The Pathological Machnism Of The Adipocyte Lipotoxicity Affect The Osteoblasts

BackgroundWith the improvement of people’s living standards, the incidence of obesity, osteoporosis, type II diabetes, insulin resistance and other diseases were increasing. Patients need to require lifelong medication and impair the quality of life. Studies showed that under these pathological conditions, the human body will produce a lot of fat tissue. And the bone marrow cavity can be observed a large accumulation of fat cells, rendering the marrow yellowed. The accumulation of fat cells in the marrow cavity inhibits osteoblast’s proliferation, differentiation and function, having serious effects on the metabolism of bone tissue. Although the proliferation and differentiation of osteoblasts important molecular framework related to the completion of the sequential occurrence has been established, specific molecular biological mechanism of fat cells produce fat toxicity on osteoblasts are still unknown. Free fatty acids may play a variety of inhibition of cell activity. Lipolysis utilize adipocytes, making intracellular lipid droplets down into free fatty acids. In most cases, the increasing free fatty acid level will produce a lot of reactive oxygen species by the mitochondrial fatty acid β-oxidation. The generation of reactive oxygen species can further activate the downstream signaling pathways cascade, impacting on apoptosis, proliferation, differentiation. Therefore, the content of free fatty acids will directly affect the biological activity of the cells.Dexamethasone is one of the most widely used in clinical glucocorticoid drugs. Long-term and large doses use of it can cause severe osteoporosis or bone necrosis. Studies showed that dexamethasone promote adipocyte lipolysis to increase the amount of free fatty acids. Metformin can delay the gastrointestinal absorption rate of glucose, improving insulin sensitivity and inhibiting the hepatic gluconeogenesis. It have a good therapeutic effect for non-insulin-dependent diabetes mellitus, while reducing the body fat level. Metformin inhibits adipocyte lipolysis.MAPKs( mitogen-activated protein kinases, MAPKs) signal transduction pathway are present in the cell of a class of serine / threonine protein kinase. The signal transduction pathway MAPKs were detected in most cells. The signal pathways play a crucial role in the cascade of extracellular stimuli delivered to the cells and the nucleus, and cause biological responses such as cell proliferation, differentiation, and apoptosis in the process. MAPKs signaling pathways play a key role in the process of bone formation. However, due to upstream which can interfere with bone formation almost all relevant molecular pathways(e.g. Ihh-PTHrP, BMP, Wnt-β-catenin, EGF, FGF). Therefore, the stimulation of different external factors can lead to the promotion or inhibition of bone formation occurs in two different results. ROS can activate intracellular MAPKs signal pathway. ObjectiveTo discuss the effects of fat cells to osteoblast proliferation, differentiation and build up the fat cells / osteoblasts co-culture system. To explore different concentrations of dexamethasone, metformin on osteoblast and fat cells proliferation, differentiation and select the appropriate dose concentration for the post-test.To analyze the changes in the level of oxidative stress in the co-culture system. To investigate the relationship between oxidative stress and free fatty acid levels. To determinate the free fatty acids levels and the effects of dexamethasone and metformin to free fatty acids;To explore the main molecular mechanisms for the biological activity of bone cells in the co-culture system. Methods and ResultsEstablishing the adipogenic, osteogenic cells co-culture system to find out the conditions for the latter test.3T3-L1 pre-selection before a fat mouse cell lines and mouse osteoblastic MC3T3-E1 cells were selected. Two cell lines were adipogenic and osteogenic for 12 days respectively. By measuring qRT-PCR for genes related to lipid PPAR-γ, Adiponectin, FABP gene and osteogenic RunX2, Col A1, osteocalcin and staining identification to identify two cell lines have been differentiated into mature adipocytes and osteoblasts, which can be used in follow-up tests and co-culture. Determining the changes in proliferation and differentiation of Osteoblasts in the co-culture system. We found that AD / OB apoptosis of bone composition significantly increased than the control group. Compared with OB / OB group, the cell proliferation in early AD / OB group was inhibited. The ability of bone formation in the latter AD / OB group also significantly reduced, which suggested that adipocytes can inhibit the biological activity of bone cells.Further study found that compared with OB / OB group, AD/OB group in the co-culture system had a significant increased in the FFAs. But the level of PAI-1、MCP-1、leptin、adiponectin、IL-6 did not change significantly. And the difference was not statistically significant.Dexamethasone, metformin can act on adipocytes and osteoblasts respectively and impact on both cell proliferation and differentiation. And there is a dose-response relationship between them. FFAs levels can be affected by dexamethasone and metformin in the co-culture system. Dexamethasone can increase the levels of FFAs. Metformin can inhibit the levels of FFAs. And there is a dose-response relationship between them. Meanwhile the two drugs also influenced the levels of PAI-1, MCP-1, leptin, adiponectin, IL-6 to some extent, but there is no statistical difference. FFAs, Dexamethasone and Metformin were added into the co-culture system.By changing the level of FFAs, we found that FFAs had a significant effect on MC3T3-E1 apoptosis, proliferation and osteogenic differentiation, which suggested that FFAs play an important role in the impact of fat cells to osteoblasts.Detecting the activity of MDA, SOD, GSH-Px and the ROS levels to analyze the changes of oxidative stress levels in the co-culture system. We found that in the AD / OB group the antioxidant levels were significantly lower, and reactive oxygen species levels were significantly higher. After adding FFAs and Dexamethasone can increase the level of active oxygen. Metformin can reduce the level of active oxygen. After adding the active oxygen inhibitors NAC, the osteogenic cell proliferation and osteogenic differentiation in AD / OB group can be improved. We considered that FFAs can increase the level of oxidative stress in osteoblasts, and affect their viability. Western bolt detection found that Akt, Erk, P38 phosphorylation levels in the AD/OB group were significantly increased. The levels were further increased after the addition of FFAs and Dexamethasone. But the levels were inhibited after adding the Metformin. This result suggests that FFAs can influence changes of osteoblasts’ activity by the Akt, Erk, P38 signal pathway. By applying LY294002(Akt signal pathway inhibitor), SB203580(P38 signal pathway inhibitor) and PD98059(Erk signal pathway inhibitor), we found that inhibiting the Erk and P38 signal pathways can significantly reduce the proliferation and the inhibition of differentiation of toxic effects by fat cells in the co-culture system. After adding NAC into the co-culture system, we observed that Akt, Erk, P38 phosphorylation levels were significantly inhibited, which suggested that fat cells inhibited the Osteoblast proliferation and differentiation by FFAs-ROS-P38/erk signal pathway. ConclusionBy studying this topic, we found that adipocytes may inhibit Osteoblast apoptosis, proliferation and differentiation in the co-culture system. And we clarified the molecular mechanisms of this process and the mechanism of FFAs-ROS-ERK/P38 signal pathway in this process. And lay the foundation for further understanding of the relationship between obesity and osteoporosis. Meanwhile in the test group,we also found that Dexamethasone play an important role in promoting the Lipotoxicity. And have a new understanding of the mechanism and etiology of the Glucocorticoid-induced osteoporosis. And the protective effect of Metformin provides a new therapeutic direction for the treatment of glucocorticoid-induced osteoporosis.