Study on the mechanism of acupuncture in improving ovarian function by promoting ovarian angiogenesis in rats based on KEAP1/PGAM5/AIFM1 pathway

ObjectiveTo observe the effect of acupuncture of “Guanyuan” （CV4）-“Sanyinjiao” （SP6） on ovarian angiogenesis in rats with diminished ovarian reserve （DOR） by regulating the kelch-like ECH-associated protein 1 （KEAP1）/phosphoglycerate mutase 5 （PGAM5）/mitochondrial apoptosis-inducing factor 1 （AIFM1） pathway， so as to explore its mechanisms underlying improvement of DOR.MethodsEighteen female SD rats were randomly divided into blank， model， and acupuncture groups， with 6 rats in each group. The DOR model was established by a single intraperitoneal injection of 75 mg/kg cyclophosphamide. After modeling， the rats of the acupuncture group received manual acupuncture stimulation of CV4 and SP6 for 1 min， with the needle retained for 20 min， once every other day， for a total of 15 times. Vaginal exfoliated cytology was used to observe the rat’s estrous cycle. The levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E2）， and anti-Mullerian hormone （AMH） in the serum were detected by ELISA. The ovarian area and the peak systolic velocity （PSV）， end-diastolic velocity （EDV）， resistance index （RI）， and pulsatility index （PI） of the ovarian artery were estimated using a small animal ultrasonography， and the PSV/EDV （S/D） was calculated. Morphological changes of ovarian tissue and follicle growth were observed by H.E. staining， and the level of reactive oxygen species （ROS） in the ovarian tissue was detected by flow cytometry. The expression levels of vascular endothelial growth factor （VEGF）， platelet endothelial cell adhesion molecule 1 （CD31）， KEAP1， PGAM5 and AIFM1 in the ovarian tissue were detected by immunofluorescence staining.ResultsCompared with the blank group， the model group had a significant decrease in the ovarian area， serum E2 and AMH contents， numbers of the primordial follicles， secondary follicles and mature follicles， and the levels of VEGF and CD31（PPPConclusionAcupuncture of CV4 and SP6 can improve the ovarian reserve function of DOR rats， which may be related to its functions in inhibiting the KEAP1/PGAM5/AIFM1 pathway of ovarian tissue， promoting ovarian angiogenesis， and ultimately improving blood perfusion.