Wang Xiaobo, Zhang Yating, Hou Ya, Jiang Hong, Kuang Tingting, Li Rui, Meng Xianli
School of Pharmacy/School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine; Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine; Research Service Office, Traditional Chinese Medicine Hospital of Meishan;
School of Pharmacy/School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine.
J Vis Exp. 2025 Apr 25(218). doi: 10.3791/66923.
Salidroside (Sal), an active ingredient of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba has been found to exert mitochondrial protective effects by improving metabolism and enhancing the energy supply of brain cells under hypoxic conditions. However, its mechanism of action has not been fully clarified. In the present study, high-performance liquid chromatography was first employed to analyze the effects of Sal on nucleotide (ATP, ADP, and AMP) levels. The cellular thermal shift assay (CETSA), a widely used molecular interaction method for validating and quantifying drug target engagement in cells and tissues across different species, was then chosen to confirm the affinity of Sal for AMPK/Sirt1/HIF-1α pathway-related proteins. The results revealed that Sal increased ATP and ADP levels in hypoxic HT22 cells while reducing AMP levels. Moreover, Sal exhibited stable binding to AMPKα, p-AMPKα, Sirt1, and HIF-1α proteins. In conclusion, Sal may exert mitochondrial protective effects by modulating the AMPK/Sirt1/HIF-1α pathway to regulate nucleotide content. This study provides a methodological reference for nucleotide content analysis in cell samples and contributes to the identification and discovery of targets for compounds derived from traditional Chinese medicine.
红景天苷（Sal）是大花红景天（Hook. f. et Thoms.）H. Ohba的一种活性成分，已发现其在缺氧条件下可通过改善代谢和增强脑细胞能量供应发挥线粒体保护作用。然而，其作用机制尚未完全阐明。在本研究中，首先采用高效液相色谱法分析红景天苷对核苷酸（ATP、ADP和AMP）水平的影响。然后选择细胞热位移分析（CETSA），这是一种广泛用于验证和量化不同物种细胞和组织中药物靶点结合的分子相互作用方法，以确认红景天苷对AMPK/Sirt1/HIF-1α通路相关蛋白的亲和力。结果显示，红景天苷可增加缺氧HT22细胞中的ATP和ADP水平，同时降低AMP水平。此外，红景天苷与AMPKα、p-AMPKα、Sirt1和HIF-1α蛋白表现出稳定结合。总之，红景天苷可能通过调节AMPK/Sirt1/HIF-1α通路来调节核苷酸含量，从而发挥线粒体保护作用。本研究为细胞样本中核苷酸含量分析提供了方法学参考，并有助于鉴定和发现中药衍生化合物的靶点。
