Zoledronate regulates osteoblast differentiation in high glucose microenvironment via p38 MAPK signaling pathway

doi:10.11958/20220368

Abstract

Abstract:

Objective To investigate the effect of zoledronate (ZOL) on the differentiation of mouse pre-osteoblast MC3T3-E1 cells under high glucose microenvironment and the regulatory effect of p38 mitogen-activated protein kinases (p38 MAPK) signaling pathway. Methods MC3T3-E1 cells were cultured in vitro and divided into the low glucose (LG) group, the LG+ZOL group, the high glucose (HG) group and the HG+ZOL group. ZOL concentration was 0.1 μmol/L. The gluse concentrations of LG and HG were 5.5 mmol/L and 16.5 mmol/L. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell proliferation. The cell cytoskeleton was detected by phalloidin staining. The alkaline phosphatase (ALP) activity was detected by kit. The mineralized nodule was observed by alizarin red staining. The fluorescence expression intensity of Wnt5a and p38 MAPK was detected by immunofluorescence staining. The HG+ZOL+ p38 MAPK inhibitor (SB203580) group was also set in the study. SB203580 concentration was 10 μmol/L. The protein expressions of Wnt5a, p38 MAPK, phosphorylation p38 mitogen-activated protein kinases (p-p38 MAPK), bone morphogenetic protein 2 (BMP2) and collagen type Ⅰ (COL Ⅰ) were detected with Western blot assay. Results There was no significant difference in cell proliferation between the four groups (P>0.05). Compared with the LG group, the clarity of cytoskeleton, ALP activity, the generation of mineralized nodules, the fluorescence expression intensity of Wnt5a and p38 MAPK, the expression levels of Wnt5a, p38 MAPK, p-p38 MAPK, BMP2 and COL Ⅰ protein were significantly higher in the LG+ZOL group (P<0.05), while the HG group showed opposite changes in the above indexes (P<0.05). Compared with the HG group, the clarity of cytoskeleton, ALP activity, the generation of mineralized nodules, the fluorescence expression intensity of Wnt5a and p38 MAPK, the expression levels of Wnt5a, p38 MAPK, p-p38 MAPK, BMP2 and COL Ⅰ protein were significantly higher in the HG+ZOL group (P<0.05). Compared with the HG+ZOL group, the expression levels of Wnt5a, p38 MAPK, p-p38 MAPK, BMP2 and COL Ⅰ protein were lower in the HG+ZOL+SB203580 group (P<0.05). Conclusion High glucose can inhibit the osteogenic differentiation of cells, and ZOL can effectively improve the inhibitory effect, which may be related to p38 MAPK signaling pathway.

Key words: diphosphonates, p38 mitogen-activated protein kinases, osteoblasts, cell differentiation, Wnt-5a protein, osteoporosis, zoledronate, high glucose microenvironment

CLC Number:

R587.2

Figures/Tables 7

Fig. 1 The proliferation levels of cells detected by MTT assay at different time points

Fig. 2 The cell cytoskeleton clarity of cells detected by phalloidin staining

Fig. 3 Comparison of the ALP activity between the four groups of cells

Fig. 4 The generation of mineralized nodules of cells detected by alizarin red staining

Fig. 5 The fluorescence expression intensity of Wnt5a and p38 MAPK detected by immunofluorescence staining

Tab.1 Relative expression of Wnt5a, p38 MAPK, p-p38 MAPK, BMP2 and COL Ⅰ proteins in cells

组别	Wnt5a	p38 MAPK	p-p38 MAPK	BMP2	COL Ⅰ
LG组	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
LG+ZOL组	1.33±0.08^a	1.38±0.18^a	1.81±0.25^a	1.21±0.07^a	1.25±0.08^a
HG组	0.64±0.09^ab	0.57±0.08^ab	0.49±0.09^ab	0.64±0.05^ab	0.81±0.06^ab
HG+ZOL组	1.10±0.11^bc	0.88±0.05^bc	0.85±0.08^bc	1.14±0.09^ac	1.05±0.07^bc
HG+ZOL+SB203580组	0.54±0.06^abd	0.52±0.10^abd	0.43±0.07^abd	0.35±0.05^abcd	0.70±0.07^abd
F	53.354^＊＊	35.789^＊＊	56.242^＊＊	110.142^＊＊	34.826^＊＊

Fig.6 Relative expression of Wnt5a, p38 MAPK, p-p38 MAPK, BMP2 and COL Ⅰ proteins in cells

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