摘要:
目的 研究巴戟天丸防治D-半乳糖（D-gal）损伤成骨细胞骨丢失的作用及机制。 方法 采用新生24 h Wistar大鼠提取的原代成骨细胞，利用D-gal对细胞进行干预，并给予巴戟天丸提取物行药物治疗。分别采用MTT法和碱性磷酸酶试剂盒评价细胞的增殖和分化水平；采用DCFH-DA荧光探针对成骨细胞内活性氧（ROS）水平进行测定。采用Western blotting法对磷酸化蛋白激酶B（p-AKT）、蛋白激酶B（AKT）、血红素氧合酶1（HO-1）、醌NADPH脱氢酶1（NQO1）等氧化相关蛋白的表达进行检测，并采用免疫荧光法测定细胞核因子E2相关因子2（Nrf2）的核内表达水平。 结果 巴戟天丸能显著提高D-gal干预细胞的增殖水平和ALP活性，并显著降低细胞内ROS水平。巴戟天丸能够显著促进细胞AKT蛋白的磷酸化，提高HO-1、NQO1的表达水平，进而激活PI3K/AKT信号通路。此外，巴戟天丸提取物还可显著促进成骨细胞核内Nrf2的表达，激活Nrf2信号通路，促进骨形成。 结论 本研究首次明确了巴戟天丸可防治D-半乳糖损伤引起的成骨细胞骨丢失，其作用机制可能与调控PI3K/AKT和Nrf2信号通路关联的氧化应激有关。
Abstract:
Objective To explore the effect and mechanism of Bajitianwan on preventing D-galactose (D-gal)-induced osteoblast bone loss. Methods Osteoblasts isolated from 24 h old Wistar rats were injured by D-gal and intervened with Bajitianwan extract. The osteoblastic proliferation and differentiation were determined by MTT and alkaline phosphatase (ALP), respectively. The cell reactive oxygen species (ROS) levels were detected by DCFH-DA fluorescent probes. The expression of cellular oxidation-related protein nuclear factor erythroid 2-related factor 2 (Nrf2), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase 1 (NQO1) were detected by Western blotting. The intranuclear expression of Nrf2 protein was measured by immunofluorescence. Results Bajitianwan extract had significantly increased the osteoblastic proliferation and differentiation, and significantly reduced the intracellular ROS level. Bajitianwan extract had activated the PI3K/AKT pathway via activating the phosphorylation of AKT in osteoblasts, and promoted NQO1 and HO-1 expression. In addition, Bajitianwan had significantly promoted the expression of Nrf2 in the nucleus of osteoblasts, activating Nrf2 signaling pathway, and further promoted bone formation. Conclusion This study confirmed that Bajitianwan could prevent D-gal injured osteoblastic bone loss for the first time. The mechanism might be related to the regulation of oxidative stress associated PI3K/AKT and Nrf2 signaling pathway.
