Yin Lu-Yun, Feng Hui-Min, Qiu Fang, Yan Jing, Jin Xiao-Fei
The Second Clinical College of Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China.
Zhen Ci Yan Jiu. 2024;49(9):933-942. doi: 10.13702/j.1000-0607.20230396.
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·d on the 1 day and 8 mg·kg·d from the 2 to 15 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 (<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 (<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 (<0.01, <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 (<0.01, <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 (<0.05, <0.01), and in down-regulating the level of MDA and the number of atretic oocytes <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.
观察从“秩边”（BL54）透刺至“水道”（ST28）对卵巢早衰（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共激活因子1α（PGC - 1α）/核因子E2相关因子2（Nrf2）信号通路的影响，以探讨其改善POI的作用机制。
将48只雌性SD大鼠平均随机分为空白对照组、POI模型组、浅刺组和透刺组（从“秩边”［BL54］透至“水道”［ST28］）。采用腹腔注射环磷酰胺建立POI模型（第1天50 mg·kg·d，第2至15天8 mg·kg·d，共15天）。造模成功后，浅刺组大鼠将毫针垂直刺入BL54，进针深度约5 - 8 mm，留针30 min；透刺组大鼠将毫针垂直刺入BL54区域，然后斜刺至同侧ST28，进针深度约12 - 15 mm，留针30 min（双侧穴位同时针刺）。每日治疗1次，每周6次，共4周。干预后，采用酶联免疫吸附测定法检测血清卵泡刺激素（FSH）、黄体生成素（LH）、雌二醇（E2）和抗苗勒管激素（AMH）含量；采用比色法检测卵巢组织中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性及丙二醛（MDA）含量。苏木精 - 伊红（H.E.）染色后观察卵巢组织的病理变化。分别采用免疫组织化学、实时荧光定量PCR和蛋白质免疫印迹法检测卵巢组织中SIRT1、PGC - 1α、Nrf2 mRNA及蛋白的免疫活性和表达水平。
造模后，大鼠发情周期紊乱，血清FSH、LH水平显著升高，E2水平明显低于空白对照组（<0.01），表明POI模型成功建立。与空白对照组相比，模型组血清FSH、LH、卵巢MDA含量及闭锁卵泡数增加（<0.01），血清E2、AMH含量、卵巢SOD和CAT活性、生长卵泡数、卵巢SIRT1、PGC - 1α、Nrf2蛋白及mRNA的免疫活性和表达水平降低（<0.01，<0.05）。干预后，BL54 - ST28透刺可逆转血清FSH、LH升高水平及卵巢MDA含量、闭锁卵泡数，以及E2、AMH含量、卵巢SOD和CAT活性、生长卵泡数、卵巢SIRT1、PGC - 1α、Nrf2蛋白及mRNA的降低（<0.01，<0.05），浅刺除对血清FSH、LH、E2和AMH含量有影响外，对其他指标无明显改善作用。透刺在上调血清AMH、卵巢SOD和CAT水平、生长卵泡数及卵巢SIRT1、PGC - 1α、Nrf2蛋白及mRNA表达，下调MDA水平和闭锁卵泡数方面效果明显优于浅刺（<0.05，<0.01）。
BL54透刺至ST28可增加POI大鼠卵巢生长卵泡数，减少闭锁卵泡数，调节血清激素水平，减轻卵巢氧化应激水平，其机制可能与激活卵巢SIRT1/PGC - 1α/Nrf2信号通路有关。
