Advanced glycation end products influence osteoclast-induced bone resorption

doi:10.3969/j.issn.2095-4344.2016.20.003

Abstract

Abstract:

BACKGROUND: The effects of advanced glycation end products (AGEs) on osteoclast-induced bone resorption is controversial and the underlying mechanisms remain unclear. Most of the studies indicate that AGEs can enhance bone resorption, while some others show the opposite effects.
OBJECTIVE: To investigate the effects of AGEs on osteoclast-induced inorganic matrix dissolution and organic component degradation and the underlying mechanisms.
METHODS: RAW 264.7 cells were induced to generate osteoclasts, and AGEs (50-400 µg/mL) or control-bovine serum albumin (100 µg/mL) was added since the beginning of the induction. The effect of AGEs on bone resorption was evaluated by analyzing the area of resorption pits on the Osteo Assay Surface plates and the expression of cathepsin K. Furthermore, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, nuclei per osteoclasts and the expression of integrin ανβ3 were detected.
RESULTS AND CONCLUSION: The area of resorption pits and expression of cathepsin K in AGEs groups were significantly decreased compared with the control group, and this inhibiting effect became more obvious with the increase of AGEs concentration. TRAP staining also showed that number of TRAP-positive multinucleated cells and nuclei per osteoclast were significantly reduced in an AGE dose-dependent manner. Quantitative PCR revealed that the expression of integrin ανβ3 decreased significantly with the extension of AGEs incubation time. These data indicate that AGEs can exert inhibitory effects on organic and inorganic matrix degradation induced by osteoclasts. The underlying mechanism may be involved in the inhibitory effects of AGEs on directed differentiation and cell fusion of osteoclast precursor cells, and migration and adhension of osteoclasts.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Glycosylation End Products, Advanced, Osteoclasts, Tissue Engineering

CLC Number:

R318

Li Zi-qing1, Wang Hai-xing1, He Pei-heng1, Luo Guo-tian1, Xiao Yin-bo1, Huang Shuai1, Li Xing1, Sheng Pu-yi1, Li Chao-hong2, Xu Dong-liang1. Advanced glycation end products influence osteoclast-induced bone resorption[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(20): 2907-2914.

Figures/Tables 2

2 结果 Results 2.1 晚期糖基化终末产物修饰蛋白的体外合成效果按Valcourt等[22]方法制备的晚期糖基化终末产物经过1周孵育后外观呈均一黄褐色，通透无沉淀；而对照用牛血清白蛋白溶液孵育后呈现无色透明状(图1A)。荧光光谱分析发现，于波长370 nm下激发时，晚期糖基化终末产物在波长440-460 nm间出现特征性峰值(图1B)，符合晚期糖基化终末产物荧光特性[13，32]。 2.2 晚期糖基化终末产物对破骨细胞骨吸收功能的影响 2.2.1 晚期糖基化终末产物抑制破骨细胞对无机骨基质的溶解破骨细胞诱导完成后，正常组无机骨基质被大量溶解吸收，而晚期糖基化终末产物组随着晚期糖基化终末产物浓度的增加，骨吸收总面积呈减少趋势，且在浓度超过100 mg/L后趋势显著(P < 0.05)；牛血清白蛋白组骨吸收总面积未见减少(图2)。将骨吸收总面积进一步分为部分吸收面积和完全吸收面积后发现，晚期糖基化终末产物对完全吸收面积的抑制效果更为明显，且与骨吸收总面积的趋势基本一致(图2B)。 2.2.2 晚期糖基化终末产物抑制破骨细胞组织蛋白酶K表达量 Western blot结果显示，所有晚期糖基化终末产物干预的细胞中组织蛋白酶K表达水平较正常组均显著下降(P < 0.05)，且呈现明显的浓度依赖性，牛血清白蛋白组组织蛋白酶K表达量较正常组无明显变化(图3A和B)。进一步实时定量PCR检测发现，晚期糖基化终末产物对组织蛋白酶K mRNA的抑制效应在破骨细胞诱导早期即已出现(晚期糖基化终末产物加入24 h后)，且抑制效果随晚期糖基化终末产物作用时间的延长而加剧(图3C)。此结果表明晚期糖基化终末产物可导致破骨细胞组织蛋白酶K的表达下调，从而使其降解Ⅰ型胶原蛋白的能力下降。 2.3 晚期糖基化终末产物对破骨细胞分化的影响抗酒石酸酸性磷酸酶染色显示，正常组出现大量抗酒石酸酸性磷酸酶阳性多核细胞，提示破骨细胞诱导良好(图4A)。随着晚期糖基化终末产物作用浓度的增强，抗酒石酸酸性磷酸酶阳性细胞数量逐渐减少，当晚期糖基化终末产物浓度超过100 mg/L后，抗酒石酸酸性磷酸酶阳性细胞减少程度明显(P < 0.05)。牛血清白蛋白组抗酒石酸酸性磷酸酶阳性细胞数量较正常组也略有下降，但差异无显著性意义(图4B)。此结果提示，晚期糖基化终末产物能阻碍破骨前体细胞向成熟破骨细胞进行定向分化，且呈浓度依赖性。进一步分析发现，破骨细胞胞体内的细胞核数量随晚期糖基化终末产物浓度的上升而呈下降趋势，且在晚期糖基化终末产物超过100 mg/L后，下降程度显著，差异有显著性意义(P < 0.05)，而牛血清白蛋白对照组则没有此种表现(图4C)。此结果表明晚期糖基化终末产物可能通过抑制细胞融合过程从而阻碍破骨细胞的分化。 2.4 晚期糖基化终末产物对破骨细胞迁移和黏附能力的影响为探究晚期糖基化终末产物是否影响破骨细胞迁移和黏附功能，加入100 mg/L浓度的晚期糖基化终末产物作用24，48及72 h。qPCR结果显示，晚期糖基化终末产物作用48 h后，整合素ανβ3的mRNA表达量较正常组显著降低(P < 0.05；图5)，提示晚期糖基化终末产物对破骨细胞迁移和黏附能力可能存在抑制效应。"

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