Effect Of Glyburide On The Proliferation、Differentiation And Apoptosis Of Mouse Embryonic Osteoblasts Line MC3T3-E1 In High Glucose

Background and Purpose In recent years more and more scholars pay attention to the effect of sulfonylurea drugs on osteoporosis. Several large clinical studies find that the incidence of fractures on patients who use different hypoglycemic drugs are significantly different, and the highest fracture risk exist in patients who use thiazolidinediones, insulin followed, then is metformin, the lowest incidence of fractures happen to the people who apply sulfonylurea drugs [1-3]. Some studies have found there are no significant correlation between the incidence of fractures and sulfonylureas [4-6], recently statistics show that the risk of fracture in patients who are given sulfonylureas actually increase, significantly higher than metformin[7-8 ]. Direct impact regarding sulfonylurea drugs on bone tissue have not been reported abroad, while Pan Ma,domestic scholar, reports that under high glucose glimepiride can promote mandibular osteoblasts of rat in vitro to intake glucose by increasing the expression of GLUT-1 and GLUT-4,and directly accelerate the proliferation and differentiation of rat bone cells by phosphatidylinositol 3-kinase / protein kinase B pathway. Moreover glimepiride can improve the activity of the alkaline phosphatase (ALP) and increase expression levels of type I collagen(COL-1) and osteocalcin(OCN) m RNA [9-11]. Glipizide and gliquidone can promote the proliferation of MC3T3-E1 cell and expression of type I collagen and inhibit its apoptosis[12-13]. Glyburide as a second-generation sulfonylurea drugs has strong hypoglycemic effect, applying to clinical treatment widely, and glyburide in foreign commonly is used in patients with gestational diabetes mellitus.This experiment aim to research the influence of different concentrations of glyburide in high glucose(16.5mmol / l) on the proliferation, differentiation and apoptosis of mouse embryos osteoblast cell line MC3T3-E1 in vitro. Methods After glibenclamide intervening cells 48 h, CCK-8 assay is used to detect the proliferation rate of MC3T3-E1 cell, we apple real-time quantitative PCR to detect the expression levels of differentiation markers(COL-1, OPN),flow cytometry is used to measure the apoptosis rate, and we apply Western blot to analysis the expression of apoptosis- related proteins(Bax, Bcl-2). Results 1.CCK-8 assay Compared with the control group, the proliferation rate of MC3T3-E1 cell gradually increase with glyburide concentration increasing,and 1μmol/l glyburide group, 10μmol/l glyburide group and 20μmol/ l glyburide group compared with the control group, proliferation rates increase by 13.1%, 24.2% and 31.3%, and the differences are statistically significant(P <0.05).2.Real-time quantitative PCR As the study shows the expression of COL-1 、OPN m RNA increase along with glibenclamide concentration raising in a certain range(P <0.05).3.Flow cytometry With glyburide concentration increasing, the early apoptotic rate of MC3T3-E1 cell in the control group, 1μmol / l glyburide group, 10μmol / l glibenclamide group, 20μmol / l glibenclamide are19.63%, 11.20%, 7.9%, 4.7%, the differences are statistically significant(P <0.05).4. Western Blot Compared with the control group, with the increasing concentration of glibenclamide the expression of Bcl-2 protein gradually enhances, While Bax protein expression gradually decreases, and the value of Bcl-2/Bax increases gradually(P <0.05). Conclusion Under high glucose glibenclamide could promote the proliferation and differentiation of MC3T3-E1 cells and inhibit its apoptosis.This action have a concentration-dependent manner within a certain range.