Ameliorative Effects of the Neng-Jing-Huo Essential Oil Blend on H<sub>2</sub>O<sub>2</sub>-induced Oxidative Stress Injury in NRK-52E Cells via the AMPK/Nrf2/HO-1 Signaling Pathway

Oxidative stress-induced renal tubular epithelial cell apoptosis or dysfunction promotes the progression of acute kidney injury and chronic kidney disease. Neng-Jing-Huo (NJH), an essential oil mixture of Gaultheria procumbens, Zingiber officinale, Bulnesia sarmientoi, Artemisia vulgaris, and Styrax benzoin, exhibits antioxidant potential. However, its specific action mechanisms remain unclear. Therefore, in this study, we aimed to investigate the protective effects of NJH against hydrogen peroxide (H₂O₂)-induced oxidative stress injury using NRK-52E renal tubular epithelial cells and assess the underlying mechanisms. Cell viability, reactive oxygen species level, mitochondrial membrane potential, apoptosis, and aging were evaluated via MTT assay, DCFH2-DA, JC-1, TUNEL, and red beta-D-galactopyranoside staining, respectively. Additionally, mRNA and protein expression levels were analyzed via semi-quantitative reverse transcription-PCR and western blotting, respectively. Notably, NJH alleviated H₂O₂-induced DNA damage and mitochondrial dysfunction and inhibited reactive oxygen species accumulation by activating the AMPK/Nrf2/HO-1 signaling pathway. Furthermore, NJH inhibited oxidative stress-induced apoptosis by upregulating the Bcl-2 levels and downregulating the Bax levels, thereby preventing the activation of caspase-9 and caspase-3. Senescence-associated β-galactosidase activity was significantly increased by H₂O₂; however, this effect was dose-dependently attenuated by NJH. The findings highlight NJH as a potential candidate for the prevention and treatment of oxidative stress injury-associated kidney disorders.