[Effect of electroacupuncture on ACSL4/LPCAT3/ALOX15 signaling pathway of ferroptosis in rats with premature ovarian insufficiency].

OBJECTIVES: To observe the effect of electroacupuncture (EA) on long-chain acyl-CoA synthetase 4 (ACSL4)/ phosphatidylcholine acyltransferase 3 (LPCAT3)/ arachidonic acid lipoxygenase 15 (ALOX15) signaling pathway in the ovarian tissue in rats with premature ovarian insufficiency (POI), so as to explore its mechanisms underlying improvement of POI from the perspective of ferroptosis.

METHODS: Forty-eight female SD rats were randomly divided into control, POI model, EA and hormone replacement treatment (medication) groups, with 12 rats in each group. The POI model was established by intraperitoneal injection of cyclophosphamide (50 mg/kg on the 1st day, then 8 mg·d-1·kg-1 for 14 d). EA (2 Hz) was applied to "Guanyuan"(CV4), bilateral "uterus"(EX-CA1) and "Sanyinjiao"(SP6) for 20 min each time. Rats of the medication group were treated by gavage of estradiol valerate for 4 consecutive days, with medroxyprogesterone acetate additionally administered on the 4th day. The treatment of both EA and medication was conducted once daily, with 4 d + one day's rest being a cycle, lasting for 4 cycles (20 d). After the intervention, the serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-mullerian hormone (AMH) contents were detected by ELISA. The H.E. staining was used to observe the morphological changes of the ovarian tissue. The contents of serum iron (Fe), glutathione (GSH), malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were detected by colorimetry. The level of reactive oxygen species (ROS) in the ovarian tissue was detected by flow cytometry. The ultrastructure of mitochondria in the ovarian tissue was observed by transmission electron microscope. The mRNA and protein expression levels of ACSL4, LPCAT3, and ALOX15 in the ovarian tissue were detected separately by real-time fluorescence quantitative PCR and Western blot.

RESULTS: Compared with the control group, the model group had a significant increase in the serum FSH, LH, Fe, MDA contents, and ovarian ROS mean fluorescence intensity, and the mRNA and protein expression levels of ACSL4, LPCAT3 and ALOX15 (P<0.01), and a striking decrease in the contents of serum E2, AMH, GSH, and SOD activity (P<0.01) in the model group. After the treatment, both the increased contents of serum FSH, LH, Fe, MDA contents, and ovarian ROS mean fluorescence intensity, and ACSL4, LPCAT3 and ALOX15 mRNA and protein expressions and the decreased contents of serum E2, AMH, GSH and SOD activity were completely reversed in the EA and medication groups (P<0.01). No significant differences were found between the EA and medication groups in up-regulating and down-regulating all the related indexes mentioned above. Morphological observation showed decreased number of growing follicles at all levels, increased number of atretic follicles, abnormal structure of follicles, disordered arrangement of the granulosa cell layer, increase of damage of the mitochondria and decrease or even disappearance of the mitochondrial cristae in the ovarian tissues in the model group, which was apparently milder in the injury state in both EA and medication groups.

CONCLUSIONS: EA can regulate hormone levels and improve ovarian function in POI rats, which may be related to its function in regulating ACSL4/LPCAT3/ALOX15 signaling pathway, reducing lipid peroxide accumulation and inhibiting ferroptosis.