唑来膦酸盐通过p38 MAPK信号通路调控高糖微环境下成骨细胞分化

doi:10.11958/20220368

天津医药 ›› 2022, Vol. 50 ›› Issue (12): 1239-1245.doi: 10.11958/20220368

唑来膦酸盐通过p38 MAPK信号通路调控高糖微环境下成骨细胞分化

侯甜(), 秦雅芝, 张妍, 温国琛, 张啸, 董伟^△()

华北理工大学口腔医学院（邮编063210）

收稿日期:2022-03-22 修回日期:2022-07-18 出版日期:2022-12-15 发布日期:2022-12-30
通讯作者: 董伟 E-mail:2428693909@qq.com;970484328@qq.com
作者简介:侯甜（1997），女，硕士在读，主要从事骨组织工程方面研究。E-mail：2428693909@qq.com
基金资助:
河北省高等学校科学技术研究项目(QN2020438)

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

HOU Tian(), QIN Yazhi, ZHANG Yan, WEN Guochen, ZHANG Xiao, DONG Wei^△()

School of Stomatology, North China University of Science and Technology, Tangshan 063210, China

Received:2022-03-22 Revised:2022-07-18 Published:2022-12-15 Online:2022-12-30
Contact: DONG Wei E-mail:2428693909@qq.com;970484328@qq.com

侯甜, 秦雅芝, 张妍, 温国琛, 张啸, 董伟. 唑来膦酸盐通过p38 MAPK信号通路调控高糖微环境下成骨细胞分化[J]. 天津医药, 2022, 50(12): 1239-1245.

HOU Tian, QIN Yazhi, ZHANG Yan, WEN Guochen, ZHANG Xiao, DONG Wei. Zoledronate regulates osteoblast differentiation in high glucose microenvironment via p38 MAPK signaling pathway[J]. Tianjin Medical Journal, 2022, 50(12): 1239-1245.

摘要/Abstract

摘要：

目的探讨唑来膦酸盐（ZOL）对高糖微环境下小鼠前成骨细胞MC3T3-E1成骨分化的影响及p38丝裂原活化蛋白激酶（p38 MAPK）通路的调节作用。方法体外培养MC3T3-E1细胞并分为低糖（LG）组、LG+ZOL组、高糖（HG）组、HG+ZOL组。ZOL为0.1 μmol/L，LG、HG的葡萄糖浓度分别为5.5 mmol/L和16.5 mmol/L。采用四甲基偶氮唑蓝（MTT）法检测细胞增殖水平；鬼笔环肽染色观察细胞骨架；试剂盒检测细胞碱性磷酸酶（ALP）活性；茜素红染色检测细胞矿化结节生成情况；免疫荧光法检测Wnt5a、p38 MAPK的荧光表达强度。另设HG+ZOL+p38 MAPK通路抑制剂（SB203580）组，SB203580为10 μmol/L。Western blot检测5组细胞中Wnt5a、p38 MAPK、磷酸化p38丝裂原活化蛋白激酶（p-p38 MAPK）、骨形态发生蛋白2（BMP2）和Ⅰ型胶原蛋白（COLⅠ）的表达水平。结果 4组细胞增殖水平差异无统计学意义（P>0.05）。与LG组比较，LG+ZOL组细胞骨架清晰程度，ALP活性，茜素红矿化结节生成，Wnt5a、p38 MAPK蛋白荧光表达强度，Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COLⅠ蛋白表达水平明显升高；HG组上述指标变化相反（P<0.05）。与HG组相比，HG+ZOL组细胞骨架清晰程度，ALP活性，茜素红矿化结节生成，Wnt5a、p38 MAPK蛋白荧光表达强度，Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COL Ⅰ蛋白表达水平明显升高（P<0.05）。与HG+ZOL组相比，HG+ZOL+SB203580组Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COL Ⅰ的蛋白表达水平降低（P<0.05）。结论高糖对细胞成骨分化起抑制作用，ZOL可有效改善其抑制作用，其机制可能与p38 MAPK通路相关。

关键词: 二膦酸盐类, p38丝裂原活化蛋白激酶类, 成骨细胞, 细胞分化, Wnt-5a蛋白, 骨质疏松, 唑来膦酸盐, 高糖微环境

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

中图分类号:

R587.2

图/表 7

图1 MTT法检测不同时间点细胞的增殖水平

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

图2 鬼笔环肽染色检测细胞骨架的清晰度及细胞数量 A：免疫荧光染色下各组细胞骨架图（×400）；B：各组MC3T3-E1细胞数量比较，1~4分别为LG组、LG+ZOL组、HG组、HG+ZOL组；a与LG组比较，b与LG+ZOL组比较，c与HG组比较，P<0.05。

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

图3 各组细胞ALP活性的比较 1~4分别为LG组、LG+ZOL组、HG组、HG+ZOL组；a与LG组比较， b与LG+ZOL组比较，c与HG组比较，P<0.05。

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

图4 茜素红染色法检测细胞的矿化结节的生成 A：各组细胞染色后镜下表现；B：各组细胞茜素红染色情况；C：各组细胞矿化结节数量比较；1~4分别为LG组、LG+ZOL组、HG组、HG+ZOL组；a与LG组比较，b与LG+ZOL组比较，c与HG组比较，P<0.05。

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

图5 免疫荧光染色检测Wnt5a和p38 MAPK的荧光表达强度 A：免疫荧光染色下各组MC3T3-E1细胞Wnt5a和p38 MAPK表现；B、C分别为各组Wnt5a和p38 MAPK平均荧光强度比较，1~4分别为LG组、LG+ZOL组、HG组、HG+ZOL组；a与LG组比较，b与LG+ZOL组比较，c与HG组比较，P<0.05。

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

表1 细胞中Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COL Ⅰ蛋白表达（n=3，$\bar{x}\pm s$）

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^＊＊

图6 细胞中Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COL Ⅰ 蛋白表达 1~5分别为LG组、LG+ZOL组、HG组、HG+ZOL组、HG+ZOL+SB203580组。

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

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唑来膦酸盐通过p38 MAPK信号通路调控高糖微环境下成骨细胞分化

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

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