RESUMO
The DLTIDDSYWYRI motif (Ln2-P3) of human laminin-2 has been reported to promote PC12 cell attachment through syndecan-1; however, the in vivo effects of Ln2-P3 have not been studied. In Schwann cells differentiated from skin-derived precursors, the peptide was effective in promoting cell attachment and spreading in vitro. To examine the effects of Ln2-P3 in peripheral nerve regeneration in vivo, we developed a dual-component poly(p-dioxanone) (PPD)/poly(lactic-co-glycolic acid) (PLGA) artificial nerve graft. The novel graft was coated with scrambled peptide or Ln2-P3 and used to bridge a 10 mm defect in rat sciatic nerves. The dual-component nerve grafts provided tensile strength comparable to that of a real rat nerve trunk. The Ln2-P3-treated grafts promoted early-stage peripheral nerve regeneration by enhancing the nerve regeneration rate and significantly increased the myelinated fibre density compared with scrambled peptide-treated controls. These findings indicate that Ln2-P3, combined with tissue-engineering scaffolds, has potential biomedical applications in peripheral nerve injury repair.
Assuntos
Laminina/química , Regeneração Nervosa/efeitos dos fármacos , Próteses Neurais , Peptídeos/farmacologia , Nervos Periféricos/fisiopatologia , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Dioxanos/química , Humanos , Imuno-Histoquímica , Implantes Experimentais , Inflamação/patologia , Ácido Láctico/química , Laminina/farmacologia , Masculino , Dados de Sequência Molecular , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Células PC12 , Peptídeos/química , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/patologia , Nervos Periféricos/ultraestrutura , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Ratos , Ratos Sprague-Dawley , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Resistência à Tração/efeitos dos fármacosRESUMO
AIM: Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs). METHODS: Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed. RESULTS: Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage. CONCLUSION: These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.
