| Osteoporosis (OP) is a disease characterized by a decrease in bone mass and a deterioration in bone microarchitecture, which leads to an enhanced fragility of the skeleton and a greater risk of fracture. Previous study found that the prevalence of osteoporosis in China was range from 6.6% to 19.3%. China has entered the aging society with an increase incidence of osteoporosis and aggravating symptoms. If unsolved, osteoporosis will bring serious consequences for the medical treatment, society and economy. Therefore, the prevention and treatment of osteoporosis has become one of the most important health problems in the world. It is important to study the pathogenesis and mechanism of osteoporosis and to develop new drugs to prevent and treat the disease. Selecting and establishing appropriate animal model and cell model are critical to study the pathogenesis of osteoporosis.Human differentiated embryonic chondrocyte expressed gene 1 (DEC1), as a growth and development related gene, was isolated from human cartilage cells in 997 years It is involved in a number of physiological processes including chondrogenesis, neurogenesis, immune response, circadian rhythms, lipogenesis, and carcinogensis. However, DEC1 plays a role in the pathogenesis of osteoporosis and the effect of DEC1 on the function of osteoblasts has not been reported.In this study, the function of DEC1 on the activity of osteoblasts is studied in vivo and in vitro, and the possible mechanism of DEC1 is also discussed. In the first part of this paper, we first construct the osteoporosis model mice with two different ways, the one is bilateral oophorectomy, and the other is prednisolone subcutaneous implantation. We found that the expression of DEC1 was significantly decreased in the bone marrow cavity of the osteoporosis model mice compared to the sham operated mice. When we construct the DEC1 gene knockout mice, we found that there was an obvious decrease in bone mass compared to the wild type mice. Based on the results above, we speculate there is a close relationship between DEC1 gene and skeletal development. We next carry out the second part of the work. We establish the glucocorticoids induced osteoporosis cell model, and icariin is given to the cells as a positive control. We found that high protein expression of DEC1 stimulates the osteogenisis. In addition, DEC1 overexpression and knockdown experiments in SaoS-2 cells confirm the results that DEC1 directly regulates osteoblastic differentiation. When the molecular mechanism is examined, we find that DECl regulates the activity of osteoblasts through the PI3K/Akt/GSK3β/β-catenin pathway.Part I in vitro:The effects of DEC1 in two different osteoporosis mice models and the bone phenotype in DEC1 gene deficient miceObjective:The prevention and treatment of osteoporosis has become one of the world’s major health problems, because of the increase incidence of osteoporosis and the aggravating symptoms. Previous studies have shown that DEC1 promotes cartilage cell growth and differentiation. In this part, we explores the expression pattern of DEC1 in osteoporosis mice, in order to broaden the horizon of DEC1 in the field of bone metabolism, to reveal the pathogenesis of osteoporosis in new ways, to provide new ideas for the treatment of osteoporosis in clinic.Methods:(1) We constructed two different osteoporosis mice model:A.8 week old female mice with bilateral oophorectomy to imitate postmenopausal osteoporosis; B. 8 week old male mice with subcutaneous implantion of 5mg,60d prednisolone sustained-release pellets to imitate glucocorticoids induced osteoporosis. Different models of mice were given a small dose of estradiol (Estradiol, E2) as replacement therapy, or the treatment of the drug icariin. After the model was successfully executed, the mice were sacrificed to detect the morphology, immunology and micro-CT. (2) DEC1 gene deficiency mice were constructed to detect the changes of bone mass in the limbs.Results:(1) In both of the osteoporosis model mice, the protein expression of DEC1 in the bone marrow cavity was decreased compared to the sham operation group, (p<0.05). (2) Compared to the osteoporosis group, the protein expression of DEC1 was increased in the treatment group (p<0.05). (3) Compared to the wild type mice, DEC1 gene deficiency mice showed typical osteoporosis phenotype (p<0.05).Conclusion:The decrease of DEC1 expression is related to the incidence of osteoporosis in mice.Part II in vitro:DEC1 promotes osteogenesis via PI3K/Akt/GSK3β/β-catenin signaling pathwayObjective:In the first part of this paper, the data show that the reduction of DEC1 in the animal models is directly related to the decrease of bone mass. The second part of the content verifies the effect of glucocorticoids on the osteogenic activity in the cell level, and studies the role of DEC1 interacting with glucocorticoids. We are in order to clarify the role of DEC 1 in osteogenic differentiation in the molecular level.Methods:(1) We treated the osteoblast like cell lines SaoS-2 with high dose of dexamethasone (Dexamethasone, DEX) to inhibite the osteogenic capacity of osteoblasts. The different bone phenotype markers and the protein level of DEC1 were detected; icariin treated the model cells, and the relevant indicators were in the detection. (2) Saos-2 cell were treated with dexamethasone, and the impotant signaling β-catenin and its upstream signaling were detected. (3) Overexpression of DEC1 in Saos-2 cells, bone phenotype markers and the related signal pathway were detected. (4) Knockdown of DEC 1 in SaoS-2 cells, bone phenotype markers and the related signal pathway were detected.Results:(1) Dexamethasone disturbs osteogenic differentiation in SaoS-2 cells characterized by the declined alkaline phosphatase activity, the decreased alkaline phosphatase staining intensities, the reduced Runx2 protein expression and the diminished extracellular matrix mineralization. At the same time, DEC1 decreased along with dexamethasone in a dose dependent manner (P<0.05). (2) Icarrin protects against the dexamethasone induced osteoporosis by ameliorating the decreased osteoblastic phenotypes and the loss of DEC1 induced by dexamethasone was also abrogated by icariin (p<0.05). (3) Dexamethasone inhibited the protein expression and nuclear translocation of β-catenin, and there was a positive correlation between the expression of DEC1 and P-catenin in the nucleus (p<0.05). (4) Overexpression of DEC1 promotes osteogenic differentiation of SaoS-2 cells. Overexpression of DEC1 stimulated P-catenin translation into the nucleus and activated its upstreamfactors, such as PIK3CA, Akt, and GSK3β (p<0.05). (5) DEC1 knockdown inhibited the expression of osteogenic phenotype, the activation of β-catenin, and its upstream signals (p<0.05).Results:DEC1 is an important regulator in osteogenic differentiation, and DEC1 realizes its function through PIK3CA/Akt/GSK3β/β-catenin signaling pathway. |
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