坤泰胶囊对卵巢储备功能低下大鼠的治疗作用及对卵巢GDF-9、Egr-1表达的影响

Effects of Kuntai Capsules on expression of ovarian GDF-9 and egr-1 in rats with diminished ovarian reserve

目的探讨坤泰胶囊对注射用环磷酰胺致卵巢储备功能低下大鼠的治疗作用及机制。方法将60只12周龄SD雌性大鼠随机分为对照组、模型组和坤泰胶囊高、中、低剂量(1.2、0.6、0.3 g/kg)组、结合雌激素片组,ip注射用环磷酰胺建立卵巢储备功能低下大鼠模型,各给药组于造模14 d后分别ig给药,连续给药36 d。观察大鼠一般状态、体质量改变,定期行阴道涂片观察大鼠动情周期的变化;酶联免疫吸附法(ELISA)检测大鼠血清性激素抗穆勒管激素(AMH)、卵泡刺激素(FSH)、黄体生成激素(LH)、雌二醇(E_2)水平变化;光镜下观察卵巢形态与卵泡数;实时荧光定量PCR(qRT-PCR)检测大鼠卵巢生长分化因子9(GDF-9)、早期生长反应因子1(Egr-1)mRNA表达水平的变化;免疫组化法检测大鼠卵巢GDF-9、Egr-1蛋白表达水平的变化。结果与模型组比较,坤泰胶囊组大鼠精神逐渐好转,饮食恢复正常,毛色有光泽,大便色深;体质量渐增,差异显著(P<0.05);动情周期紊乱得到明显改善(P<0.05);卵巢存在成熟卵泡,卵泡腔内卵子生长良好;大鼠血清FSH、LH水平明显降低,E_2、AMH水平明显升高,差异有统计学意义(P<0.05);GDF-9、Egr-1蛋白及基因表达明显增加,差异有统计学意义(P<0.05),且作用均呈剂量相关性,高剂量组治疗效果与结合雌激素片无明显差距。结论坤泰胶囊能够修复大鼠受损的卵巢组织结构,提高成熟卵泡存在数量,改善大鼠动情周期及激素水平,机制可能与增加GDF-9、Egr-1表达相关。

Objective To explore the therapeutic effects and mechanism of Kuntai Capsules on ovarian reserve dysfunction in rats induced by cyclophosphamide injection. Methods The 60 12-week-old SD female rats were randomly divided into control group, model group, Kuntai Capsules high, middle, and low dose（1.2, 0.6, and 0.3 g/kg） groups, and estrogen group. Cyclophosphamide peritoneal injection was used to establish the model of ovarian reserve dysfunction. Rats in each drug group were ig administrated successively for 36 d after 14 d of modeling. During the experiment, the behavior and the changes of body mass of ratswere observed. Regular vaginal smear was done to observe the changes of estrus cycle in rats. Serum sex hormone levels changes of antimullerian hormone（AMH）, and follicle-stimulating hormone（FSH）, luteinizing hormone（LH）, and Estradiol（E2） were detected by enzyme-linked immunosorbent assay（ELISA）. The ovary morphology and follicular number were observed under light microscope. Real-time fluorescence quantitative PCR（qRT-PCR） was used to detect the changes in mRNA expression levels of growth differentiation factor 9（GDF-9） and early growth response gene 1（Egr-1） in rats. The expression levels of GDF-9 and Egr-1 in rats were measured by immunohistochemistry. Results Compared with the model group, the spirit of the rats in the Kuntai Capsules group gradually improved, the diet was restored to normal, the hair color was luster, the color of the stool was deep, the body mass was increasing, the difference was significant（P 〈 0.05）; The disorder of the estrous cycle was obviously improved（P 〈 0.05）; The ovary had mature follicles and the follicles grew well in the follicle cavity; The level of FSH and LH in the serum of rats decreased significantly, the level of E2 and AMH increased significantly（P 〈 0.05）, and the expression of GDF-9, Egr-1 protein and gene increased significantly（P 〈 0.05）, and the effect was in a dose-dependent manner. There were no significant therapeutic effects between Kuntai Capsules high dose group and Conjugated Estrogens Tablets group. Conclusion Kuntai Capsules could repair damaged ovarian tissue of rats structure, increase the number of mature follicles, and improve estrous cycle and hormone level in rats, which may be related to the increased expression of GDF-9 and Egr-1.