ABSTRACT
Objective@#Yingying, Email: yywdentist@163.com, Tel: 86⁃28⁃85503579 【Abstract】 Objective To explore the effect of 1,25(OH) 2 D 3 on the regulation of bone metabolism in a high⁃glucose environment and to provide evidence for the possible regulatory mechanism of 1,25(OH) 2 D 3 on osteoblasts in a high⁃glu⁃ cose environment.@* Methods@#The osteoblast cell line MC3T3⁃E1 was cultured in 3 groups: ① control group, cultured in low⁃glucose (5.5 mmol/L) DMEM; ② high⁃glucose group: cultured in high⁃glucose (22 mmol/L) DMEM; ③ high⁃glu⁃ cose +1,25(OH) 2 D 3 group: high⁃glucose DMEM + 1,25(OH) 2 D 3 medium culture. The CCK⁃8 method was used to detect cell proliferation in each group; Annexin V and FITC apoptosis kits were used to detect apoptosis; Alizarin red was used to semiquantitatively analyze cell differentiation; qRT⁃PCR was used to detect forkhead transcription factor⁃1 (forkhead transcription factor 1, FoxO1) mRNA expression. Immunofluorescence was used to observe the changes in FoxO1 pro⁃ tein expression and its relative position in the nucleus.@* Results@#ence was used to observe the changes in FoxO1 pro⁃ tein expression and its relative position in the nucleus. Results Our analysis showed that compared with those in the control group, the osteoblast apoptosis and proliferation in the high⁃glucose group were improved, while differentiation was inhibited (P < 0.05); at the same time, the mRNA expression of FoxO1(P = 0.006) was reduced. The immunofluores⁃ cence results showed that more FoxO1 was inside the nucleus (P < 0.001). Compared with those in the high⁃glucose group, excessive proliferation was inhibited, apoptosis was reduced, and osteogenic differentiation was improved in the high⁃glucose +1,25(OH) 2 D 3 group (P < 0.05); furthermore, FoxO1 mRNA was decreased (P = 0.006), and the transfer of FoxO1 protein was blocked (P < 0.001).@* Conclusion @#re, FoxO1 mRNA was decreased (P = 0.006), and the transfer of FoxO1 protein was blocked (P < 0.001). Conclusion We found that 1,25(OH) 2 D 3 may prevent the transfer of FoxO1 to the cell nucleus, inhibit the abnormal proliferation and apoptosis of osteoblasts in a high⁃glucose environment, and re⁃ verse the inhibitory effect of high glucose on the differentiation of osteoblasts.
ABSTRACT
ABSTRACT We previously revealed the involvement of extracellular regulated protein kinases 1/2 (ERK1/2) in interleukin-6 (IL-6) secretion induced by cyclic compressive force (CCF) in MLO-Y4 cells. In this study, we investigated the contributions of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways to IL-6 secretion by stimulating MLO-Y4 cells with CCF. At 80% confluence, different magnitudes (1000μstrain, 2000 μstrain and 4000 μstrain), frequencies (0.5 Hz, 1.0 Hz and 2.0 Hz) and durations (10 min, 30 min, 1 h, 3 h and 6 h) of CCF were loaded onto cells using a four-point bending system. Flow Cytometry (FCM) analysis was used to analyze cell mortality rates after CCF loading. p38 and p65 phosphorylation as well as IκBα degradation in MLO-Y4 cells were detected by Western blotting (WB). Changes in IL-6 secretion after inhibitor treatment were assessed by enzyme-linked immunosorbent assays (ELISAs). Cellular viability was over 90 percent after CCF. p38 and p65 phosphorylation increased under all conditions, whereas IκBα protein levels decreased. However, phosphorylation and degradation were not completely dependent on the loading magnitude, frequency or duration. Furthermore, p38 inhibition using the specific inhibitor SB203580 reduced both p38 phosphorylation and IL-6 secretion. Similarly, NF-κB inhibition using BAY 11-7082 decreased p65 phosphorylation and IL-6 secretion but increased the concentration of IκBα. These findings reveal significant roles for the p38 and NF-κB signaling pathways in IL-6 secretion induced by CCF in MLO-Y4 cells.