo , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Fibroblastos , Membrana Sinovial , Fragmentos Fc de Inmunoglobulinas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Receptores Tipo II del Factor de Necrosis TumoralRESUMEN
The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signalling that cause collateral damage to protective signalling cascades. The single domain chain firstly discovered in Camelidae displays fully functional ability in antigen-binding against variable targets, which has been seemed as attractive candidates for the next-generation biologic drug study. In this study, we established a simple prokaryotic expression system for a dual target-directed single domain-based fusion protein against the interleukin-6 receptor and human serum, albumin, the recombinant anti-IL-6R fusion protein (VHH-0031). VHH-0031 exhibited potent anti-inflammatory effects produced by LPS on cell RAW264.7, where the major cytokines and NO production were downregulated after 24 h incubation with VHH-0031 in a dose-dependent manner. In vivo, VHH-0031 presented significant effects on the degree reduction of joint swelling in the adjuvant-induced arthritis (AIA) rat, having a healthier appearance compared with the dexamethasone. The expression level of JNK protein in the VHH-0031 group was significantly decreased, demonstrating that VHH-0031 provides a low-cost and desirable effect in the treatment of more widely patients.
Asunto(s)
Antiinflamatorios/inmunología , Artritis Experimental/tratamiento farmacológico , Interleucina-6/antagonistas & inhibidores , Albúmina Sérica Humana/antagonistas & inhibidores , Anticuerpos de Dominio Único/inmunología , Animales , Antiinflamatorios/uso terapéutico , Especificidad de Anticuerpos , Artritis Experimental/inmunología , Citocinas/metabolismo , ADN Complementario/genética , Dexametasona/uso terapéutico , Evaluación Preclínica de Medicamentos , Inducción Enzimática/efectos de los fármacos , Humanos , Interleucina-6/inmunología , Lipopolisacáridos/toxicidad , MAP Quinasa Quinasa 4/biosíntesis , MAP Quinasa Quinasa 4/genética , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Óxido Nítrico/metabolismo , Conformación Proteica , Células RAW 264.7 , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Albúmina Sérica Humana/inmunología , Anticuerpos de Dominio Único/genéticaRESUMEN
Objective To determine the effect of ropivacaine on peripheral neuropathy in diabetic rats and its possible mechanism. Methods Forty-eight Sprague-Dawley rats were randomly divided into six groups: nondiabetic control group, nondiabetic group A (0.25% ropivacaine), nondiabetic group B (0.75% ropivacaine), diabetic control group (diabetic peripheral neuropathy (DPN) +artificial cerebrospinal fluid), diabetic group A (DPN+0.25% ropivacaine), and diabetic group B (DPN + 0.75% ropivacaine), with eight rats in each group. Within an hour of the last administration, the sciatic motor nerve conduction velocity (MNCV) of each group was measured, and the morphological changes of rat sciatic nerve were observed by HE, Weil's staining and electron microscopy. The expression of transient receptor potential vanilloid (TRPV1) in the spinal cord dorsal horn of rats was analyzed by immunohistochemistry, and the expression of Calcitonin gene-related peptide (CGRP) protein in the spinal cord was analyzed by Western blot. Results Compared with the nondiabetic control group, elevated blood glucose, decreased weight and reduced average mechanical withdrawal threshold (MWT), additionally, the sciatic nerves showed significantly slowed conduction velocity (both P<0.001) and damaged pathological structure, the expression of TRPV1 and CGRP were decreased (both P<0.001) in the diabetic groups. Compared with the diabetic control group, down-regulation of TRPV1 and CGRP in spinal cord was significant for the diabetic groups A and B treated with 0.25 and 0.75% ropivacaine, the higher concentration of ropivacaine correlated with a greater change. Conclusion Ropivacaine can significantly block sciatic nerve conduction velocity in DPN rats in a concentration-dependent manner, which may be related to the expression of the TRPV1-CGRP pathway.
