Xia Tian-Shuang, Xu Sheng-Yan, Lai Li-Yong, Jiang Yi-Ping, Wang Na-Ni, Xin Hai-Liang
School of Pharmacy, Naval Medical University, Shanghai, 200433, China.
Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, Zhejiang, 310007, China.
J Food Drug Anal. 2024 Dec 15;32(4):506-519. doi: 10.38212/2224-6614.3508.
Bitter acids (BA) are main component of Humulus lupulus L. (hops). They are known for beer brewing and have various biological and pharmacological properties, especially the bone-protective effect confirmed by our previous in vivo study. Here we aimed to elucidate the anti-senior osteoporosis (SOP) effect of BA on osteoblasts and explore its underlying mechanism. In vitro SOP model was established by D-galactose (D-gal) injured osteoblasts, and the bone formation markers and apoptosis level were measured. mCherry-EGFP-LC3 adenovirus infection and autophagic markers including beclin1 and LC3 proteins were detected to investigate the autophagy level in osteoblasts. To further verify whether BA play the bone-protective role through regulating autophagy, the autophagy inhibitor 3-MA was used, and the cell proliferation, ALP activity, bone mineralization, apoptosis rate and SA-β-gal staining areas were measured. Finally, the protein expressions of AKT/mTOR signaling pathway were detected by Western blotting, and AKT agonist SC79 and mTOR agonist MHY1485 were used to further study the mechanism of BA on AKT/mTOR-mediated autophagy. The results showed that BA stimulated osteoblastic differentiation and inhibited apoptosis proteins Bcl-2/Bax in D-gal-treated osteoblasts. BA also increased the expression of autophagic markers beclin1 and LC3-II/LC3-I in D-gal-treated osteoblasts. mCherry-EGFP-LC3 autophagic double fluorescent adenovirus showed BA promoted the generation of autolysosomes and autophagosomes in D-gal-injured osteoblasts, indicating that BA might prevent osteoblastic bone loss through activating autophagy. Autophagy inhibitor 3-MA was used to further verify whether BA played the bone-protective role via regulating autophagy. The results revealed the promotion effects of BA on proliferation, ALP activity, and mineralized nodule formation in D-gal-injured osteoblasts were eliminated after autophagy blocking with 3-MA, and the inhibitory effects of BA on apoptosis rate and SA-β-gal staining areas were also eliminated. Moreover, BA reduced the phosphorylation levels of AKT, mTOR, p70S6K, and 4EBP in AKT/mTOR pathway, and the promotion of BA on the autophagic markers was blocked after the activation of AKT and mTOR by SC79 and MHY1485. In conclusion, it was the first time to demonstrate that BA improved cell activities and bone formation in aging osteoblasts, and revealed the mechanism of BA against SOP in osteoblasts was activating AKT/mTOR-mediated autophagy.
苦味酸（BA）是啤酒花的主要成分。它们因用于啤酒酿造而闻名，并且具有多种生物学和药理学特性，尤其是我们之前的体内研究证实的骨保护作用。在此，我们旨在阐明BA对成骨细胞的抗老年骨质疏松症（SOP）作用，并探索其潜在机制。通过D-半乳糖（D-gal）损伤成骨细胞建立体外SOP模型，并检测骨形成标志物和凋亡水平。检测mCherry-EGFP-LC3腺病毒感染以及包括beclin1和LC3蛋白在内的自噬标志物，以研究成骨细胞中的自噬水平。为了进一步验证BA是否通过调节自噬发挥骨保护作用，使用了自噬抑制剂3-MA，并检测细胞增殖、碱性磷酸酶（ALP）活性、骨矿化、凋亡率和衰老相关β-半乳糖苷酶（SA-β-gal）染色面积。最后，通过蛋白质免疫印迹法检测AKT/mTOR信号通路的蛋白表达，并使用AKT激动剂SC79和mTOR激动剂MHY1485进一步研究BA对AKT/mTOR介导的自噬的作用机制。结果表明，BA刺激D-gal处理的成骨细胞的成骨分化并抑制凋亡蛋白Bcl-2/Bax。BA还增加了D-gal处理的成骨细胞中自噬标志物beclin1和LC3-II/LC3-I的表达。mCherry-EGFP-LC3自噬双荧光腺病毒显示BA促进了D-gal损伤的成骨细胞中自溶酶体和自噬体的产生，表明BA可能通过激活自噬来预防成骨细胞的骨质流失。使用自噬抑制剂3-MA进一步验证BA是否通过调节自噬发挥骨保护作用。结果显示，在用3-MA阻断自噬后，BA对D-gal损伤的成骨细胞增殖、ALP活性和矿化结节形成的促进作用被消除，并且BA对凋亡率和SA-β-gal染色面积的抑制作用也被消除。此外，BA降低了AKT/mTOR通路中AKT、mTOR、p70S6K和4EBP的磷酸化水平，并且在通过SC79和MHY1485激活AKT和mTOR后，BA对自噬标志物的促进作用被阻断。总之，首次证明BA改善了衰老成骨细胞的细胞活性和骨形成，并揭示了BA在成骨细胞中对抗SOP的机制是激活AKT/mTOR介导自噬。
