Abstract: BACKGROUND: Most of the formulas for the clinical treatment of premature ovarian insufficiency have evolved from the basic formula of Liuwei Dihuang Pills, and have achieved good therapeutic efficacy. Currently, most of the experimental studies on Liuwei Dihuang Pills focus on morphological observations and physiological and biochemical detection of in vivo animal models, while fewer studies on molecular mechanisms have been reported.
OBJECTIVE: To explore the molecular mechanism of Liuwei Dihuang Pills in the treatment of premature ovarian insufficiency based on the receptor gamma coactivator-1 alpha/mitochondrial transcription factor A/reactive oxygen species pathway. 
METHODS: Premature ovarian insufficiency model was established in mice by intraperitoneal injection of cyclophosphamide 120 mg/kg combined with busulfan 12 mg/kg, and then Liuwei Dihuang Pill suspension was used to intervene in premature ovarian insufficiency mice. After 12 weeks of intervention, the levels of follicle-stimulating hormone, luteinizing hormone, estradiol, anti-Mullerian hormone, 8-hydroxydeoxyguanosine, total antioxidant capacity and reactive oxygen species in serum of mice were detected by ELISA method. The morphological changes in mouse ovaries were observed by hematoxylin-eosin staining. The ultrastructure of mouse follicular granulosa cells and the apoptosis of granulosa cell mitochondria were observed by transmission electron microscopy. The expression levels of receptor gamma coactivator-1 alpha and mitochondrial transcription factor A in mouse ovarian granulosa cells were detected by immunohistochemistry.
RESULTS AND CONCLUSION: Compared with the model group, serum levels of follicle-stimulating hormone, luteinizing hormone, reactive oxygen species, and 8-hydroxydeoxyguanosine were decreased in the experimental group (P < 0.05), and the levels of estradiol, anti-Mullerian hormone, and total antioxidant capacity were increased (P < 0.05). Hematoxylin-eosin staining showed that in the model group, there were more atretic follicles and corpus luteum forms, some secondary follicles, and interstitial fibrosis and hyperplasia; in the experimental group, a large number of atretic follicles, few corpus luteum forms, primordial follicles were observed at the edges but there were few secondary follicles and no mature follicles. Transmission electron microscopy showed that the organelles in ovarian granulosa cells of mice in the experimental groups were relatively intact. Immunohistochemical results showed that compared with the model group, the expression level of receptor gamma coactivator-1 alpha in the ovarian tissue of mice increased slightly in the experimental group at the 4th week, and there was no significant change at the 8th and 12th weeks. The expression level of mitochondrial transcription factor A in the ovarian tissues of mice in the experimental group was transiently increased at the 4th week, and then slightly decreased, which were all significantly different from those of the model group. To conclude, Liuwei Dihuang Pills inhibit ovarian granulosa cell apoptosis in mice with premature ovarian insufficiency to a certain extent through the receptor gamma coactivator-1 alpha/mitochondrial transcription factor A/reactive oxygen species signaling pathway, thereby improving the endocrine function of the ovary, enhancing the antioxidant capacity, and attenuating the degree of oxidative stress damage.

Key words: Liuwei Dihuang Pill, premature ovarian insufficiency, transcription activator, mitochondrial transcription factor A, reactive oxygen species