[Electroacupuncture promotes expression of glutathione related regulatory enzymes in ovary tissue of rats with diminished ovarian reserve].

To observe the effect of electroacupuncture (EA) on ovarian function and expression of glutathione (GSH) related regulatory enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) protein and gene in rats with diminished ovarian reserve (DOR), so as to explore its mechanisms underlying up-regulation of antioxidant stress ability.A total of 30 female SD rats with normal estrous cycle were randomly divided into blank control, model and EA groups, with 10 rats in each group. The DOR model was established by gavage of tripterygium wilfordii polyglycoside suspension (50 mg·kg-1·d-1) for 14 consecutive days, while the rats in the blank group were given equal volume of 0.9% sodium chloride solution. One hour after daily gavage, EA (1.0 mA, 100 Hz) was applied alternately to bilateral "Shenshu"(BL23), and "Zhongwan"(CV12)+"Guanyuan"(CV4) for 10 min, for 14 consecutive days. Estrous cycles of rats in each group were observed and recorded daily during intervention.After the intervention, H.E.staining was used to observe histopathological changes of the ovarian tissue. The contents of serum sex hormones ［follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), estradiol (E2)］ and oxidative damage markers ［8-hydroxydeoxyguanosine (8-OHDG) and nitrotyrosine (NTY)］ were determined by ELISA. The contents of GSH and oxidized glutathione (GSSG) in the liver tissue were determined by colorimetry, and their ratios were calculated. Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the immunoactivity and gene expression levels of γ-GCS and GR in the ovarian tissues, respectively.Compared with the blank group, the model group had a marked increase in the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents (P<0.01) and a considerable decrease in the levels of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number as well as the expression of γ-GCS and GR mRNAs (P<0.05, P<0.01). After EA intervention, the increase of the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents and the decrease of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number of γ-GCS and GR as well as the expression of γ-GCS genes were all reversed (P<0.01, P<0.05). H.E. staining showed degenerative changes of the ovarian tissue, fewer follicles at every level and increase of atretic follicles, disarrangement and layer number decrease of granulosa cells, and atrophy of corpus luteum in the model group, which were relatively milder in the EA group.EA can improve ovarian function, and reduce oxidative stress damage in DOR rats, which may be associated with its functions in up-regulating the expression of γ-GCS and GR protein and gene in the ovarian tissue.目的：观察电针对卵巢储备功能减退(DOR)大鼠卵巢功能的保护作用及对谷胱甘肽(GSH)相关调控酶γ-谷氨酰半胱氨酸合成酶(γ-GCS)、谷胱甘肽还原酶(GR)蛋白及基因表达的影响，进一步探讨电针提高DOR模型大鼠抗氧化应激能力的可能机制。方法：雌性SD大鼠随机分为空白组、模型组、电针组，每组10只。采用雷公藤多苷片混悬液(50 mg·kg-1·d-1)连续灌胃14 d复制DOR模型。电针组每日灌胃1 h后隔日交替电针双侧“肾俞”和“中脘”“关元”，每次10 min，连续14 d。干预期间每日记录各组大鼠的动情周期，计算动情周期紊乱率。干预结束后，采用HE染色法观察大鼠卵巢病理形态学变化；ELISA法检测大鼠血清卵泡刺激素(FSH)、抗苗勒管激素(AMH)、雌二醇(E2)、8-羟基脱氧鸟苷(8-OHdG)、硝基酪氨酸(NTY)的含量；比色法检测肝脏组织谷胱甘肽(GSH)及氧化型谷胱甘肽(GSSG)含量，并计算两者比值；免疫组织化学法及荧光定量PCR法分别检测卵巢组织γ-GCS、GR蛋白及基因表达水平。结果：与空白组比较，模型组大鼠的动情周期紊乱率升高(P<0.01)；卵巢组织卵泡颗粒细胞层次紊乱，闭锁卵泡增多，黄体固缩；血清FSH、8-OHdG、NTY含量升高(P<0.01)，AMH、E2含量降低(P<0.05，P<0.01)；肝脏组织GSH、GSSG含量降低(P<0.05，P<0.01)，GSH/GSSG降低(P<0.01)；卵巢组织γ-GCS、GR蛋白及基因表达降低(P<0.01)。与模型组比较，电针组动情周期紊乱率下降(P<0.01)；卵巢卵泡颗粒细胞层排列整齐，闭锁卵泡减少，黄体发育良好；血清FSH、8-OHdG、NTY含量降低(P<0.01，P<0.05)，AMH、E2含量升高(P<0.05，P<0.01)；肝脏组织GSH、GSSG含量升高(P<0.05，P<0.01)，GSH/GSSG升高(P<0.05)；卵巢γ-GCS、GR蛋白表达升高(P<0.01)，γ-GCS mRNA表达升高(P<0.05)。结论：电针可调节DOR大鼠的动情周期，改善性激素水平，减轻机体氧化损伤程度，增加卵巢组织γ-GCS、GR蛋白及基因表达，提高肝脏GSH水平，提示电针可能通过调节卵巢GSH相关调控酶蛋白及mRNA表达，提高机体抗氧化应激能力，改善卵巢功能。.