经皮穴位电刺激对早发性卵巢功能不全大鼠卵巢颗粒细胞凋亡的作用机制

Mechanism of action of transcutaneous electric acupoint stimulation on ovarian granulosa cell apoptosis in premature ovarian insufficiency rats

目的 探讨经皮穴位电刺激(TEAS)对早发性卵巢功能不全(POI)大鼠卵巢颗粒细胞凋亡的作用机制。方法 将40只SPF级SD雌性大鼠按照随机数字表法分成4组,对照组、模型组、TEAS组和激素组。除对照组外,其余各组均使用雷公藤多苷片连续灌胃14 d建立POI大鼠模型。造模成功后,TEAS组给予电子针疗仪刺激20 min,1次/d,共干预2周;激素组给予戊酸雌二醇溶液1 mg/(kg·d)灌胃,对照组和模型组仅给予相同时间的抓取和固定,不予任何治疗。采用酶联免疫吸附试验检测大鼠血清卵泡刺激素(FSH)、雌二醇(E_(2))、抗米勒管激素(AMH)水平,TUNEL检测卵巢颗粒细胞凋亡情况,Western blot法检测卵巢组织微小RNA-23a(miR-23a)、SMAD同源物5(SMAD5)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白的表达。结果 与对照组比较,模型组大鼠血清E_(2)、AMH水平降低,FSH水平升高(P<0.05);颗粒细胞凋亡率均升高(P<0.05);与模型组比较,TEAS组及激素组大鼠血清E_(2)、AMH水平均升高,FSH水平均降低,颗粒细胞凋亡率均下降(P<0.05)。与对照组比较,模型组大鼠卵巢SMAD5蛋白表达减少,而mi R-23a、Caspase-3蛋白表达增加(P<0.05);与模型组比较,TEAS组及激素组大鼠卵巢SMAD5蛋白表达增加,mi R-23a、Caspase-3蛋白表达减少(P<0.05)。结论 TEAS能够有效调节POI模型大鼠血清激素水平,可能通过mi R-23a/SMAD5信号通路,下调凋亡调控蛋白Caspase-3的表达,从而减少颗粒细胞的过度凋亡,改善卵巢功能。

Objective To investigate the mechanism of action of transcutaneous electric acupoint stimulation(TEAS)on ovarian granulosa cell apoptosis in premature ovarian insufficiency(POI)rats.Methods Forty SD female rats with regular estrous cycle were randomly divided into control group,model group,TEAS group,and hormone group,with 10 rats in each group.Except the control group,the POI rat model was established in the other groups by continuous intragastric administration of tripterygium glycoside tablets.After successful modeling,TEAS group was stimulated with an electronic needle therapy device for 20 minutes,once a day for two weeks.Meanwhile,the Hormone group was given estradiol valerate solution 1 mg/(kg·d)by intragastric administration.The control group and model group were only given the same time to grasp and fixation.Serum levels of follicle stimulating hormone(FSH),estradiol(E_(2))and anti-mullerian hormone(AMH)were detected by ELISA kit,and apoptosis of ovarian granulosa cells was detected by TUNEL.The expressions of miR-23a,SMAD5,and cysteine aspartate proteinase 3(Caspase-3)in ovarian tissue were determined by Western blot.Results Compared with the control group,the serum levels of E_(2) and AMH in model group were decreased,the level of FSH was increased(P<0.05),and the apoptosis rate of granulosa cells was increased(P<0.05).Compared with the model group,the serum E_(2) and AMH levels were increased,FSH level was decreased,and granulosa cell apoptosis rates was decreased in the TEAS group and the hormone group(P<0.05).Compared with the control group,the expression of SMAD5 protein in ovary of model group was decreased,while the expression of miR-23a and Caspase-3 protein were increased(P<0.05).Compared with the model group,the expression of SMAD5 protein in ovary of the TEAS group and the hormone groups were increased,and the expression of miR-23a and Caspase-3 protein were decreased(P<0.05).Conclusion TEAS can effectively regulate serum hormone levels in POI model rats,possibly by down-regulating the expression of Caspase-3 protein through miR-23a/SMAD5 signaling pathway,thereby reducing the apoptosis rate of granulosa cells and regulating ovarian function.