Effects Of Aspirin On Proliferation, Differentiation, Mineralization Of MC3T3 Cells And Its Possible Mechanism

Background: Osteoporosis is a chronic disease, defined as a skeletal disorder characterized by decreases in bone mass and microarchitectural alterations which result in bone fragility and increased risk of fractures. The morbidity is higher in postmenopausal women and older men. So far, the pathogenesis of osteoporosis is still unclear. Aspirin as an antipyretic analgesic and antiplatelet drug is widely used in clinical, recently, many studies suggest that aspirin has the anti-osteoporosis effect, but the specific mechanism is unclear. OPG/RANKL/RANK signaling pathway plays an important role in coupling osteoblasts and osteoclasts, which lead to be thought this pathway as a crucial process of osteoporosis.Objective: To study the effects of Aspirin on proliferation, differentiation, mineralization of MC3T3 cells and explore the possible mechanism of aspirin working on MC3T3 cells through OPG/RANKL/RANK signaling pathways.Methods: Cultivate MC3T3 cells by using different concentrations of aspirin induced liquid culture(0, 0.25, 0.5, 1, 1.5, 2, 5, 10mmol/L), then, 1. MTT experiment should be conducted to test the influence on proliferation of MC3T3 cells in different concentration of aspirin; 2. Alkaline phosphatase staining and quantitative analysis should be tested to show the contribution of different concentrations of aspirin for bone differentiation; 3. Results of alizarin red staining reflect mineralization of different concentrations of aspirin; 4. Western Blot and Real-time PCR technologies are used to explore the possible mechanism of aspirin on MC3T3 cells.Results: 1. The results of MTT experiments show that the OD values of low concentration of aspirin(0.25, 0.5, 1, 1.5, 2mmol/L) groups have no difference than the control group(P>0.05), while the OD values of high concentration of aspirin(5,10 mmol/L) groups are significantly lower than the control group(P<0.01); 2. The result of alkaline phosphatase staining shows the group of 1.5mmol/L aspirin has much more positive particles than other experimental groups; 3. The quantitative analysis of alkaline phosphatase indicates that the content of alkaline phosphatase in each concentration of aspirin group(0.5, 0.5, 1, 2, 3mmol/L) is more than the control group(P<0.01), the increase of 1.5mmol/L Aspirin group is most significantly(P<0.01); 4. Alizarin red staining showed groups of 1.5mmol/L Aspirin accelerated calcium nodule than other drug groups and the control group, the increase of 1.5mmol/L Aspirin group is most significantly(P<0.01); 5. The results of Western Blot show that the content of OPG protein, Col1 a protein in 1.5mmol/L Aspirin group are much more than the control group(P<0.01), the content of RANKL protein is lower than the contol group(P<0.01); 6. The results of Real-time PCR experiment suggest 1.5mmol/L aspirin group promote the content of OPGmRNA and decrease the level of RANKLmRNA(P<0.01).Conclusions: 1. Each low concentration of aspirin has no effect on MC3T3 cell proliferation, while high concentrations of aspirin inhibit proliferation of MC3T3 cells; 2. The osteogenetic effects and mineralization of 1.5mmol/L aspirin group is greater than other drug groups and control group significant; 3. Aspirin can stimulate the OPG expression and suppress RANKL expression to prevent osteoporosis through OPG/RANKL/RANK signaling pathways.