| Objective To investigate the effects of high glucose on the proliferation, differentiation and mineralization in osteoblast(OB) and the role of P13K/Akt signal pathway. To disscuss the intervene of resveratrol on high glucose inducing oxidative stress during PI3K/Akt singal pathway.Methods 1.Neonatal Wistar rat calvarium OB were isolated, cultured, and observed the morphologic and biological characters of OB. The OB were identified by the alkaline phosphatase (ALP) and the mineralization nodes staining.2. OB which were in logarithmic phase growth of generation one or two were collected and modulated the cell density at we needed, cells were incubated at 37℃in a humidified atmosphere of 5% CO2. OB were cultured in a-MEM with no serum for 24 hours after adherent, then discard the supernatant. We randomized OB for normal glucose(NG group,5.6mmol/L), middle glucose(MG group,11.2mmol/L), high glucose(HG group, 22.4mmol/L), normal glucose plus resveratrol(NG+Res group, Res 10μmol/L), normal glucose plus LY294002(NG+LY group, LY 10μmol/L), normal glucose plus resveratrol and LY294002 (NG+Res+LY group, Res 10μmol/L, LY294002 10μmol/L), middle glucose plus resveratrol (MG+Res group, Res 10μmol/L), middle glucose plus LY294002(MG+LY group, LY 10μmol/L), middle glucose plus resveratrol and LY294002 (MG+Res+LY group, Res 10μmol/L, LY294002 10μmol/L), high glucose plus resveratrol(HG+Res group, Res 10μmol/L), high glucose plus LY294002(HG+LY group, LY 10μmol/L), high glucose plus resveratrol and LY294002 (HG+Res+LY group, Res 10μumol/L, LY294002 10μmol/L). Measured MTT values (12h,24h, 48h), activity of ALP (7d) and mineralized nodule ROS (60 min,120 min), SOD, MDA (12h, 24h,48h,72h) of each group. Detected the expression of p-Akt(Ser 473) by Western blotting (48h).Results 1.OB proliferation showed an increasing trend with increasing time (p<0.05, p<0.01). OB proliferation was decreasing with increasing glucose concentration(p<0.05, p<0.01).In the same time and glucose concentration, resveratrol promoted cell proliferation, the OD values of NG+Res group,MG+Res group and HG+Res group increased significantly than other groups(p<0.05, p<0.01); LY294002 inhibit cell proliferation, the OD values of NG+LY group,MG+LY group and HG+LY group decreased significantly than other groups(p><0.05, p<0.01); resveratrol improved inhibition of OB proliferation by LY294002, the OD values of NG+Res+LY group,MG+ Res+LY group and HG+ Res+LY group increased significantly than NG+LY group,MG+LY group and HG+LY group respectively (p<0.05, p<0.01).2. ALP avtivity decreased with increasing concentrations of glucose (p<0.05, p<0.01). In the same glucose concentration, resveratrol ptomoted ALP activity, the ALP activitiy of NG+Res group, MG+Res group, HG+Res group increased significantly than other groups(p<0.05, p<0.01); LY294002 inhibit ALP activity, the ALP activities of NG+LY group,MG+LY group and HG+LY group decreased significantly than other groups(p<0.05,p<0.01); resveratrol improved inhibition of ALP activity by LY294002, the ALP activity of NG+Res+LY group,MG+ Res+LY group and HG+ Res+LY group increased significantly than NG+LY group,MG+LY group and HG+LY group respectively (p<0.05, p<0.01). 3. With increased of glucose concentration, the number of mineralized nodule decreased (p<0.05, p0.01). In the same concentration of glucose, the number of mineralized nodule of NG+Res group, MG+Res group, HG+Res group increased significantly than other groups(p<0.05, p<0.01).4. Resveratrol decreased production of ROS, the expression of ROS in NG+Res group, MG+Res group, HG+Res group decreased than other groups, the expression of ROS in 120 min increased than 60 min.5. The content of MDA increased with increasing time (p<0.05, p<0.01). In the same glucose concentration, the content of MDA of NG+Res group, MG+Res group, HG+Res group decreased than other groups(p<0.05, p0.01); the content of MDA of NG+LY group,MG+LY group and HG+LY group increased significantly than other groups(p<0.05, p0.01); the content of MDA of NG+Res+LY group,MG+ Res+LY group and HG+ Res+LY group decreased significantly than NG+LY group,MG+LY group and HG+LY group respectively (p<0.05. p0.01).6. The content of SOD decreased with increasing time (p<0.05, p<0.01). In the same glucose concentration, the content of SOD of NG+Res group, MG+Res group, HG+Res group increased than other groups(p<0.05, p0.01); the content of SOD of NG+LY group,MG+LY group and HG+LY group decreased significantly than other groups (p<0.05, p0.01l); the content of MDA of NG+Res+LY group,MG+ Res+LY group and HG+ Res+LY group increased significantly than NG+LY group,MG+LY group and HG+LY group respectively (p<0.05. p0.01).7. In the same glucose concentration, the expression of p-Akt in Res group increased significantly than other groups (p0.01); the expression of p-Akt protein in Res+LY group increased significantly than LY group(p0.01). With increasing of concentrations of glucose, the expression of p-Akt(Ser473) protein was showed decreased trend.Conclusion 1. Resveratrol can mitigate the effect that high glucose inhibited the proliferation, differentiation and mineralization in osteoblast(OB), and the mechanism was related to the activation of PI3K/Akt signal pathway.2.Resveratrol amiliarated oxdative stress via activating of PI3K/Akt signal pathway. |
