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di-(2-Ethylhexyl) phthalate (DEHP) has potential reproductive toxicity. Bu-Shen-Tian-Jing formulations (BSTJFs) are beneficial for female reproductive capacity. However, BSTJF2 has much lower cytotoxicity than BSTJF1. To investigate the effects of BSTJFs on ovarian granulosa cells exposed to DEHP and determine the potential molecular mechanisms. Human granulosa-like tumor cell line (KGN) cells were divided into control, DEHP, BSTJF1 and BSTJF2 groups. The DEHP group were given 1 μM DEHP for 24 h. They were then given BSTJF1 at 200 μg/mL or BSTJF2 at 100 μg/mL for 24 h. The control group was treated with the same concentration of DMSO (0.1%). Oxidative stress and mitochondrial function were measured. The mRNA and protein expression levels of HDAC3 and HSP90AA were determined. Integrative network pharmacology analysis of BSTJF2 was also performed. DEHP (1 μM) significantly suppressed the proliferation of KGN cells by 17%, significantly increased ROS levels by 28% and MDA levels by 47%, significantly decreased MMP levels by 22% and mtDNA copy by 30%. DEHP significantly increased protein expression of HDAC3 by 21%and HSP90AA by 64%. All these changes were significantly reversed by BSTJFs. Integrative network pharmacology analysis revealed HSP90AA was a key target (degree = 8). Both RGFP966 and BSTJF2 significantly reversed the increased expression of HDAC3 and HSP90AA, attenuated oxidative stress, and mitochondrial damage which were induced by DEHP. BSTJFs might have therapeutic potential on oxidative stress and mitochondrial damage through the HDAC3/HSP90AA pathway which encourages further clinical trials. [ABSTRACT FROM AUTHOR]

Mitochondrial function and glucose metabolism play important roles in bidirectional signaling between granulosa cells (GCs) and oocytes. However, the factors associated with mitochondrial function and glucose metabolism of GCs in polycystic ovary syndrome (PCOS) are poorly understood, and their potential downstream effects on oocyte quality are still unknown. The aim of this study was to investigate whether there are alterations in mitochondrial-related functions and glucose metabolism in ovarian GCs of women with PCOS and the role of Sirtuin 3 (SIRT3) in this process. Here, we demonstrated that women with PCOS undergoing in vitro fertilization and embryo transfer had significantly lower rates of metaphase II oocytes, two-pronuclear fertilization, cleavage, and day 3 good-quality embryos. Germinal vesicle- and metaphase I-stage oocytes from women with PCOS exhibited increased mitochondrial reactive oxygen species (ROS), decreased mitochondrial membrane potential, and downregulation of glucose-6-phosphate dehydrogenase. GCs from women with PCOS presented significant alterations in mitochondrial morphology, amount, and localization, decreased membrane potential, reduced adenosine triphosphate (ATP) synthesis, increased mitochondrial ROS and oxidative stress, and insufficient oxidative phosphorylation (OXPHOS) together with decreased glycolysis. SIRT3 expression was significantly decreased in GCs of PCOS patients, and knockdown of SIRT3 in KGN cells could mimic the alterations in mitochondrial functions and glucose metabolism in PCOS GCs. SIRT3 knockdown changed the acetylation status of NDUFS1, which might induce altered mitochondrial OXPHOS, the generation of mitochondrial ROS, and eventually defects in the cellular insulin signaling pathway. These findings suggest that SIRT3 deficiency in GCs of PCOS patients may contribute to mitochondrial dysfunction, elevated oxidative stress, and defects in glucose metabolism, which potentially induce impaired oocytes in PCOS. [Display omitted] • Women with polycystic ovary syndrome have impaired oocyte quality. • Granulosa cells of polycystic ovary syndrome show altered mitochondrial functions. • Granulosa cells of polycystic ovary syndrome show defects in glycolysis. • Sirtuin 3 deficiency exists in granulosa cells of polycystic ovary syndrome. • Sirtuin 3 regulates acetylation status of NDUFS1 and insulin signaling pathway. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS), as a common endocrine disease in reproductive-age women, which is characterized by both reproductive and metabolic disorders. Cang-Fu-Dao-Tan Formula (CFDTF) is an effective and relatively safe treatment for PCOS. However, the underlying mechanism is poorly understood. To explore the effective compounds and mechanisms of CFDTF in treating PCOS based on UPLC/Q-TOF-MS/MS, network pharmacology and molecular experiments. The UPLC/Q-TOF-MS/MS and TCMSP, SwissTargetPrediction databases were used to identify the active ingredients of CFDTF. Then GeneCards, Disgenet, Drugbank databases were used to obtain the PCOS related targets. Based above, the Drug-component-target (D-C-T) network and protein-protein-interaction (PPI) network were built to analysis the key targets. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis were performed to find the potential mechanisms. Finally, molecular docking analysis, molecular dynamics (MD) simulations and molecular experiments were used to confirm the interactions among the active compounds, targets and explore the potential mechanisms. A total of 20 compounds were identified by UPLC/Q-TOF-MS/MS, and 136 active compounds by TCMSP from CFDTF. After removing the duplicate results, there were 370 targets related to both CFDTF and PCOS, among which, MAPK3, AKT1, RELA, EGF, TP53 and MYC were proved to have high interactions with the components. The mechanisms of CFDTF against PCOS were related to PI3K-Akt, mTOR, MAPK signaling pathways, and the in vitro experiments proved that the CFDTF positively regulated the cell proliferation and inhibited the apoptosis levels in PCOS cell model. The combination of UPLC/Q-TOF-MS/MS, systematic network pharmacology and molecular experiments identified that the quercetin, hesperidin, and glycyrrhizin disaccharide are the TOP 3 effective compounds of CFDTF in treating PCOS and the potential mechanisms may involve in regulating proliferation and apoptosis of granulosa cells. • Cang-Fu-Dao-Tan Formula (CFDTF) is an effective and relatively safe treatment for polycystic ovary syndrome (PCOS). • The quercetin, hesperidin, and glycyrrhizin disaccharide are the TOP 3 effective compounds of CFDTF in treating PCOS. • MAPK3, AKT1, RELA, EGF, TP53 and MYC were proved to have high interactions with the active components. • The potential mechanisms may involve in regulating proliferation and apoptosis of granulosa cells. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine and metabolic disorder. Chinese herbal medicine has some advantages in the treatment of PCOS with its unique theoretical system and rich clinical practice experiences. The present study was to investigate the potential mechanisms of Bu-Shen-Jian-Pi Formula (BSJPF) on the treatment of PCOS. The combination of ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS) rapid analysis, network pharmacology, molecular docking analysis and bio-experiments were firstly conducted to identify the main effective components of BSJPF, and to predict the potential mechanisms. The ovarian granulosa cell line (KGN) was treated with testosterone to construct the PCOS model in vitro, and the cells were further treated with the lyophilized powder of BSJPF. The levels of proliferation, autophagy and apoptosis were detected to explore the mechanisms of BSJPF on treating PCOS. Firstly, thirty-six active compounds were identified in BSJPF and thirty-one potential targets on PCOS were found. Then, PI3K and PDK1 were verified to have good binding activity with the active compounds through molecular docking analysis. In bio-experiments, BSJPF significantly alleviated the arrested proliferation of KGN cells in G0/G1 phase and reduced the active levels of autophagy and apoptosis of KGN cells induced by testosterone. Additionally, the inhibition of autophagy diminished apoptosis, while the repression apoptosis enhanced autophagy. Finally, BSJPF significantly decreased the FOXO1 expression levels induced by testosterone, especially for nuclear FOXO1, and significantly activated the PI3K/AKT pathway. BSJPF significantly alleviated the activated autophagy and apoptosis in KGN induced by testosterone through PI3K/AKT1/FOXO1pathway, which is an effective treatment for PCOS. [Display omitted] [ABSTRACT FROM AUTHOR]