Mechanistic Insights into Salidroside's Mitochondrial Protection <em>via</em> AMPK/Sirt1/HIF-1α Pathway in Hypoxic HT22 Cells

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.