Effects Of High Glucose Concentration On Proliferation Of RAW264.7 Cells And RANKL-induced Osteoclastogenesis

Part 1:The effects of high glucose on mouse monocytes(RAW264.7)Objective:To investigate the effects of different concentrations of glucose on the proliferation,apoptosis and cell cycle of RAW264.7(osteoclast precursor cells)cultured in vitro.Method:1.RAW264.7 cells were selected and divided into five groups,cultured with different concentrations of glucose(5.5,11,22,33,44mM),Glc 5.5mM group as Control group.To examine the following indexes:(1)Cell proliferation(EDU assay and MTT method).(2)The cell cycle and cell apoptosis rate(flow cytometry).(3)The level of ROS(fluorescence microscopy).2.RAW264.7 cells were selected and divided into four groups,set the normal CON group(Glc 5.5mM),high glucose HG group(Glc 22mM),and the addition of antioxidant NAC(5mM)treatment of the HG+NAC group and CON+NAC group.To examine the following indexes:(1)cell proliferation(MTT method).(2)the level of ROS(fluorescence microscopy).(3)The expression level of cyclin D1 protein(Western blot).Result:1.(1)The results of EdU assay and MTT method showed that high glucose(22mM)can promote the proliferation of RAW264.7 cells compared with the Control group significantly(P<0.01).(2)Cell cycle analysis indicated that Glc 22 mM group mainly for the percentage of cells in G1 phase were decreased,the proportion of cells in S phase were increased,the synthesis of DNA were increased compared with Control group(P<0.01).(3)Cell apoptosis detection results showed that the apoptosis rate of RAW264.7 were increased with the increase of glucose concentration,Glc 33 mM group and Glc 44 mM group were significant compared with the Control group(P<0.01).(4)The ROS test results showed that the level of ROSincreased gradually with the elevated of glucose concentration.2.(1)The results showed that after the addition of NAC treatment,the level of cells proliferation of HG+NAC group obviously lower than HG group(P<0.01).(2)Western blot assay found the expression levels of cyclin D1 in HG+NAC group were lower than HG group(P<0.01).(3)ROS results showed that the levels of ROS in HG+NAC group were lower than HG group significantly(P<0.01),NAC can reduce the increased of ROS levels induced by high glucose.Conclusion:High glucose(22mM)can significantly promote the proliferation of RAW264.7(osteoclast precursor cells),and its mechanism may be related to the oxidative stress induced by high glucose.Part 2:The effects of high glucose on the proliferation and differentiation of osteoclast induced by RANKLObjective:The effects of different concentrations of glucose on the proliferation and differentiation of osteoclast cells induced by RANKL.Method:RAW264.7 cells were divided into six groups,five group were added 100ng/ml of RANKL induced and a vehicle group without RANKL,cultured with different concentrations of glucose(5.5,11,22,33,44mM),Glc 5.5mm group was the control group.To examine the following indexes:(1)Cell proliferation level(MTT method).(2)The assay of positive mature osteoclast cells(TRAP).(3)The assay of bone resorption area(OAAS).(4)The expression level of cyclin D1 protein(Western blot).Result:(1)MTT assay showed that proliferation Glc 22 mM can promote the proliferation of osteoclast cells significantly compared with the control group(P<0.01).(2)Western blot results also show that Glc 22 mM group can increase the expression level of Cyclin D1 protein significantly compared with the control group(P<0.01).(3)TRAP assay staining showed that Glc 22 mM can increase the number of trap positive osteoclast cells significantly compared with the control group(P<0.01).(4)OAAS staining results show that Glc 22 mM can enlarge the bone resorption area of osteoclast cells significantly compared with the control group(P<0.01).Conclusion:High glucose(22mM)can significantly promote the proliferation and differentiation of osteoclast induced by RANKL,which may be one of the important pathogenesis of diabetic osteoporosis.