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  • Title: [Effect of electroacupuncture on autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency].
    Author: Zhang Y, Huang S, Shi X, Zhang D, Li X, She Y.
    Journal: Zhongguo Zhen Jiu; 2024 Jun 12; 44(6):676-84. PubMed ID: 38867630.
    Abstract:
    OBJECTIVE: To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. METHODS: Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. RESULTS: The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. CONCLUSION: EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB. 目的：观察电针对早发性卵巢功能不全（POI）模型大鼠卵巢颗粒细胞自噬的影响，探讨电针改善POI的作用机制。方法：从32只雌性SD大鼠中随机选取8只作为空白组，剩余大鼠作为造模组腹腔注射环磷酰胺15 d（第1天剂量为50 mg/kg，第2天到第15天剂量为8 mg/kg）建立POI模型，将造模成功的大鼠随机分为模型组、电针组和雌二醇组，每组8只。电针组大鼠予电针双侧“公孙”，连续波，频率2 Hz，电流强度0.1~1 mA，每日1次，每次20 min，共14 d。雌二醇组大鼠予戊酸雌二醇溶液（0.01 mg/mL）灌胃（10 mL/kg），每日1次，共14 d。造模前后采用快速革兰氏染色观察大鼠动情周期变化。干预后，采用HE染色法观察大鼠卵巢组织形态；ELISA法检测大鼠血清卵泡刺激素（FSH）、雌二醇（E2）、抗苗勒管激素（AMH）、抑制素B（INHB）含量；免疫荧光染色法观察大鼠卵巢颗粒细胞p62阳性表达；透射电镜观察大鼠卵巢颗粒细胞结构，并比较自噬小体和自噬溶酶体数量；Western blot法和实时荧光定量PCR法检测大鼠卵巢组织p62、Beclin-1、微管相关蛋白1A/1B-轻链3（LC3）蛋白和mRNA表达。结果：空白组大鼠阴道涂片结果呈规律的周期变化；造模组大鼠阴道涂片表现为动情周期延长或周期停滞，多处于动情前期或动情间期，卵巢整体萎缩，整体结构紊乱，颗粒细胞减少。与空白组比较，模型组大鼠血清FSH含量升高（P<0.01），E2、AMH和INHB含量降低（P<0.01）；卵巢颗粒细胞p62阳性表达减少（P<0.01）；卵巢颗粒细胞明显肿胀，线粒体轻中度肿胀，粗面内质网略显扩张，高尔基体肥大，卵巢自噬小体和自噬溶酶体数量增加（P<0.01）；卵巢组织p62蛋白及mRNA表达降低（P<0.01），Beclin-1、LC3蛋白表达及mRNA表达升高（P<0.01）。与模型组比较，电针组和雌二醇组大鼠血清FSH含量下降（P<0.01），E2、AMH和INHB含量升高（P<0.01）；卵巢颗粒细胞p62阳性表达增加（P<0.01）；卵巢颗粒细胞损伤程度有所缓解，细胞器形态较为完整，卵巢自噬小体和自噬溶酶体数量减少（P<0.01）；卵巢组织p62蛋白和mRNA表达升高（P<0.01），Beclin-1、LC3蛋白和mRNA表达降低（P<0.01）。结论：电针“公孙”可通过减少自噬小体和自噬溶酶体数量，上调p62表达，下调Beclin-1、LC3表达，从而抑制卵巢颗粒细胞自噬，调节大鼠血清FSH、E2、AMH和INHB水平，改善POI卵巢储备功能。.
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