Objectives: To investigate the effect of Chinese medicine He's Yangchao recipe on premature ovarian insufficiency (POI) and its relationship with mitochondrial function of ovarian granulose cells in an animal model.
Methods: Thirty-six female C57BL/6J mice were randomly divided into blank control group, model group, low-, medium- and high-dose He's Yangchao recipe treatment group and coenzyme Q10 (Q10) treatment group (positive control). The POI model was induced by a single intraperitoneal injection of cyclophosphamide (90 mg/kg). The animals were sacrificed after 21 days. Primary granulose cells were obtained from POI mice and treated with He's Yangchao recipe, ERβ inhibitor PHTPP, and He's Yangchao recipe+PHTPP in vitro for 24 h, respectively. Ovarian histopathological changes were observed by hematoxylin-eosin (HE) staining, ATP levels were detected by luciferase assay, mtDNA copy numbers were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), mitochondrial structure changes were observed by transmission electron microscopy, protein and mRNA expression levels of estrogen receptor β (ERβ), peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), mitochondrial transcription factor A (TFAM), and superoxide dismutase 2 (SOD2) were detected by Western blotting and qRT-PCR.
Results: The ovarian tissue in model group exhibited few secondary and tertiary follicles, whereas the He's Yangchao recipe groups and Q10 group had abundant secondary and tertiary follicles. Compared with the blank control group, ATP and mtDNA levels in model group decreased (P<0.01), mitochondrial crista disappeared or abnormal vacuolated structure increased; the protein and mRNA levels of ERβ, PGC1α, TFAM, and SOD2 decreased (all P<0.01). ATP production increased in granulose cells of high-dose He's Yangchao recipe group and Q10 group; mtDNA copy numbers increased (P<0.05 or P<0.01); abnormal mitochondrial structure was reduced; the protein and mRNA expressions of ERβ, PGC1α, TFAM, and SOD2 increased (P<0.05 or P<0.01). Compared with the PHTPP intervention group, the proportion of normal mitochondrial structure in the granulose cells of He's Yangchao recipe + PHTPP group was higher; ATP content increased (P<0.05 or P<0.01); mtDNA copy numbers increased (P<0.05 or P<0.01); the protein and mRNA expression of ERβ, PGC1α, TFAM and SOD2 increased (P<0.05 or P<0.01).
Conclusions: He's Yangchao recipe can regulate mitochondrial biogenesis through ERβ/PGC1α/TFAM pathway to improve ovarian function in POI mice.
目的: 探究何氏养巢方（简称“养巢方”）提高卵巢颗粒细胞线粒体功能的机制。方法: 取36只6~8周龄C57BL/6J雌鼠随机分为空白对照组，模型对照组，养巢方小、中、大剂量组和阳性对照组。均予环磷酰胺90 mg/kg单次腹腔注射以建立原发性卵巢功能不全（POI）模型后，分组灌胃21 d后处死。另取6只6~8周龄C57BL/6J雌鼠造模后获取原代颗粒细胞，分为养巢方组、PHTPP（一种雌激素受体阻滞剂）组、养巢方+PHTPP组，分别予养巢方含药血清或PHTPP或同时给予两者干预培养24 h。分别采用苏木精-伊红（HE）染色观察卵巢组织病理学变化；荧光素酶法检测颗粒细胞腺苷三磷酸（ATP）水平；定量逆转录聚合酶链反应（qRT-PCR）检测线粒体DNA（mtDNA）的拷贝数；透射电镜观察线粒体结构变化；蛋白质印迹法检测雌激素受体β（ERβ）、过氧化物异酶体增殖物激活受体γ共激活因子1α（PGC1α）、线粒体转录因子A（TFAM）、超氧化物歧化酶2（SOD2）蛋白表达；qRT-PCR检测Erβ、Pgc1α、Tfam、Sod2 mRNA表达。结果: 模型对照组卵巢组织次级及三级卵泡较少，养巢方各剂量组及阳性对照组次级及三级卵泡较多。与空白对照组比较，模型对照组颗粒细胞ATP和mtDNA水平均下降（均P<0.01），线粒体嵴消失或空泡化结构异常比例增加，ERβ、PGC1α、TFAM、SOD2蛋白及其mRNA表达均下降（均P<0.01）。养巢方中、大剂量组及阳性对照组颗粒细胞内ATP生成增多、mtDNA拷贝数增加（P<0.05或P<0.01）；颗粒细胞内结构异常线粒体减少，ERβ、PGC1α、TFAM、SOD2蛋白及其mRNA表达均增加（P<0.05或P<0.01）。体外实验中，与PHTPP组比较，养巢方+PHTPP组线粒体结构正常比例更高，线粒体ATP含量增加（P<0.05或P<0.01），mtDNA拷贝数增加（P<0.05或P<0.01）；ERβ、PGC1α、TFAM、SOD2蛋白及其mRNA表达均上升（P<0.05或P<0.01）。结论: 养巢方可以通过ERβ/PGC1α/TFAM通路提高线粒体生物发生从而改善POI小鼠卵巢功能。.
Keywords: Estrogen receptor β; He’s Yangchao recipe; Mice; Mitochondria; Mitochondrial transcription factor A; Peroxisome proliferator-activated receptor γ coactivator 1α; Premature ovarian insufficiency.