Rosmarinic acid activates the Nrf2/HO-1 axis and suppresses NF-κB to protect against gentamicin-induced acute kidney injury

BACKGROUND: Rosmarinic acid (RA), a polyphenolic ester abundant in Melissa officinalis, exhibits potent antioxidant properties. While the traditional use of M. officinalis for detoxification is well documented, the molecular mechanisms by which RA protects against drug-induced acute kidney injury (AKI), particularly whether it actively induces endogenous cytoprotective pathways, remain incompletely defined. PURPOSE: This study investigated whether RA activates the Nrf2/HO-1 antioxidant pathway to confer nephroprotection in a rat model of gentamicin-induced AKI, and explored additional anti-inflammatory and mitochondrial protective effects. METHODS: RA was extracted from M. officinalis and characterized by HPLC-DAD, LC-Q-TOF-MS NMR, and FTIR (purity: 85.0 ± 3.2%; DPPH IC₅₀ = 12.5 µM) (Results shown in Supplementary Figures S3 to S6). Male Wistar rats received gentamicin (100 mg/kg/day, i.p.) alone or with RA (50 or 100 mg/kg/day, p.o.) for 7 days. A vehicle control group (0.5% CMC with residual diethyl ether) was included. Renal function, oxidative stress markers, Nrf2/HO-1 pathway activation (plus NQO1 and GCLC by qPCR), pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), NF-κB activation, 8-OHdG (DNA oxidation), and mitochondrial membrane potential were assessed. Histopathological damage was scored by two blinded pathologists (interobserver κ = 0.86). Acute oral toxicity followed OECD 425. RESULTS: Gentamicin induced severe renal dysfunction, oxidative stress, DNA damage, and inflammation. RA (100 mg/kg) significantly attenuated these effects: creatinine ↓68%, urea ↓59%, MDA ↓58%, and restored GSH, SOD, CAT, GPx. RA enhanced nuclear Nrf2 accumulation (2.5-fold), HO-1 expression (2.1-fold), and upregulated NQO1 (2.3-fold) and GCLC (2.0-fold). RA also reduced TNF-α (↓72%), IL-1β (↓65%), IL-6 (↓68%), nuclear NF-κB (↓61%), and 8-OHdG (↓58%), while restoring mitochondrial membrane potential. Quantitative histopathology confirmed that RA reduced tubular necrosis scores from 2.8 to 0.9 (p < 0.01). The vehicle control group showed no effect on any parameter (Supplementary Table S2). RA alone showed no toxicity. CONCLUSION: Rosmarinic acid exerts dose-dependent nephroprotection against gentamicin-induced AKI through dual mechanisms: direct radical scavenging, activation of the Nrf2/HO-1 pathway (including NQO1 and GCLC), suppression of NF-κB-mediated inflammation, and mitochondrial protection. These findings position RA as a mechanistically validated natural product candidate for adjunctive therapy, while acknowledging the need for bioavailability and pharmacokinetic studies.