RESUMO
Puerarin has attracted increasing attention because of its beneficial effects on antiosteoporosis, but the molecular mechanisms underlying its actions on osteoblasts are not fully understood. The current study aimed to investigate the effect of puerarin on the cell viability and differentiation of mouse MC3T3E1 osteoblastlike cells in vitro and its underlying mechanisms. The results indicated that 0.01, 0.1 and 1 mg/ml puerarin significantly promoted the viability of osteoblasts, enhanced alkaline phosphatase (ALP) activity and increased the expression of transforming growth factorß1, Smad2, Smad3 and Runtrelated transcription factor (Runx)2. Micro (mi)RNA target prediction programs predicted that miR204 may directly target Runx2. Following treatment with 0.1 mg/ml puerarin for 48 h, the expression level of miR204 was downregulated. Besides, miR204 dramatically repressed the luciferase activity of wildtype Runx2 3'UTR transfected cells, but not that of the mutant ones. Overexpression of miR204 in osteoblasts significantly decreased the protein expression of Runx2, while inhibition of miR204 enhanced Runx2's expression. In addition, overexpression of miR204 inhibited the cell viability and ALP activity of osteoblasts, while inhibition of miR204 had the opposite effect. The results suggested that puerarin may promote MC3T3E1 osteoblastlike cell viability and differentiation, which may be related to the downregulation of miR204 and the following activation of Runx2.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Isoflavonas/farmacologia , MicroRNAs/genética , Regulação para Cima/efeitos dos fármacos , Regiões 3' não Traduzidas/efeitos dos fármacos , Fosfatase Alcalina/genética , Animais , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Proteína Smad3/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.