RESUMO
OBJECTIVE: Glucocorticoids (GCs)-induced osteoblast apoptosis has been identified as an important cause of GCs related osteonecrosis of the femoral head (ONFH). Glycogen synthase kinase 3ß (GSK3ß) has been proved to mediate dexamethasone (Dex)-induced osteoblast apoptosis. This study aimed to investigate the underlying mechanism of GSK3ß in Dex-induced osteoblast apoptosis. METHODS: Osteoblast cells were transfected with lentivirus expressing GSK3ß-shRNA, and a DNA microarray was performed to analyze gene expression after Dex treatment with or without GSK3ß-shRNA. Some differentially expressed genes were further validated by quantitative real-time-PCR (qRT-PCR). RESULTS: 460 genes were up-regulated (at least 2-fold) with Dex treatment but down-regulated (at least 2-fold) with GSK3ß-shRNA treatment. In addition, 315 genes were down-regulated (at least 2-fold) with Dex treatment but up-regulated (at least 2-fold) with GSK3ß-shRNA treatment. Among these genes, the apoptosis-related genes Hoxb8, Kif18a, Dock8, Dlk1, Tnfsf14, Casq2, Bcl2l14 and mechanosensation-related gene Piezo2 were selected for further qRT-PCR analysis. 7 of 8 genes (Piezo2, Hoxb8, Kif18a, Dlk1, Tnfsf14, Casq2, Bcl2l14) showed the same tendency between gene chip results and qRT-PCR results. The microarray data also showed that apoptotic pathway, MAPK pathway, TGFß pathway and Wnt pathway might be related to the mechanism of GSK3ß in Dex-induced osteoblast apoptosis. CONCLUSION: Our findings indicate that GSK3ß-shRNA treatment can alter various genes expression levels and change diverse signaling pathways involved in Dex-induced osteoblast apoptosis. Furthermore, Piezo2, Hoxb8, Kif18a, Dlk1, Tnfsf14, Casq2 and Bcl2l14 genes may play an important role in the GSK3ß-mediated osteoblast apoptosis process.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/genética , Osteoblastos/efeitos dos fármacos , Animais , Apoptose/genética , Proteínas de Ligação ao Cálcio , Calsequestrina/genética , Calsequestrina/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Mecanotransdução Celular , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismoRESUMO
OBJECTIVE: To evaluate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on preventing rats from glucocorticoid-induced osteonecrosis of femoral head (GCONFH) in the early stage in vivo and to investigate the possible mechanism of hUC-MSCs in regulating the balance of osteogenesis and adipogenesis. METHODS: All rats were randomly divided into 3 groups: control group (C group), model group (M group), and intervention group (I group). The model of GC-ONFH was developed by a sequential administration of lipopolysaccharide and methylprednisolone. The rats in the I group were treated with caudal vein injection of hUC-MSCs. Six weeks later, the blood samples were obtained to measure the activity of alkaline phosphatase (ALP) and the content of triglyceride (TG) in serum, and the femoral heads were harvested and observed by hematoxylin-eosin staining, Micro-CT, Western blot and real-time quantitative polymerase chain reaction. RESULTS: After intervention of hUC-MSCs, the necrosis rate of femoral head decreased from 83% (10/12) to 33% (4/12), the rate of empty bone lacuna was significantly decreased, the activity of ALP increased significantly, the content of TG decreased significantly, the bone density increased obviously, the expression of RUNX2 and Col I increased significantly and the expression of PPARγ decreased significantly. CONCLUSION: These results revealed that caudal vein injection of hUC-MSCs can effectively reduce the incidence of GC-ONFH in rats by increasing ALP activity and reducing TG content in serum, increasing bone mineral density, promoting the expression of RUNX2 and Col I, and inhibiting the expression of PPARγ.
Assuntos
Necrose da Cabeça do Fêmur/terapia , Glucocorticoides/efeitos adversos , Transplante de Células-Tronco Mesenquimais/métodos , Cordão Umbilical/citologia , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/metabolismo , Necrose da Cabeça do Fêmur/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , PPAR gama/genética , PPAR gama/metabolismo , Distribuição Aleatória , Ratos , Resultado do Tratamento , Triglicerídeos/sangue , Regulação para CimaRESUMO
Nowadays, the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head (ONFH). Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level. Glycogen synthase kinase 3ß (GSK3ß) is an important regulator of cellular differentiation and apoptosis pathway, which can modulate the balance between osteoblasts and osteoclasts. Several studies have reported about its function in osteoporosis, but little is known about it in osteonecrosis. In our study, lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model. The phosphorylation of GSK3ß Ser-9 was decreased in the model. Western blotting examination of ß-catenin, Bcl-2, Bax and caspase-3 revealed that the osteoblasts were apoptotic. In dexamethasone (Dex)-incubated primary osteoblasts, the expression profile of GSK3ß phosphorylation and apoptotic factors were consistent with those in the rat ONFH model. To further investigate the regulation of osteonecrosis caused by GSK3ß, the expression and function of GSK3ß were inhibited in Dex-incubated primary osteoblasts. The knockdown of GSK3ß by siRNA decreased the expression of Bax and cleaved caspase-3, but increased Bcl-2 and ß-catenin. On the other hand, selective inhibition of GSK3ß function by LiCl counteracted the activation of caspase-3 induced by Dex. Our work is the first study about the GSK3ß phosphorylation in ONFH, and provides evidence for further therapeutic methods.