加味地黄汤调控Bcl-2相关线粒体凋亡信号通路改善小鼠卵巢储备功能的研究  

Modified Dihuang Decoction improves ovarian reserve in mice by regulating Bcl-2-related mitochondrial apoptosis pathway

作　　者：张双 周惠芳[1] 刘玉楠[1] 刘贝 袁艺祯 单进军 纪建建[2] ZHANG Shuang;ZHOU Hui-fang;LIU Yu-nan;LIU Bei;YUAN Yi-zhen;SHAN Jin-jun;JI Jian-jian(Affiliated,Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;Institute of Pediatrics,Nanjing University of Chinese Medicine,Nanjing 210023,China;Zhangjiagang Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine^Suzhou 215600,China)

机构地区：[1]南京中医药大学附属医院,江苏南京210029 [2]南京中医药大学中医儿科研究所,江苏南京210023 [3]南京中医药大学附属张家港医院,江苏苏州215600

出　　处：《中国中药杂志》2021年第24期6493-6501,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81973898,81774354);张家港市科技计划项目(ZKS2010)。

摘　　要：研究B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)相关线粒体凋亡信号通路在加味地黄汤改善小鼠卵巢储备功能低下中的作用。成年雌性BALB/c小鼠48只,随机分为空白组,模型组,加味地黄汤高、中、低剂量组,芬吗通组。空白组予等体积生理盐水,其余组予12 mg·kg^(-1)环磷酰胺联合1.2 mg·kg^(-1)白消安单次腹腔注射制备模型。从第36天开始,加味地黄汤高、中、低剂量组分别灌胃加味地黄汤64.74、43.16、21.58 g·kg^(-1),芬吗通组灌胃芬吗通(雌二醇片0.13 mg·kg^(-1)连续2 d,第3天起予雌二醇地屈孕酮片1.43 mg·kg^(-1)连续3 d,以此重复3个周期),空白组、模型组灌胃等体积生理盐水,每日1次,连续15 d。观察小鼠一般情况及动情周期改变,酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测血清性激素水平,苏木精-伊红(hematoxylin-eosin,HE)染色观察卵巢形态学改变,Western blot法检测卵巢含半胱氨酸的天冬氨酸蛋白水解酶-9(cysteinyl aspartate specific proteinase-9,caspase-9)、活化型含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved cysteinyl aspartate specific proteinase-3,cleaved caspase-3)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、Bcl-2、超氧化物歧化酶-2(superoxide dismutase-2,SOD-2)、谷胱甘肽过氧化物酶-1(glutathione peroxidase-1,GPx-1)蛋白表达,实时荧光定量PCR法检测卵巢Bax、Bcl-2 mRNA的表达。结果显示,与空白组比较,模型组小鼠体质量下降,动情周期紊乱;血清卵泡刺激素(follicle stimulating hormone,FSH)、黄体生成素(luteinising hormone,LH)水平升高,雌二醇(estradiol,E_(2))、抗苗勒管激素(anti-mullerian hormone,AMH)、抑制素B(inhibin B,INHB)水平降低;卵巢内生长卵泡数量及颗粒层明显减少,闭锁卵泡数量增多;卵巢caspase-9、cleaved caspase-3、Bax蛋白表达上调,Bcl-2、SOD-2、GPx-1蛋白表达下调,Bax mRNA表达上调,Bcl-2 mRNA表达下调。与模型组比较,加味地黄汤作用后DOR小The present study investigated the effect of Modified Dihuang Decoction in improving ovarian reserve in mice through the Bcl-2-related mitochondrial apoptosis pathway.Forty-eight adult female BALB/c mice were randomly divided into the following six groups with eight mice in each group:a blank group,a model group,a femoston group(three cycles of treatment with 0.13 mg·kg^(-1)estradiol tablets for 2 days and 1.43 mg·kg^(-1)estradiol and dydrogesterone tablets for 3 days),and high(64.74 g·kg^(-1))-,medium(43.16 g·kg^(-1))-,and low-dose(21.58 g·kg^(-1))Modified Dihuang Decoction groups.Mice in other groups except the blank group received a single intraperitoneal injection of 12 mg·kg^(-1)cyclophosphamide and 1.2 mg·kg^(-1)busulfan to induce a model of diminished ovarian reserve(DOR),while those in the blank group received an equal volume of normal saline.Mice were treated with corresponding drugs for 15 d from the 36 th day,once per day,and the mice in the blank group and the model group were treated with an equal volume of normal saline.The general condition and oestrous cycle were observed.The serum hormone levels were detected with the enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovaries were observed by HE staining.Western blot was used to detect the protein expression of cysteinyl aspartate specific proteinase-9(caspase-9),cleaved caspase-3,Bcl-2 associated X protein(Bax),Bcl-2,superoxide dismutase-2(SOD-2),and glutathione peroxidase-1(GPx-1).The mRNA expression of Bax and Bcl-2 was detected by real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR).The results showed that compared with the blank group,the model group showed body weight loss,disordered oestrous cycle,elevated serum levels of follicle-stimulating hormone(FSH)and luteinizing hormone(LH),reduced serum levels of estradiol(E_(2)),anti-mullerian hormone(AMH),and inhibin B(INHB),the declining number of ovarian follicles and granulosa layers,increased number of atretic follicles,up-regulated pro

关 键 词：加味地黄汤 卵巢储备功能低下 细胞凋亡 BCL-2 BAX 氧化应激 

分 类 号：R285.5[医药卫生—中药学]