基于氧化应激探讨疏肝补肾法对卵巢早衰大鼠颗粒细胞凋亡的影响  

Effect of Soothing Liver and Tonifying Kidney on Apoptosis of Granulosa Cells in Rats with Premature Ovarian Failure based on Oxidative Stress

作　　者：王敏[1,2] 张斐 王洋 WANG Min;ZHANG Fei;WANG Yang(Research Base of Traditional Chinese Medicine Syndrome,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;College of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]福建中医药大学中医证研究基地,福建福州350122 [2]福建中医药大学中医学院,福建福州350122

出　　处：《中医药学报》2024年第5期30-37,共8页Acta Chinese Medicine and Pharmacology

基　　金：国家自然科学基金青年科学基金项目(82004257);福建省自然科学基金项目(2021J01933)。

摘　　要：目的:探讨疏肝补肾法对卵巢早衰大鼠颗粒细胞凋亡的作用机制。方法:将48只SD大鼠随机分为正常组、模型组、柴胡疏肝散合左归饮低剂量组、柴胡疏肝散合左归饮中剂量组、柴胡疏肝散合左归饮高剂量组、雌激素组,每组8只。除正常组外,其余大鼠腹腔注射环磷酰胺连续15 d建立卵巢早衰大鼠模型,同时采用慢性不可预知温和应激方式建立肝郁证模型,连续21 d。造模成功后,柴胡疏肝散合左归饮低、中、高剂量组分别灌服柴胡疏肝散合左归饮混悬液3.39 g·kg^(-1)、6.77 g·kg^(-1)、13.55 g·kg^(-1),雌激素组灌胃戊酸雌二醇(补佳乐)0.105 mg·kg^(-1),正常组和模型组灌胃等体积的灭菌水,连续干预3周。采集阴道脱落细胞监测大鼠动情周期变化,行为学检测肝郁证造模情况,HE染色观察卵巢组织病理学情况,ELISA法检测血清雌二醇(E_(2))、促卵泡激素(FSH)、抗缪勒管激素(AMH)、抑制素B(INHB)含量,TUNEL法检测卵巢颗粒细胞凋亡指数,q-PCR和Western Blot法检测卵巢组织Bcl-2、Bax、活性氧(ROS)、SOD-2 mRNA和蛋白表达量。结果:与正常组相比,模型组大鼠动情周期紊乱,卵巢组织结构不清晰,颗粒细胞层明显变薄,旷场实验移动总距离和蔗糖水消耗率降低(P<0.05,P<0.01),血清FSH水平升高,E_(2)、AMH、INHB水平显著降低(P<0.01),颗粒细胞凋亡指数升高(P<0.01),Bcl-2、SOD-2的mRNA和蛋白表达量降低,Bax的mRNA和蛋白表达量升高,ROS的mRNA表达量升高(P<0.05,P<0.01)。与模型组相比,各给药组卵巢组织病理形态和行为学实验结果有所改善,血清FSH水平降低,E_(2)、INHB水平升高(P<0.05,P<0.01),Bax、ROS的mRNA表达水平降低,柴胡疏肝散合左归饮中、高剂量组和雌激素组颗粒细胞凋亡指数降低(P<0.01)、Bcl-2蛋白表达水平升高,柴胡疏肝散合左归饮高剂量组和雌激素组Bax蛋白表达降低、SOD-2和Bcl-2的mRNA和蛋白表达升高(P<0.05,P<0.01)。与柴胡疏�Objective:To investigate the possible mechanism of soothing the liver and tonifying the kidney on the apoptosis of granulosa cells in rats with premature ovarian failure.Methods:Forty-eight SD rats were randomly divided into normal group,model group,bupleurum liver-soothing powder plus left-restoring beverage(BLP+LRB)low dose,medium dose,and high dose group,and estrogen group,with 8 rats in each group.Except for the normal group,the rats in other groups were intraperitoneally injected cyclophosphamide for 15 days to establish a model of premature ovarian failure,while the chronic unpredictable mild stress mode was used for 21 days to establish a model of liver depression.After successful modeling,BLP+LRB low,medium and high dose groups were administrated with BLP+LRB suspension 3.39 g·kg^(-1),6.77 g·kg^(-1) and 13.55 g·kg^(-1),respectively,the estrogen group was administrated with estradiol valerate(bujiale)0.105 mg·kg^(-1),and the normal group and model group were administrated with equal volume of sterilized water for 3 weeks.Exfoliated vaginal cells were collected and the changes of estrous cycle in rats were monitored.The modeling of liver depression syndrome was detected by behavior.The histopathology of the ovary was observed by HE staining,the contents of serum estradiol(E_(2)),follicle stimulating hormone(FSH),anti-Muller-tube hormone(AMH),and Inhibin B(INHB)were detected by ELISA,and the apoptosis index of ovarian granulosa cells was detected by TUNEL method.The mRNA and protein expressions of Bcl-2 Associated X gene(Bax),B-cell lymphoma-2 gene(Bcl-2),reactive oxygen species(ROS)and superoxide dismutase-2(SOD-2)in ovarian tissue were detected by q-PCR and Western Blot.Results:Compared with the normal group,the estrous cycle of rats in the model group was in disorder,the ovarian tissue structure was unclear,the granulosa cell layer was obviously thinner,the total distance of open field experiment movement and sucrose water consumption rate decreased(P<0.05,P<0.01),the serum FSH level increased,the lev

关 键 词：卵巢早衰 氧化应激 疏肝补肾法 颗粒细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]