The Effects Of HU-308 On The Biological Properties Of MC3T3-E1 Osteoblasts Under High Glucocorticoids Concentrations

Objective:Glucocorticoids(GC)are widely used in clinical practice because of their anti-immunity,anti-inflammatory,anti-toxin,anti-shock and other effects,but there are also many side effects.For example,osteoporosis caused by long-term use can affect implants.The surrounding bone is combined.Dexamethasone is currently the most commonly used glucocorticoid in the clinic.Studies have shown that dexamethasone can inhibit osteoblast proliferation at concentrations of 10-6 mmol/L and 10-7 mmol/L.HU-308 is a CB2 cannabinoid receptor agonist.In recent years,scholars have found that HU-308 can affect bone formation,increase bone mass and maintain bone mass,thereby promoting bone union around the implant.However,its influence on the biological properties of osteoblasts remains controversial.Therefore,this study investigated the effect of HU-308 on the biological properties of osteoblasts under the action of high concentration of glucocorticoids(ie dexamethasone),and explored the possible mechanism of HU-308 enhancing the osseointegration of implants in patients with long-term glucocorticoids.A new method has been provided to improve the success rate of long-term glucocorticoid patients.Method:MC3T3-E1 cells were cultured in the high concentration of glucocorticoid(10-6 mol/L)and then added the HU-308.Cells in concentration of 10-10,10-9,10-8,10-7 and 10-6 mol/L of HU-308 were the experimental groups,and that in only glucocorticoid as the control group.The CCK-8 used to detect cell proliferation,ALP activity and mRNA expression of Runx2 and OPG.The morphology of the cytoskeleton was observed by confocal laser scanning microscope when the MC3T3-E1 cells were cultured for 24hResults:1.The results of CCK-8 showed that there were statistically significant between the control group and the experimental groups(P<0.05).OD values in different concentrations between the experimental group compared with no statistical significance in 1 d and 7 d(P>0.05),but the cell proliferation were effetely promoted in the experimental group(10-9,10-8,10-7 and 10-6 mol/L)in 4 d(P<0.05).2.The activity of alkaline phosphatase(ALP)showed that the activity of ALP in MC3T3-E1 cells increased with the increase of cell culture time,the difference was statistically significant(P<0.05);1d,4d,7d after incubation In the control group and the experimental group,there was no significant difference between the groups at the same time point(P>0.05).3.Alizarin red staining showed that the number of stained nodules in the control group was small.In the experimental group(HU-308 concentrations of 10-10 mol/L,10-9 mol/L,10-8 mol/L,10-7 mol/L,10-6 mol/L)compared with the control group,The number of stained nodules increases and the volume increases.However,there was no significant difference in the stained nodules between the experimental groups.4.RT-qPCR was used to detect the expression of OPG and Runx2 mRNA after 7 days of osteogenic induction:compared with the control group,the expression of OPG and Runx2 mRNA were observed at the concentration of 10-10mol/L and 10-9mol/L HU-308.The up-regulation was observed,and the up-regulation effect was enhanced with the decrease of concentration(P<0.05).The expression of OPG and Runx2 mRNA was inhibited by high concentration of HU-308,and the difference was statistically significant(P<0.05).5.Staining of the cytoskeleton morphology showed that MC3T3-E1 cells in the control group had a small extension and spreading area,no clear actin fibers were observed,and a clear network structure inside the cells was not observed.As the concentration of HU-308 increased,the cell volume increased and the cell stretch range increased.It was gradually clear that the cytoskeleton was meshed and the internal structure was clearer.The fiber shape and the internal structure of the cells were clearly observed in the two groups with HU-308 concentrations of 10-7 mol/L and 10-6 mol/L.Conclusion:HU-308 promotes cells proliferation of MC3T3-E1 under the high concentration of glucocorticoid,but the differentiation is not obvious.The low concentration of HU-308 expression of Runx2 and OPG.It was obviously observed that with the concentration of HU-308 increased,cells spreading area increased and the internal frame structure is more clear.All the results showed that the HU-308 may be advantageous to the implant bone union for osteoporosis patients caused by the long-term use of glucocorticoid.