背景:Angelicin,在补骨脂中发现,可以通过阻止破骨细胞的形成来帮助预防骨质疏松症,尽管确切的机制尚不清楚。
方法:我们使用去卵巢骨质疏松模型大鼠和RAW264.7细胞评价当归素对破骨细胞氧化应激水平的影响。使用H&E染色和micro-CT研究了股骨骨量的变化。通过DHE荧光标记研究ROS含量。使用蛋白质印迹法检测破骨细胞相关基因和蛋白质的表达,免疫组织化学,抗酒石酸酸性磷酸酶染色,和实时定量PCR。还使用MTT测定法评估了当归素对破骨细胞发育的影响,双荧光素测定,染色质免疫沉淀,免疫沉淀和KAT6AsiRNA转染。
结果:用当归素治疗的大鼠具有相当高的骨矿物质密度和较少的破骨细胞。当归素受RANKL刺激时,在体外阻止RAW264.7细胞分化成破骨细胞。实验显示ROS水平降低,细胞内KAT6A显著上调,HO-1和Nrf2在当归素处理后。破骨细胞特有基因的表达,如MMP9和NFATc1也下调。最后,KAT6AsiRNA转染增加细胞内ROS水平,同时降低KAT6A,Nrf2和HO-1蛋白在破骨细胞中的表达。然而,在没有KAT6AsiRNA转染的情况下,当归素大大抵消了破骨细胞的这种作用。
结论:当归素增加了KAT6A的表达。这种增强的KAT6A表达有助于激活Nrf2/HO-1抗氧化应激系统并降低破骨细胞中的ROS水平。从而抑制氧化应激水平和破骨细胞形成。
BACKGROUND: Angelicin, which is found in Psoralea, can help prevent osteoporosis by stopping osteoclast formation, although the precise mechanism remains unclear.
METHODS: We evaluated the effect of angelicin on the oxidative stress level of osteoclasts using ovariectomized osteoporosis model rats and RAW264.7 cells. Changes in the bone mass of the femur were investigated using H&E staining and micro-CT. ROS content was investigated by DHE fluorescence labelling. Osteoclast-related genes and proteins were examined for expression using Western blotting, immunohistochemistry, tartrate-resistant acid phosphatase staining, and real-time quantitative PCR. The influence of angelicin on osteoclast development was also evaluated using the MTT assay, double luciferin assay, chromatin immunoprecipitation, immunoprecipitation and
KAT6A siRNA transfection.
RESULTS: Rats treated with angelicin had considerably higher bone mineral density and fewer osteoclasts. Angelicin prevented RAW264.7 cells from differentiating into osteoclasts in vitro when stimulated by RANKL. Experiments revealed reduced ROS levels and significantly upregulated intracellular
KAT6A, HO-1, and Nrf2 following angelicin treatment. The expression of genes unique to osteoclasts, such as MMP9 and NFATc1, was also downregulated. Finally,
KAT6A siRNA transfection increased intracellular ROS levels while decreasing
KAT6A, Nrf2, and HO-1 protein expression in osteoclasts. However, in the absence of KAT6A siRNA transfection, angelicin greatly counteracted this effect in osteoclasts.
CONCLUSIONS: Angelicin increased the expression of
KAT6A. This enhanced
KAT6A expression helps to activate the Nrf2/HO-1 antioxidant stress system and decrease ROS levels in osteoclasts, thus inhibiting oxidative stress levels and osteoclast formation.