“阴三针”对卵巢早衰小鼠卵巢功能及卵巢TNF-α、Caspase-3蛋白表达的影响

Effect of‘Yin-Three-Needle’on Ovarian Function and Protein Expressions of TNF-αand Caspase-3 in Mice with POF

目的:通过观察卵巢早衰(POF)模型小鼠血清中雌二醇(E_(2))、卵泡刺激素(FSH)、促黄体生成素(LH)和下丘脑中β-内啡肽(β-EP)水平的变化、卵巢组织形态变化及TNF-α、Caspase-3蛋白在卵巢中的表达,探讨卵巢早衰的生理病理变化及“阴三针”对卵巢早衰的改善、治疗作用。从而以“经脉所过,主治所及;腧穴所在,主治所及”的针灸理论为基础,以“阴三针”为治疗组方,为临床上电针延缓生殖轴衰老的作用机制提供依据。方法:用雷公藤多苷片45 mg/kg灌胃模拟出卵巢早衰小鼠模型,6周后将小鼠随机分为空白对照组、模型组、电针组、坤泰胶囊药物组与假电针组。干预3周后,利用酶联免疫吸附法(ELISA)检测血清中E_(2)、FSH、LH水平及下丘脑中β-EP水平;HE染色观察卵巢组织形态的变化;免疫印迹法检测TNF-α、Caspase-3蛋白在卵巢中的表达。结果:与空白对照组相比,POF模型小鼠血清中FSH、LH升高,E_(2)和下丘脑中β-EP显著下降,卵巢颗粒细胞层减少、生长卵泡和黄体减少及闭锁细胞增多,卵巢中TNF-α蛋白的表达显著升高,Caspase-3蛋白的表达无显著变化;与POF模型组小鼠相比,电针组小鼠血清中LH降低,E_(2)和下丘脑中β-EP提高,卵巢颗粒细胞层增多、闭锁细胞减少、生长卵泡和黄体明显增加,卵巢中TNF-α蛋白的表达显著降低,Caspase-3蛋白的表达无显著变化。结论:本研究结果表明“阴三针”可以促进下丘脑、垂体和卵巢分泌相关激素,抑制颗粒细胞凋亡,使卵巢功能得到改善或恢复,最终达到下丘脑、垂体对卵巢及卵巢对下丘脑、垂体的正负反馈调节。

Objective:To explore the physiological and pathological changes of premature ovarian failure(POF)and the improving and therapeutic effect of‘Yin-three-needle’on POF,by observing the the changes of estradiol(E_(2)),follicle-stimulating hormone(FSH)and luteinizing hormone(LH)in serum and beta-endorphin(β-EP)in hypothalamus,as well as ovarian tissue morphology changes and protein expressions of TNF-αand Caspase-3 in ovarian.To further provide theoretical basis for the mechanism of EA in delaying the aging of reproductive axis in clinical practice of‘Yin-three-needle’based on the theory of acupuncture and moxibustion in which‘where meridian passes,the indications are involved;where acupoints locates,the indications are reached’.Methods:The mice model of POF was simulated by 45mg/kg tripterygium wilfordii polyglycoside tablets through intragastric administration.After 6 weeks,the modeling mice were randomly divided into the model group,the electro-acupuncture(EA)group,the Kuntai Capsule group and the sham EA group,and the normal mice was taken as the blank control.After 3 weeks of intervention,the levels of E_(2),FSH and LH in serum and the content ofβ-EP in hypothalamus were determined by ELISA.HE staining was used to observe the morphological changes of ovarian tissue.The protein expressions of TNF-αand Caspase-3 in ovary were detected by Western blot.Results:Compared with those in the blank control group,the serum levels of FSH and LH were increased,the serum level of E_(2)andβ-EP in hypothalamus were significantly decreased,ovarian granulosa cell was reduced,follicular growth and luteal growth were decreased,atresia cells were increased,and the protein expression of TNF-αin ovary was significantly increased,but the protein expression of Caspase-3 was not significantly changed in the model group.Compared with those in the model group,the serum level of LH was decreased,the serum level of E_(2)andβ-EP in hypothalamus were increased,ovarian granulosa cell layer was increased,atresia cells were decreased,follicle growth and luteum were increased significantly,the protein expression of TNF-αin ovary was decreased significantly,but the protein expression of Caspase-3 was not significantly changed in the EA group.Conclusion:The results of this experiment show that the‘Yin-three-needle’can promote hypothalamus,pituitary and ovary secreting related hormones,inhibit apoptosis of granulosa cells,improve or restore ovarian function,and finally achieve positive and negative feedback regulation of hypothalamus-pituitary to ovary and ovary to hypothalamus-pituitary.