Osthol effects on the proliferation and differentiation of osteoblasts based on endoplasmic reticulum stress

doi:10.3969/j.issn.2095-4344.0802

Abstract

Abstract:

BACKGROUND: Osthol has been reported to promote osteogenesis by increasing osteoblast proliferation, but the anti-osteoporosis mechanism underlying osthol is poorly understood.
OBJECTIVE: To observe the effect of osthol on the proliferation and differentiation of rat osteoblasts in vitro and to explore its mechanism of anti-osteoporosis effect.
METHODS: Rat osteoblasts were isolated by secondary enzyme digestion and identified by alkaline phosphatase staining and mineralized nodule staining. There were five groups: blank control, solvent control, β-estradiol as well as low-, medium- and high-dose osthol groups. Cell proliferation was detected by cell counting kit-8 assay, and cell differentiation was evaluated by detection of alkaline phosphatase and mineralized nodule staining. The expression levels of GRP78, PDI and CHOP were detected by western blot assay.
RESULTS AND CONCLUSION: Osthol at the concentrations of 1x10-4 and 1x10-5 mol/L could inhibit osteoblast proliferation. 1x10-4 mol/L osthol could increase the activity of alkaline phosphatase in osteoblasts and enhance osteoblastic mineralization. Meanwhile, 1x10-4 mol/L osthol was able to down-regulate the expression level of GRP78 and up-regulate the expression levels of PDI and CHOP. To conclude, osthol can promote osteoblast differentiation and inhibit osteoblast proliferation probably by endoplasmic reticulum stress.

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: Cnidium, Osteoblasts, Endoplasmic Reticulum, Tissue Engineering

CLC Number:

R318

Zheng Su-yang1, Ma Yong1, 2, Guo Yang1, Wang Li-ning1, Pan Ya-lan1. Osthol effects on the proliferation and differentiation of osteoblasts based on endoplasmic reticulum stress[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(12): 1817-1822.

Figures/Tables 2

2.1 成骨细胞的鉴定 2.1.1 形态学观察在倒置显微镜下观察，刚接种的成骨细胞呈球形，悬浮于培养基中(图1A)；24 h后经过换液，存活细胞已完全贴壁展开，此时细胞形态不规则，呈三角形或多角形，有较多突起(图1B)。生长期细胞分离相多见，细胞突起互相连接(图1C)。汇合时，细胞呈铺路石状(图1D)。 2.1.2 碱性磷酸酶染色经偶氮偶联法染色后，胞浆中有许多紫红色颗粒，使成骨细胞胞体呈现紫红色，胞质丰富而清晰。使用甲基绿复染细胞核，可以清晰地看到单个细胞核，呈卵圆形，含有1-3个核仁(图2A，B)。 2.1.3 矿化结节染色采用茜素红法行矿化结节染色，染色前矿化结节表现为暗黄色颗粒状不透光结晶，茜素红染色后矿化结节呈红色(图2C)。 2.2 蛇床子素对成骨细胞增殖的影响含药完全培养基干预后24，48 h，各浓度蛇床子素均能抑制成骨细胞增殖；药物干预后72 h，10-4 mol/L与10-5 mol/L的蛇床子素可以抑制成骨细胞增殖；药物干预后24，48，72 h，10-8 mol/L的β-雌二醇均能抑制成骨细胞增殖，但差异无显著性意义，而10-4 mol/L与10-5 mol/L的蛇床子素抑制成骨细胞增殖效应强于10-8 mol/L的β-雌二醇(表1)。 2.3 蛇床子素对成骨细胞分化的影响 2.3.1 蛇床子素对成骨细胞碱性磷酸酶活力的影响含药完全培养基干预24，48，72 h后，10-4 mol/L的蛇床子素均能提高成骨细胞内碱性磷酸酶活力，而10-8 mol/L的β-雌二醇虽然可以提高成骨细胞内碱性磷酸酶活力，但差异无显著性意义。10-4 mol/L的蛇床子素促进成骨细胞碱性磷酸酶活力的效应强于10-8 mol/L的β-雌二醇(表2)。 2.3.2 蛇床子素对成骨细胞矿化能力的影响 10-4 mol/L的蛇床子素可以增强成骨细胞的矿化能力，而10-8 mol/L的β-雌二醇及10-5 mol/L、10-6 mol/L的蛇床子素虽然也能增强成骨细胞的矿化能力，但与溶剂对照组相比差异无显著性意义(表3)。 2.4 内质网应激相关蛋白检测 10-4 mol/L的蛇床子素可以减少GRP78蛋白表达，增加PDI蛋白表达；10-6 mol/L的蛇床子素可以降低PDI蛋白的表达。CHOP只有在高浓度(10-4 mol/L)蛇床子素组有明显表达，其余各组表达量极小(图3)。"

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