A Study On GSK-3?/?-catenin Pathway Mediating Protection Of Estrogen On Bone Formation Damages Of Osteoblast MC3T3-E1 Induced By High Glucose

Background and objective:Osteoporosis?OP?is a kind of metabolic bone disease characterized by the low bone mass and micro organizational structure destruction,eventually leading to bone fragility and easy to fracture.In the Osteoporosis,osteoblasts'function is inhibited,and this is a very important reason for the OP.Diabetes is a chronic elevated blood glucose levels as a significant feature of the comprehensive metabolic diseases,mainly duing to the lack of insulin or action defects.Long-term hyperglycemia can make the body systemic function changes,and the impact of hyperglycemia on bone is prominent.This changes eventually lead to osteoporosis.Estrogen is the hormone drugs to stimulate bone formation in wide range of applications.Estrogen can promote a variety of bone cell proliferation and differentiation.Previous researches have found that estrogen could promote osteoblast proliferation.In the present study,we clarify the focus on diabetic osteoporosis and the relationship between estrogen and PI3K/Akt/GSK-3?/?-catenin signal pathway.The purposes are to explore the mechanism of estrogen therapy in diabetic osteoporosis and seek the new therapeutic targets.Methods:MC3T3-E1 cells were cultured at different concentrations of glucose?5.5mmol/L,11mmol/L,22mmo/L,33mmol/L,44mmol/L?in vitro for 24h,and cell viability was deteced.Compared with the control group,33mmol/L of glucose was used in the injury model.Subsequently,The cells were treated with 10^-8mol/L,10^-7mol/L,and 10^-6mol/L of estradiol and 5mmol/L LiCl for 24h and 48h,respectively.The cell viability was measured by Wst-1 method.Cell differentiation and mineralization were assayed by alkaline phosphatase?ALP?staining and Alizarin Red staining.The expression levels of the PI3K,Akt,GSK-3?and?-catenin proteins were detected by Western blots.Results1.With the concentration of glucose increasing,MC3T3-E1 cells'viability reduced gradually,but after the addition of LiCl,the cell viability significantly increased compared with the model group?P<0.05?.Compared with the control group,?-catenin protein levels were decreased with high glucose at different concentrations;in addition,ALP staining results showed that the number of positive cells in high glucose decreased significantly?P<0.05?,but the addition of LiCl remarkably restored the changes induced by high glucose?P<0.05?;Similar results were obtained from Alizarin red staining.2.Compared with the control group,MC3T3-E1 cells'viability was reduced gradually,however,estradiol can significantly increase the cell viability in a concentration-dependent manner?P<0.05?.ALP staining results also showed that the number of positive cells in high glucose decreased significantly?P<0.05?,but after the addition of estradiol,the positive cells were increased?P<0.05?;Alizarin red staining results showed that hyperglycemia can inhibit the mineralization of MC3T3-E1,and estradiol were able to reverse these changes;Western blot results further displayed that high glucose resulted in down-regulation of PI3K,p-Akt,p-GSK-3?and?-catenin,but estradiol can reverse these alterations.Unexpectedly,addition of two specific estrogen receptor inhibitors did not affect estradiol-induced changes,suggesting that estradiol play an important role through a non-classical pathway.Conclusion1.High glucose can inhibit the activity of GSK-3?/?-catenin pathway,and injure the function of MC3T3-E1.2.17-?estradiol can activate PI3K/Akt and the GSK-3?/?-catenin pathway,and reverse the damage of MC3T3-E1 cells induced by high glucose,which may not be mediated by ER?and ER?.