Effects of penetration needling from "Zhibian" (BL54) to "Shuidao" (ST28) on SIRT1/PGC-1α/Nrf2 signaling pathway in rats with premature ovarian insufficiency

Abstract

To observe the effect of penetration needling from "Zhibian" (BL54) to "Shuidao"(ST28) on silencing information regulator 1 (SIRT1) /peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) /nuclear factor E2 related factor 2 (Nrf2) signaling in rats with premature ovarian insufficiency (POI), so as to explore its mechanisms underlying improvement of POI. A total of 48 female SD rats were equally and randomly allocated to blank control, POI model, shallow needling and penetration needling (from "Zhibian" ［BL54］ to "Shuidao" ［ST28］) groups. The POI model was established by intraperitoneal injection of cyclophosphamide (50 mg·kg-1·d-1 on the 1st day and 8 mg·kg-1·d-1 from the 2nd to 15th day, for a total of 15 days). After successful modeling, for rats of the shallow needling group, a filiform needle was inserted into BL54 to a depth about 5-8 mm, and then retained for 30 min. And for rats of the penetration needling group, a filiform needle was inserted into BL54 area and advanced to the unilateral ST28 to a depth about 12-15 mm, and then retained for 30 min (bilateral acupoints were used at the same time). The treatments were conducted once daily, 6 times a week for 4 weeks. After the interventions, the contents of serum follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-Müllerian hormone (AMH) were detected using ELISA, and the activity of superoxide dismutase (SOD), catalase (CAT) and content of malondialdehyde (MDA) in the ovarian tissue were detected using colorimetry. Histopathological changes of the ovarian tissue were observed after H.E. staining. The immunoactivities and expression levels of SIRT1, PGC-1α, and Nrf2 mRNA and protein in the ovarian tissues were detected using immunohistochemistry, quantitative real-time PCR and Western blot, respectively. After modeling, the rats' estrus cycles were disordered, contents of serum FSH and LH levels significantly increased, and the E2 level markedly decreased compared with those of the blank control group (P<0.01), indicating that the POI model was successfully established. Relevant to the blank control group, the model group had an increase in serum FSH and LH, ovarian MDA contents, and the number of atretic oocytes (P<0.01), and a decrease in serum E2 and AMH contents, ovarian SOD and CAT activities, number of growing oocytes, immunoactivities and expressions of ovarian SIRT1, PGC-1α and Nrf2 protein and mRNA (P<0.01, P<0.05). Following interventions, both the increased levels of serum FSH and LH and ovarian MDA contents, and the number of atretic oocytes, and the decreased levels of E2 and AMH contents, ovarian SOD and CAT activities, number of growing oocytes, immunoactivities and expressions of ovarian SIRT1, PGC-1α and Nrf2 protein and mRNA were reversed by penetration needling of BL54-ST28 (P<0.01, P<0.05), but not by shallow needling, except serum FSH, LH, E2 and AMH contents. The effects of penetration needling were obviously superior to those of shallow needling in up-regulating the levels of serum AMH, ovarian SOD and CAT, number of growing oocytes, and the expressions of ovarian SIRT1, PGC-1α and Nrf2 protein and mRNA (P<0.05, P<0.01), and in down-regulating the level of MDA and the number of atretic oocytes (P<0.05). Penetration needling stimulation of BL54 to ST28 can increase the number of ovarian growing oocytes and reduce the number of atretic oocytes, regulate the serum hormone levels and relieve the ovarian oxidative stress level in POI rats, which may be associated with its functions in activating ovarian SIRT1/PGC-1α/Nrf2 signaling pathway.