Study On The Effect Of High Glucose On The Function Of Estrogen Receptor In MC3T3-E1 Cells

Purpose:Our laboratory found that high glucose and estrogen can regulate bone formation through β-catenin pathway.As reported in the existing part that ER and β-catenin play synergistic effect in osteoblasts.So the purpose of this study is to observe the effect of estrogen,and regulate the expression of ER in osteoblasts and its interaction withβ-catenin.Method:Western-blot method was used to observe the expression of ERα and ERβ in osteoblast precursor cell line MC3T3-E1 under different concentration and duration.Select the appropriate concentration and action time to observe the effect of high glucose and estrogen alone or combined on the expression,distribution and transcription activity of ER protein.Immunofluorescence assay was used to detect the distribution of ER and the activity of ER was detected by double luciferase reporter gene assay.At the same time,MTT and alizarin red staining were used to detect the effects of high glucose and estrogen on the proliferation and mineralization of osteoblasts.LiCl was used to inhibit the degradation of β-catenin,and to observe the effect of LiCl on the function of ER induced by high glucose.Result:The expression of estrogen receptor protein with glucose concentration increasing with dose-dependent effect;Adding estradiol in 33mmol/L with high glucose,the expression level of estrogen receptor estrogen group decreased.The results of immunofluorescence assay showed that the expression and nuclear distribution of estrogen receptor in high glucose group(33mmol/L)and estradiol group was higher than that in normal group(5.5mmol/L).After high glucose combined with estradiol,the number of receptors and the effect of nucleus were higher than those in the high glucose or estradiol group.Luciferase experiment results showed that estrogen receptor transcriptional activity was decreased in high glucose.Estradiol group canincrease the transcriptional activity of ER.The transcriptional activity of Estradiol combined with high glucose group is decreased than estradiol group.According to MTT,alizarin red staining showed that MC3T3-E1 cell activity and mineralization ability were inhibited after induction by high glucose(33mmol/L).The activity and mineralization ability decreased than estradiol group in MC3T3-E1 cell.After adding ER(33mmol/L)and LiCl(5mmol/L),the expression level was significantly lower than that in the high glucose group.LiCl can increase the transcription activity of ER induced by high glucose.Conclusion:1.High glucose can up regulate the expression of ER and down regulate the activity of ER,which may be related to the down regulation of β-catenin expression.2.Estrogen can up regulate ER protein expression and transcriptional activity,which can be abolished in high glucose.