Advanced glycation end products modulate osteoclastic acidification by inhibiting the expression of V-ATPase a3 and ClC-7

doi:10.3969/j.issn.2095-4344.2017.12.004

Abstract

Abstract:

BACKGROUND: The effect of advanced glycation end products (AGEs) on bone resorption is controversial. Our previous study has shown that bone resorption is significantly inhibited when AGEs present with pre-osteoclast cells RAW 264.7, while the effect of AGEs on osteoclastic acidification remains unknown.
OBJECTIVE: To investigate the effect of AGEs on osteoclastic acidification and the underlying mechanism.
METHODS: RAW 264.7 cells were induced by RANKL (15 μg/L; normal group) to generate osteoclasts, and AGEs (50-400 mg/L; experimental group) or bovine serum albumin (100 mg/L; control group) were added at the beginning of the induction. The effect of AGEs on bone resorption was evaluated by analyzing the area of bone resorption on the Osteo Assay Surface plates, and the effect of AGEs on osteoclastic acidification was evaluated by acridine orange staining. Furthermore, the expression levels of V-ATPase a3 and ClC-7 were detected to investigate the underlying mechanism.
RESULTS AND CONCLUSION: The bone resorption area in the AGEs group was significantly decreased compared with the normal group (P < 0.05). Acridine orange staining revealed that the red fluorescence (620 nm) intensity in the AGEs group was significantly decreased compared with the normal group (P < 0.05), and this inhibitory effect became obvious with the increase of AGEs concentration. Immunocytochemistry, western blot assay and PCR findings showed that the expression levels of V-ATPase a3 and ClC-7 in the AGEs group were decreased significantly compared with the normal group (P < 0.05). To conclude, AGEs exert inhibitory effect on osteoclastic acidification, probably by inhibiting the expression of V-ATPase a3 and ClC-7.

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

Wang Hai-xing1, Li Zi-qing1, Xiao Yin-bo1, Zhang Zi-ji1, Zhang Yang-chun1, Yang Xing1, Li Chao-hong2, Sheng Pu-yi1. Advanced glycation end products modulate osteoclastic acidification by inhibiting the expression of V-ATPase a3 and ClC-7[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(12): 1826-1832.

Figures/Tables 2

2.1 晚期糖基化终末产物对破骨细胞骨吸收的影响破骨细胞诱导第5天，正常组的骨基质被大量溶解吸收，而晚期糖基化终末产物组的骨吸收总面积随着刺激浓度的增加呈减少趋势，且在浓度超过100 mg/L后趋势显著(P < 0.05)；牛血清白蛋白组的骨吸收总面积相较正常组未见减少(图1)。此结果提示，晚期糖基化终末产物作用于破骨细胞前体可显著抑制骨吸收功能。 2.2 晚期糖基化终末产物对破骨细胞泌酸功能的影响晚期糖基化终末产物对破骨细胞泌酸功能的影响通过吖啶橙染色加以探究。吖啶橙是一种荧光色素染料，该染料的发射荧光波长与其浓度相关，在低浓度时发出绿色荧光 (图2A)，而在高浓度(高于100 nmol/L)时转换为橙红色荧光[21]。吖啶橙可透过细胞膜，但在酸性环境中被质子化后会丧失膜通透性，因此吖啶橙会在活细胞的酸性囊泡中高浓度聚积而发出橙红色荧光(图2B)。在本研究中，吖啶橙染色显示正常组的破骨细胞胞质及细胞边缘出现大量发出橙红色荧光的酸性囊泡，提示破骨细胞泌酸功能良好。随着晚期糖基化终末产物浓度的增高，破骨细胞的橙红色荧光强度逐渐减弱而绿色荧光强度逐渐增强，牛血清白蛋白组与正常组相比差异无显著性意义(图3A)。利用多功能酶标仪对各组的红色荧光强度 (620 nm发射波长)进行定量检测发现，当晚期糖基化终末产物浓度高于100 mg/L时，破骨细胞的红色荧光强度相较正常组显著下降(P < 0.05)，而牛血清白蛋白组相较正常组差异无显著性意义(图3B)。此结果表明晚期糖基化终末产物可干扰破骨细胞转运囊泡的正常酸化，减弱其胞外泌酸功能，进而影响破骨细胞的骨基质溶解吸收能力。 2.3 晚期糖基化终末产物对破骨细胞V-ATPase a3及ClC-7表达水平的影响 2.3.1 晚期糖基化终末产物抑制V-ATPase a3的表达免疫细胞化学染色的结果显示，晚期糖基化终末产物组的V-ATPase a3表达量较正常组有所下降，在浓度超过 100 mg/L后抑制效应显著(P < 0.05)，牛血清白蛋白组相较正常组差异无显著性意义(图4A，B)；实时定量PCR的结果显示，100 mg/L的晚期糖基化终末产物刺激破骨细胞前体24，48及72 h后V-ATPase a3的mRNA表达量均较正常组显著下降(P < 0.05)(图4C)。 2.3.2 晚期糖基化终末产物抑制ClC-7的表达 Western Blot结果显示，晚期糖基化终末产物组的氯离子通道ClC-7表达量随着刺激浓度的增高逐渐下降，在浓度超过100 mg/L时差异有显著性意义(P < 0.05)，牛血清白蛋白组的ClC-7表达与正常组相比差异无显著性意义(图5A，B)；实时定量PCR结果提示，100 mg/L的晚期糖基化终末产物刺激破骨细胞前体24，48及72 h后的mRNA表达量较正常组有所下降，在 48 h和72 h时差异有显著性意义(P < 0.05，图5C)。"

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