Cistanche attenuates acute kidney injury by inhibiting ferroptosis through the disruption of Keap1-Nrf2 interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferroptosis, an iron-dependent regulated cell death, critically contributes to acute kidney injury (AKI) progression. Emerging evidence suggests ferroptosis inhibition as a therapeutic strategy against AKI. Cistanche (Cis), a kidney-tonifying herb in traditional Chinese medicine, exhibits antioxidant activity via nuclear factor erythroid 2-related factor 2 (Nrf2) activation. However, the precise molecular mechanism underlying cistanche-mediated Nrf2 activation remains undefined, and its nephroprotective potential in AKI is incompletely characterized.

AIM OF THE STUDY: To investigate the renoprotective effects of cistanche against AKI and elucidate its mechanism through the Keap1-Nrf2-ferroptosis axis.

MATERIALS AND

METHODS

An ischemia-reperfusion injury (IRI)-induced AKI mouse model and a hypoxia-reoxygenation-induced mRTEC cell model were constructed to evaluate the protective effects of cistanche. First, cistanche (2.055, 4.110 and 8.220 g/kg), and saline were administered for 6 days. On the 5th day, the mice were anesthetized and then bilateral renal artery clamping was performed for 45 min. Mice in the sham-operated group underwent the same operation but no arterial clamping was performed. Until 24 h after the IRI procedure, all mice were anesthetized, then blood samples and kidney tissue were collected. Subsequently, Nrf2 knockout mRTEC cells were used to verify whether Nrf2 mediated the nephroprotective effects of cistanche. Furthermore, the effect of cistanche on Keap1-Nrf2 protein-protein interaction (PPI) in the kidney of IRI mice was verified by CO-IP assay. Finally, UPLC-Q-TOF/MS technique and molecular docking were utilized to analyze the potential pharmacological components of cistanche.

RESULTS

In vivo, cistanche treatment improved renal function in IRI mice, as shown by decreased serum creatinine (SCr), blood urea nitrogen (BUN), inflammatory factors, and renal injury markers (NGAL, KIM-1), along with ameliorated ferroptosis-related abnormalities. In vitro, cistanche suppressed ferroptosis in hypoxia-reoxygenation-induced HK2 cells by activating Nrf2, an effect that was significantly diminished upon Nrf2 silencing in mRTEC cells. Mechanistically, cistanche facilitated Nrf2 nuclear translocation and inhibited ferroptosis, likely by disrupting the Keap1-Nrf2 protein-protein interaction. Furthermore, UPLC-Q/TOF-MS analysis identified key bioactive components in cistanche that may contribute to its anti-ferroptosis effects.

CONCLUSION

Overall, cistanche functions as a putative Keap1-Nrf2 PPI inhibitor primarily through its phenylethanoid glycosides. By disrupting Keap1-Nrf2 binding, it activates Nrf2 signaling, suppresses ferroptosis, and confers protection against AKI, positioning it as a promising therapeutic candidate.