Study On The Effect Of High Glucose On Osteoclastic Differentiation Of RAW264.7 Cells Induced By RANKL And Its Mechanism

Part one:Effects of high glucose on proliferation and osteoclast differentiation of RAW264.7 cells induced by RANKLObjective:This paper used murine monocyte-macrophage RAW264.7 cells to differentiation into mature osteoclasts induced by RANKL and investigate the effects of different concentration of glucose on osteoclast induced by RANKL.Method:RAW264.7 cells were cultured in vitro and cultured with 50ng/ml RANKL inducing medium to induce the cells for 6d as follows: blank control group(no RANKL),control group(glucose 5.5mmol/l + RANKL),experimental group(at different concentrations glucose 11,22,33,44mmol/l+RANKL).Cell viability was detected by MTT assay and the expression level of Cyclin D1 protein was detected by Western blot method;TRAP staining and OAAS method were used to observe the formation of TRAP+ cells and the size of the bone resorption in osteoclasts.Result:MTT results showed that compared with control group,high glucose(22m M)can promote the proliferation of osteoclast cells obviously(P<0.01);Western Blot results showed that compared with control group,high glucose(22m M)can promote the expression of Cyclin D1 of osteoclast cells obviously(P<0.01);TRAP staining results showed that compared with control group,high glucose group inhibited osteoclast differentiation in a dose dependent manner(P<0.01);the results of OAAS showd that compared with the control group,high glucose group inhibited osteoclastic bone resorption in a dose dependent manner(P<0.01).Conclusion:High glucose(22 m M)significantly can promote the proliferation of RAW264.7cells induced by RANKL,while high glucose inhibited its osteoclastogenesis in a dose-dependent manner.Part two:Explore the mechanism of high glucose on osteoclastic differentiation of RAW264.7 cells induced by RANKLObjective:This paper used murine monocyte-macrophage RAW264.7 cells to differentiation into mature osteoclasts induced by RANKL and explored the mechanism of glucose on the formation of osteoclast induced by RANKL.Method:1.RAW264.7 cells were cultured at different concentrations of glucose without RANKL or not in vitro for 24h: the expression level of PI-3K and p-Akt/Akt were detected by Western blot method.2.RAW264.7 cells were cultured in vitro for 6d,to determine whether the effects of by hypertonic,when high glucose(22m M)can promote the proliferation of osteoclast induced by RANKL significantly,that were decreased in glucose(33,44 m M):cell viability was detected by MTT method and the expression level of Cyclin D1 was deteced by Western blot method.3.RAW264.7 cells were cultured with RANKL in vitro for 24 h,to explore the role of oxidative stress in the formation of osteoclasts induced by RANKL: the level of reactive oxygen was detected by fluorescence microscopy;the contents of CAT,GPX,SOD,MDA of oxidative stress were detected.Result:1.The results of Western Blot showed that compared with control group,the expression levels of PI-3K and p-AKt protein in the high glucose group were increased in a dose-dependent manner,but the rate of increase was higher in the 44 mmol / L glucose group((P<0.01).2.In order to to determine whether the impact of by hypertonic,when high glucose(22m M)can promote the proliferation of osteoclast induced by RANKL significantly,that were decreased in glucose(33,44 m M).The results showed that the proliferation of osteoclasts was significantly higher in the high glucose(22m M)group than that in the control group(P<0.01).There was no significant difference with different high glucose and hyperosmotic concentrations of 22 m M and 33 m M in the proliferation of the cells(P>0.05).Compared with the control group,thehyperosmolar group(11,22,33,44 m M)inhibited the proliferation of osteoclasts and the expression level of Cyclin D1 protein in a dose-dependent manner(P<0.01).Thus,hypertonic inhibited cell proliferation by inhibiting of expression of Cyclin D1 of cell in a dose-dependent manner,and different concentrations of glucose promoted cell proliferation through increasing expression of PI-3K and p-AKt in a dose-dependent manner,the composition of different high glucose and hypertonic did not affect cell proliferation,that suggesting high glucose(22 m M)promoted osteoclast proliferation and decreased at 33 and 44 m M,which is related to hyperosmolar.3.The results of ROS showed that compared with control group,the levels of ROS in osteoclasts were significantly decreased in glucose(11,22,33,44 m M)(P<0.01).This indicated that high glucose could significantly inhibit levels of reactive oxygen species in osteoclast induced by RANKL.4.To observe the effects of pretreatment with high glucose(22m M)on CAT,GPX,SOD and MDA in osteoclasts before and after induction: RAW264.7 cells were cultured in vitro,compared with vehicle group,the levels of CAT,GPX and SOD were significantly increased and the levels of MDA were significantly decreased in RANKL group and high glucose(22m M)group(P<0.01).Compared with the control group,when the high glucose(22m M)and RANKL combined on RAW264.7 cells,the levels of CAT,GPX and SOD were significantly increased,and the levels of MDA were significantly decreased(P<0.01).5.To observe the effects of different high glucose pretreatment on CAT and MDA in osteoclasts induced by RANKL: Compared with control group,the contents of CAT were significantly increased and the levels of MDA were significantly decreased in 11,22,33 and 44 m M groups(P<0.01),but the levels of GPX and SOD did not change significantly.Conclusion:1.High glucose can promote the proliferation of RAW264.7 cells induced by RANKL,and its mechanism is related to up-regulation of PI3 K / Akt pathway,which is related to hyperosmolar.2.High glucose inhibits the differentiation and bone resorption of RAW264.7cells induced by RANKL,and its mechanism may be related to the inhibition ofoxidative stress.