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  • Title: [Effects of penetration needling from "Zhibian" (BL54) to "Shuidao" (ST28) on SIRT1/PGC-1α/Nrf2 signaling pathway in rats with premature ovarian insufficiency].
    Author: Yin LY, Feng HM, Qiu F, Yan J, Jin XF.
    Journal: Zhen Ci Yan Jiu; ; 49(9):933-942. PubMed ID: 39401830.
    Abstract:
    OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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. 目的: 观察“秩边透水道”针法对早发性卵巢功能不全（POI）大鼠沉默信息调节因子1（SIRT1）/过氧化物酶体增殖物激活受体-γ辅激活因子1α（PGC-1α）/核因子E2相关因子2（Nrf2）信号通路的影响，探讨“秩边透水道”针法治疗POI的氧化应激机制。方法: 随机将48只SD雌性大鼠平均分为空白组、模型组、浅刺组和秩边透水道组，每组12只。采用腹腔注射环磷酰胺制备POI大鼠模型。模型建立后，浅刺组采用0.20 mm×13 mm不锈钢毫针针刺大鼠双侧“秩边”，秩边透水道组采用0.20 mm×25 mm不锈钢毫针针刺大鼠双侧“秩边”并透向同侧“水道”，均每次30 min，每日1次，6次/周，连续4周。治疗结束后，ELISA法检测大鼠血清促卵泡激素（FSH）、黄体生成素（LH）、雌二醇（E2）和抗米勒管激素（AMH）含量；采用比色法检测卵巢组织超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、丙二醛（MDA）含量；HE染色法观察大鼠卵巢组织形态的变化并计算卵泡数目；免疫组织化学法、Western blot法检测大鼠卵巢组织SIRT1、PGC-1α、Nrf2蛋白表达；实时荧光定量PCR法检测大鼠卵巢组织中SIRT1、PGC-1α、Nrf2 mRNA表达。结果: 造模结束后，除空白组外，其余大鼠动情周期均紊乱，且FSH、LH含量较空白组升高（P<0.01），E2含量降低（P<0.01），提示造模成功。干预后，与空白组比较，模型组大鼠血清FSH、LH含量升高（P<0.01），E2、AMH含量降低（P<0.01），卵巢组织SOD、CAT活性降低（P<0.01），MDA含量升高（P<0.01），生长卵泡数目减少（P<0.01），闭锁卵泡数目增加（P<0.01），SIRT1、PGC-1α、Nrf2蛋白和mRNA表达水平均降低（P<0.01，P<0.05）。相较于模型组，两针刺组大鼠血清FSH、LH含量降低（P<0.01），E2、AMH含量升高（P<0.01）；秩边透水道组卵巢组织SOD、CAT活性上升（P<0.05）、MDA含量下降（P<0.01），生长卵泡数目增加（P<0.01），闭锁卵泡数目减少（P<0.01），SIRT1、PGC-1α、Nrf2蛋白及mRNA表达水平均上升（P<0.01，P<0.05）。与浅刺组比较，秩边透水道组大鼠血清AMH含量升高（P<0.01），卵巢组织SOD、CAT活性上升（P<0.05），MDA含量下降（P<0.05），生长卵泡数目增加（P<0.05），闭锁卵泡数目减少（P<0.05），SIRT1、PGC-1α、Nrf2蛋白及mRNA表达水平均升高（P<0.05）。结论: “秩边透水道”针法可以改善POI大鼠血清性激素水平及卵巢氧化应激水平，进而改善POI大鼠卵巢功能，其作用机制可能与SIRT1/PGC-1α/Nrf2信号通路的激活有关。.
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