Journal of Prevention and Treatment for Stomatological Diseases ›› 2020, Vol. 28 ›› Issue (1): 24-29.doi: 10.12016/j.issn.2096-1456.2020.01.004

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Study on the role of FoxO1 in the regulation of osteoblastic metabolism by 1,25(OH)2D3 in a high glucose environment

ZHOU Jiaqi1,SHU Linjing2,XIONG Yi3,ZHANG Yixin1,XIANG Lin3,WU Yingying3()   

  1. 1.State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
    2.Department of Oral Implantology, Stomatological Hospital of Chongqing Medical University, Chongqing 400016, China
    3.Department of Oral Implantology, West China Hospital of Stomatology, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Sichuan University, Chengdu 610041, China
  • Received:2019-05-15 Revised:2019-09-16 Online:2020-01-20 Published:2020-01-17
  • Contact: Yingying WU E-mail:yywdentist@163.com

Abstract:

Objective To explore the effect of 1,25(OH)2D3 on the regulation of bone metabolism in a high-glucose environment and to provide evidence for the possible regulatory mechanism of 1,25(OH)2D3 on osteoblasts in a high-glucose 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-glucose +1,25(OH)2D3 group: high-glucose DMEM + 1,25(OH)2D3 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 protein 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 immunofluorescence 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)2D3 group (P < 0.05); furthermore, 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)2D3 may prevent the transfer of FoxO1 to the cell nucleus, inhibit the abnormal proliferation and apoptosis of osteoblasts in a high-glucose environment, and reverse the inhibitory effect of high glucose on the differentiation of osteoblasts.

Key words: osteoblast, osteogenic differentiation, hyperglycemia, diabetes, forkhead transcription factor 1(FoxO1), proliferation, apoptosis, 1,25(OH)2D3, vitamin D, bone metabolism

CLC Number: 

  • R78

Table 1

Names of genes and primer sequences for qRT-PCR"

Gene Primer sequence(5′→3′)
FoxO1 F:AGTGGATGGTGAAGAGCGTG
R:GAAGGGACAGATTGTGGCGA
GAPDH F:GGTGAAGGTCGGTGTGAACG
R:CTCGCTCCTGGAAGATGGTG

Figure 1

Proliferation of osteoblasts Control vs. high glucose at 48 h *: P=0.036 (P < 0.05); high glucose vs. high glucose+1,25(OH)2D3 at 48 h $$: P=0.001 (P < 0.01); control vs. high glucose at 72 h *: P=0.012 (P < 0.05); control vs. high glucose+1,25(OH)2D3 at 72 h #: P=0.032 (P < 0.05); high glucose vs. high glucose+1,25(OH)2D3 at 72 h $$: P=0.001 (P < 0.01)"

Figure 2

Flow cytometry analysis of apoptosis"

Figure 3

Mineralized nodule formation of osteoblasts by alizarin red staining a: alizarin red staining of mineralized nodules was observed under microscope(× 400); b: semi-quantitative results of calcium mineralization deter mination by alizarin red, control vs. high glucose **: P=0.001(P < 0.01); control vs. high glucose+1,25(OH)2D3 *: P=0.033(P < 0.05); high glucose vs. high glucose+1,25(OH)2D3 **: P=0.008(P < 0.01)"

Figure 4

The distribution of FoxO1 protein in osteoblasts shown by immunofluorescence staining a:Dapi represents nucleus, FoxO1 represents FoxO1 protein, Merge represents the merged image of nucleus and FoxO1 protein (× 400); b: the percentage of FoxO1 expressed in the nucleus, control vs. high glucose **: P=0.001(P < 0.01);control vs. high glucose+1,25(OH)2D3 **: P=0.001(P < 0.01);high glucose vs. high glucose+1,25(OH)2D3 ***:P < 0.001"

Figure 5

Relative expression of FoxO1 mRNA Control vs. high glucose **, P=0.006; high glucose vs. high glucose+1,25(OH)2D3 **,P=0.006"

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