Based on the theory of "Gongsun (SP4) connects the Thoroughfare Vessel" of the "eight confluence acupoints", the effect of electroacupuncture (EA) at SP4 on Kelch-like epichlorohydrin associated protein factor 1 (Keap1)/ nuclear factor-erythroid 2 related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) signaling pathway was observed in rats with premature ovarian insufficiency (POI) for exploring its underlying mechanisms in treating POI. Thirty-two female SD rats were randomly divided into 4 groups:blank, model, EA and medication, with 8 rats in each group. The POI model was established by intraperitoneal injection of cyclophosphamide. EA (2 Hz, a strength inducing slight limb tremor) was applied to bilateral SP4 for 20 min, once a day for a total of 14 d. Rats of the medication group were treated by gavage of solution of estradiol valerate tablets (0.09 mg/kg) once a day for 14 d. During the experiment, the changes of body mass in each group were recorded and the general conditions were observed. After the interventions, the contents of follicle-stimulating hormone (FSH), estradiol (E2), anti-mullerian hormone (AMH), malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) were detected by ELISA. The morphological changes of ovarian tissue were observed under light microscope after H.E. staining. The protein and mRNA expression levels of Keap1, Nrf2 and HO-1 in the ovarian were detected by immunohistochemistry, Western blot, and PCR, respectively. After modeling, the model rats had poor mental state, reduced activity, irritability, emaciated body, dry and dull hair, decreased food intake, and occasionally yellow soft stools, black stools, and bloody secretions around the eyes and the tip of the nose. After the interventions, the mental state of rats in the EA and medication groups was recovered, and had brown oval feces, with no bloody secretion. Compared with the blank group, the body mass, levels of serum E2, AMH, SOD, GSH-Px, immunoactivity of Nrf2 and HO-1, and expression levels of Nrf2 and HO-1 proteins and mRNAs in the ovarian tissue were significantly decreased (P<0.01), and the contents of serum FSH and MDA, immunoactivities and expressions of Keap1 protein and mRNA were obviously increased (P<0.01) in the model group. Following the interventions, both the decrease in the levels of E2, AMH, SOD, GSH-Px, and the protein and mRNA expression levels of Nrf2 and HO-1 and the increase of contents of serum FSH and MDA, immunoactivities and expressions of Keap1 protein and mRNA were reversed in both EA and medication groups (P<0.01, P<0.05), without significant differences between the two groups in all the indexes mentioned above. H.E. staining showed a decrease in the primordial and growing follicles in the ovarian cortex and an increase in the atretic follicles and disordered and loose arrangement of the granular layer in the model group, and an increase in the primordial and growing follicles in the ovarian cortex, reduction in the atretics follicles, thickened granular layer and closer arrangement in the EA and medication groups. EA of SP4 can promote the restoration of the body mass, regulate the level of serum sex hormones, improve the body's antioxidant capacity, reduce the damage of oxidative stress of ovaries and promote the normal development of follicles in POI rats, which may be related to its function in regulating Keap1/Nrf2/HO-1 signaling pathway.
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