Kaempferol exerts antioxidant effects in age‐related diminished ovarian reserve by regulating the HSP90/NRF2 pathway

Abstract Kaempferol is the active ingredient of Er‐Xian decoction (EXD), a traditional Chinese medicine formula used clinically to treat ovarian dysfunction, but the mechanism of kaempferol relieving age‐related diminished ovarian reserve (AR‐DOR) is still unclear. In this study, 36 volunteers and 78 DOR patients (37 patients with EXD treatment) were enrolled in the clinical research. Meanwhile, 32‐week‐old female mice were used to establish the AR‐DOR model, and these model mice were intragastrically administered with 100 mg/kg kaempferol in the presence or absence of 200 mg/kg geranylgeranylacetone (GGA) or 1 mg/kg geldanamycin (GDA). The effects of kaempferol on serum hormone levels and oxidative stress‐related indexes were detected by enzyme‐linked immunosorbent assay. Antral follicle count (AFC) was determined by hematoxylin–eosin staining. The protein levels of HSP90 and nuclear factor erythroid 2‐related factor 2 (NRF2) were assayed by Western blot. This study displayed that the serum anti‐Mullerian hormone (AMH) level in DOR patients with EXD treatment was higher than that in DOR patients without EXD treatment. Kaempferol treatment reversed the low levels of AMH, estradiol (E2), AFC, superoxide dismutase (SOD), and catalase (CAT), as well as the high levels of follicle‐stimulating hormone (FSH), reactive oxygen species (ROS), and malonaldehyde (MDA). The results showed that HSP90 was predicted to have high affinity with kaempferol, and its expression was inhibited by kaempferol, while the expression of NRF2, the target of HSP90, was up‐regulated by kaempferol. However, the above effects of kaempferol were reversed by GGA. On the contrary, GDA enhanced the therapeutic effects of kaempferol on AR‐DOR mice. Moreover, the treatment of kaempferol resulted in a reduction in the phosphorylation level of heat shock factor 1 (HSF1), the transcription factor associated with HSP90, and an increase in the phosphorylation level of Src, a client protein of HSP90. In summary, kaempferol exerts an antioxidant effect on AR‐DOR by inhibiting HSP90 expression to up‐regulate NRF2 expression. This study provides a theoretical basis for the clinical application of kaempferol in AR‐DOR.