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骨质疏松症(OP)是一种由骨吸收和骨形成之间的关系失衡造成,以低骨量和骨组织微结构破坏为特征,导致骨质脆性增加和易于骨折的全身性骨代谢疾病。其中,成骨细胞是骨形成的功能细胞,在维持骨稳态中起到关键作用[1]。目前,高氧化应激相关的骨丢失已成为骨质疏松研究领域的热点。有研究表明,细胞保护酶是机体对抗氧化应激状态下活性氧(ROS)损伤的主要机制,其活性主要由转录因子Nrf2和FoxO调控,而二者所介导的氧化应激通路同样被证实具有调节成骨细胞氧化还原平衡以及促进骨形成分化的功能[2]。与此同时,β-淀粉样蛋白(amyloid β-protein,Aβ)的沉积可使机体ROS生成增多,进而抑制成骨细胞的增殖、成骨基质的产生及矿化[3]。由此可见,Aβ沉积偶联的氧化损伤是破坏成骨细胞骨形成,进而引发骨丢失的一大诱因。
啤酒花(Hops, Humulus lupulus L.)为桑科葎草属多年生草质蔓生藤本植物,其雌性球穗花序不仅作为啤酒酿造的添加原料,也是全球广泛应用的植物药,在欧洲广泛用于缓解更年期潮热及绝经后骨质疏松症[4]。我们前期研究发现啤酒花能够促进成骨细胞骨矿化结节的形成,降低活性氧水平,并显著改善APP/PS1转基因小鼠的骨丢失[5-6],但其对外源性Aβ损伤成骨细胞的氧化应激水平及骨形成的影响尚不明确。此外,我们首次确认了氧化应激和Aβ沉积之间的双向关联,及其在老年性骨质疏松症发病中的重要作用[7-8]。故本文拟以Aβ损伤的成骨细胞为模型,以Nrf2和FoxO1两条经典氧化应激相关通路为核心,对啤酒花的抗氧化能力及对骨形成干预作用进行探究。
Hops extract alleviates Aβ-injury to osteoblasts through antioxidant pathway
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摘要:
目的 探讨啤酒花改善β-淀粉样蛋白(Aβ)损伤成骨细胞的骨形成作用及其机制。 方法 以新生24 h Wistar大鼠所分离的成骨细胞为研究对象,用Aβ1-42寡聚体对成骨细胞进行损伤,并用啤酒花提取物进行药物干预。分别采用MTT法、碱性磷酸酶(ALP)活性检测以及茜素红染色法评价成骨细胞的增殖、分化及骨矿化水平,流式细胞仪检测成骨细胞凋亡。采用蛋白质印迹法检测骨形成相关蛋白及氧化应激Nrf2、FoxO1通路蛋白的表达水平,并用免疫荧光检测FoxO1蛋白的入核表达。 结果 啤酒花提取物可显著促进Aβ损伤成骨细胞的增殖,提高ALP活性及骨矿化结节水平,抑制细胞凋亡率,并促进骨形成相关蛋白I型胶原酶(COL-I)及骨桥蛋白(OPN)的表达。此外,啤酒花提取物可显著激活Aβ损伤成骨细胞的Nrf2和FoxO1信号通路,促进该氧化应激信号通路相关蛋白的表达,通过抗氧化维持骨代谢平衡。 结论 本研究表明啤酒花具有减轻Aβ损伤成骨细胞的作用,初步阐明其作用机制与抗氧化有关,为抗骨质疏松作用机制及药物研发提供了新思路。 Abstract:Objective To explore the effects of Humulus lupulus L. extract (HLE) and its mechanism on improving bone formation of Aβ-injured osteoblasts. Methods Osteoblasts isolated from 24 h-old Wistar rats were injured by Aβ1-42 oligomer and intervened with HLE. The proliferation, differentiation and bone mineralization of osteoblasts were determined by MTT assay, alkaline phosphatase (ALP) activity assay and alizarin red staining, respectively. The apoptosis of osteoblasts was detected by flow cytometer. The expression levels of bone formation related proteins, and proteins of Nrf2 and FoxO1 pathways were measured by Western blotting analysis. The intranuclear expression of FoxO1 protein was detected by immunofluorescence. Results HLE significantly improved the cell proliferation, ALP activity and bone mineralization, and inhibited the apoptosis of Aβ-injured osteoblasts. HLE also significantly promoted the expressions of collagen type Ι (COL-I) and osteopontin (OPN) in Aβ-injured osteoblasts. HLE notably activated the Nrf2 and FoxO1 signaling pathways in Aβ-injured osteoblasts by promoting the expressions of related proteins and maintained bone metabolism through relieving oxidative stress. Conclusion This study confirms that HLE can alleviate Aβ-injury to osteoblasts, and preliminarily clarifies the mechanism being related to antioxidation, which provides a new reference for the mechanism research and drugs development for anti-osteoporosis. -
Key words:
- Hops (Humulus lupulus L.) /
- Aβ /
- osteoblast /
- antioxidation /
- osteoporosis
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