Network pharmacological analysis and experimental verification of Zisheng Tongmai decoction in the treatment of premature ovarian failure.

Premature ovarian failure (POF) is a disease that seriously jeopardizes women's physical and mental health worldwide. Zisheng Tongmai decoction (ZSTMD), a famous Traditional Chinese Medicine (TCM) formula, has a marked effect on the clinical treatment of POF. This study investigated the potential mechanism of ZSTMD to improve POF through network pharmacology and experimental validation. The active components, key targets and potential mechanisms of ZSTMD against POF were predicted by network pharmacology and molecular docking. The POF model was induced in rats by cyclophosphamide (CTX) and subsequently gavaged with different doses of ZSTMD. KGN cells were treated with different concentrations of quercetin and CTX. Histopathological were observed via hematoxylin and eosin (H&E) staining and immunofluorescence staining. Serum estrogen levels were detected via ELISA. Protein expression was detected via Western blot. We identified quercetin as the main active ingredients targeting VEGFA. Molecular docking showed that VEGFA interacted well with the main active components of ZSTMD. In vivo experiments, ZSTMD significantly increased body weight and the ovarian index, significantly increased E2 and AMH, and decreased FSH and LH in POF rats. Histologic results showed that ZSTMD increased the number of follicles and vascular density in the ovary. It also increased VEGFA and CD31 protein expression. In vitro experiments, quercetin suppressed CTX-induced apoptosis in KGN cells and increased VEGFA protein expression. Our study demonstrated that ZSTMD improves POF by promoting angiogenesis through VEGFA target.

Transcription factor YY1 adversely governs ovarian granulosa cell growth in PCOS by transcription activation-mediated CDKN1C upregulation.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women of childbearing age, making it imperative to explore more biomarkers for PCOS. Furthermore, previous studies have reported that cyclin dependent kinase inhibitor 1 C (CDKN1C) might be associated with PCOS progression. However, the molecular mechanism of CDKN1C involved in PCOS is poorly defined. METHODS: CDKN1C and Yin-Yang-1 (YY1) expression levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay. Cell viability, proliferation, cell cycle progression, and cell apoptosis were analyzed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Caspase 3 activity was examined using a commercial kit. Binding between YY1 and CDKN1C promoter was predicted by JASPAR and verified using Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. RESULTS: CDKN1C and YY1 were highly expressed in PCOS granulosa cells (GCs). Furthermore, CDKN1C silencing could promote cell proliferation and cell cycle process and repress cell apoptosis in human ovarian granulosa cell line KGN cells. For mechanistic analysis, YY1 is directly bound to the promoter of CDKN1C and transcriptional-regulated CDKN1C expression. CONCLUSION: YY1-activated CDKN1C might block KGN cell proliferation and induce cell apoptosis, providing a possible therapeutic target for PCOS treatment.

Mitigating bisphenol A-induced apoptosis in KGN cells: the therapeutic role of 1,25-dihydroxyvitamin D3 through upregulation of PGC-1α expression and inhibition of the mitochondrial cytochrome c pathway.

PURPOSE: This study investigated the potential of 1,25-dihydroxyvitamin D3 (1,25(OH)2VD3) to mitigate bisphenol A (BPA)-induced apoptosis in human ovarian granulosa KGN cells with the aim of establishing a theoretical foundation for understanding of how vitamin D improved ovarian function in patients with polycystic ovary syndrome (PCOS). METHODS: The impact of varying concentrations of BPA and 1,25(OH)2VD3 on KGN cell viability was elucidated. It was established that BPA-induced apoptosis in KGN cells. Subsequently, KGN cells underwent pretreatment with 1,25(OH)2VD3, followed by exposure to BPA. The apoptosis rate, reactive oxygen species (ROS) levels, and mitochondrial function of the cells were meticulously assessed, along with the expression levels of genes associated with apoptosis as well as antioxidant and mitochondrial biogenesis. RESULTS: BPA induced a notable increase in apoptosis (P < 0.001) and oxidative stress (P < 0.001) in KGN cells, accompanied by a significant reduction in mitochondrial membrane potential (P < 0.001) and severe impairment of mitochondrial function. Following pretreatment of KGN cells with 1,25(OH)2VD3, there was a significant decrease in the apoptosis rate (P = 0.004), coupled with a reduction in ROS production (P = 0.002). Concomitantly, the upregulation of PGC-1α (P = 0.009) and SOD (P = 0.018) was observed, while mRNA expression of BAX (P = 0.011), Cyt c (P = 0.001), Apaf-1 (P = 0.012), caspase-9 (P < 0.001), and caspase-3 (P = 0.011) was downregulated. Notably, the mitigation of mitochondrial damage was evident through restored mitochondrial membrane potential (P < 0.001), as corroborated by electron microscope results. CONCLUSIONS: 1,25(OH)2VD3 mitigated BPA-induced damage and apoptosis in KGN cells by upregulating the expression of PGC-1α and impeding the mitochondrial cytochrome c (Cyt c) apoptotic pathway. This study established a novel theoretical foundation for utilizing vitamin D in the treatment of PCOS patients.

MicroRNA-646 inhibits the proliferation of ovarian granulosa cells via insulin-like growth factor 1 (IGF-1) in polycystic ovarian syndrome (PCOS).

INTRODUCTION: Polycystic ovarian syndrome (PCOS) is a common endocrinopathy in women. MicroRNAs (miRNAs) have been proven to play a crucial role in balancing the proliferation and apoptosis of granulosa cells (GCs) in PCOS. MATERIAL AND METHODS: The miRNA of PCOS was screened by bioinformatics analysis, and microRNA 646 (miR-646) was found to be involved in insulin-related pathways by enrichment analysis. The cell counting kit-8 (CCK-8), cell colony formation, and the 5-ethynyl-2'-deoxyuridine (EdU) assays were used to explore the effect of miR-646 on proliferation of GCs, flow cytometry was used to measure the cell cycle and apoptosis, and Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to explore the biological mechanism of miR-646. The human ovarian granulosa cells KGN were selected by measuring the miR-646 and via insulin-like growth factor 1 (IGF-1) levels and used for cell transfection. RESULTS: Overexpressed miR-646 inhibited KGN cell proliferation, and silenced miR-646 advanced it. Most cells were arrested in the S phase of cell cycle with overexpressed-miR-646, while after silencing miR-646, cells were arrested in the G2/M phase. And the miR-646 mimic raised apoptosis in KGN cells. Also, a dual-luciferase reporter proved the regulation effect of miR-646 on IGF-1, miR-646 mimic inhibited IGF-1, and miR-646 inhibitor advanced IGF-1. The cyclin D1, cyclin-dependent kinase 2 (CDK2), and B-cell CLL/lymphoma 2 (Bcl-2) levels were inhibited with overexpressed-miR-646, while silenced-miR-646 promoted their expression, and the bcl-2-like protein 4 (Bax) level was the opposite. This study found that silenced-IGF1 antagonized the promotive effect of the miR-646 inhibitor on cell proliferation. CONCLUSIONS: MiR-646 inhibitor treatment can promote the proliferation of GCs by regulating the cell cycle and inhibiting apoptosis, while silenced-IGF-1 antagonizes it.

Malvidin-3-O-Glucoside Ameliorates Cadmium-Mediated Cell Dysfunction in the Estradiol Generation of Human Granulosa Cells.

Cadmium (Cd) is a frequent environmental pollutant associated with biological toxicity that can harm female reproduction. Anthocyanins have been reported to reduce the toxicity of Cd. In the present study, the protective effects and underlying mechanisms of malvidin-3-O-glucoside (M3G) against the toxicity of Cd on female reproduction in KGN cells (human ovarian granulosa-like tumor cells) were investigated. After treating cells with 10 µmol/L cadmium chloride, the results showed that M3G lessened Cd-induced KGN cell cytotoxicity better than malvidin and malvidin-3,5-O-diglucoside. Additionally, M3G significantly decreased the Cd-induced generation of reactive oxygen species, inhibited the Cd-induced arrest of the G2/M phase of the cell cycle, and increased estradiol (E2) production. According to transcriptomic results, M3G reduced the abnormal expression of genes that responded to estrogen. Additionally, M3G promoted the endogenous synthesis and secretion of E2 by controlling the expression of CYP17A1 and HSD17B7. The current findings indicated that M3G is of great potential to prevent Cd-induced female reproductive impairment as a dietary supplement.

Small Peptides from Periplaneta americana Inhibits Oxidative Stress-Induced KGN Cell Apoptosis by Regulating Mitochondrial Function Through Bcl2L13.

This study examined the protective effects of small peptides from Periplaneta americana against H2O2-induced mitochondrial injury in human ovarian granulosa cells. The ATP level and mitochondrial membrane potential as well as the quantity and ultrastructure of mitochondria in cells were detected. Mitochondrial DNA copy number and expression levels of Bcl2L13, LC3B, and p62 were tested. Targeted silencing of Bcl2L13 expression in KGN cells. The expression levels of Bcl2L13 and LC3B as well as interaction were evaluated. The ATP level, mtDNA-CN, and MMP of the H2O2 group were significantly lower than those of the normal control group (P < 0.05), accompanied by a reduction in mitochondrial mass and mitochondrial fluorescence intensity (P < 0.05). However, the ATP level, mtDNA, and MMP in KGN cells were increased after SPPA treatment (P < 0.05). Scanning electron microscopy shows that SPPA ameliorates H2O2-induced structural damage to mitochondria. Moreover, the expression levels of Bcl2L13 and p62 in the H2O2 group were downregulated significantly compared with those of the normal control group (P < 0.05), while LC3B was upregulated (P < 0.05). After SPPA treatment, the expression levels of Bcl2L13 and p62 were upregulated (P < 0.05), while LC3B was downregulated (P < 0.05). The Co-IP results indicated that Bcl2L13 and LC3B interacted, and this interaction was weakened after cell treatment with H2O2, and dissociation between Bcl2L13 and LC3B declined after SPPA treatment. SPPA inhibits KGN cell apoptosis induced by oxidative stress via inhibition of mitochondrial injury Bcl2L13-mediated mitochondrial autophagy might participate in the regulation process.

Effects of omega-3 polyunsaturated fatty acids on cellular development in human ovarian granulosa tumor cells (KGN).

Emerging research has shown that polyunsaturated fatty acids (PUFAs) benefit human health and exert anti-cancer effects. However, there is little understanding of the specific mechanisms by which PUFAs regulate the cells of the ovarian granulosa tumor. In the current study, we investigate the effects and the possible mechanisms of PUFAs on human ovarian tumor cells development. KGN cells were treated with omega-3. Small interfering (siRNA) and specific activator were used to knock down and overexpress gene expression in KGN cells. The protein content levels were analyzed by Western blot. Cell viability, proliferation and apoptosis assay were performed to examine the cellular development. And the level of glucose uptake in KGN cells were assessed by 2-DG measurement. The results showed that omega-3 treatment reduced cell viability, proliferation and increased cell apoptosis. Further studies showed that omega-3 also reduced GLUT1/4 protein content and cellular glucose uptake. Subsequent knockdown and overexpression of OCT4 using Oct4 siRNA and O4I2 (OCT4 activator) showed that OCT4 was involved in the regulations of omega-3 on GLUT1/4 expression and cell development. Our data demonstrate that omega-3 inhibits cellular development by down-regulating GLUT1/4 expression and glucose uptake in KGN cells, which are mediated through OCT4.

The circular RNA circ_0030018/miR-136/migration and invasion enhancer 1 (MIEN1) axis promotes the progression of polycystic ovary syndrome.

The abnormal expression of circular RNAs (circRNAs) is associated with the progression of polycystic ovary syndrome (PCOS), which commonly causes infertility in women. In this study, we identified the role of circ_0030018 in PCOS. Quantitative polymerase chain reaction (qPCR) was used to detect the expression levels of circ_0030018, microRNA (miR)-136, and migration and invasion enhancer 1 (MIEN1). Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays were performed to analyze the proliferation of KGN cells. Apoptosis was analyzed using fluorescence-activated cell sorting. Transwell assays were performed to measure the migration and invasion abilities of cells. qPCR and Western blotting were used to measure the levels of E-cadherin, N-cadherin, Snail, and vimentin. The correlation of circ_0030018 or MIEN1 expression with miR-136 expression was confirmed via luciferase reporter and RNA pull-down assays. Results showed that circ_0030018 expression was upregulated in patients with PCOS and KGN cells. Knockdown of circ_0030018 suppressed the proliferation, migration, and invasion of cells, while promoting their apoptosis. circ_0030018 sponged miR-136, which targeted MIEN1. Moreover, downregulation of miR-136 abrogated the effects of circ_0030018 silencing, while the overexpression of MIEN1 reversed the miR-136-induced effect on KGN cells. In summary, loss of circ_0030018 delayed the progression of PCOS via the miR-136/MIEN1 axis.

MiR-520h inhibits viability and facilitates apoptosis of KGN cells through modulating IL6R and the JAK/STAT pathway.

This study embarked on the assessment regarding the function and mechanism of miR-520h/IL6R axis in polycystic ovary syndrome (PCOS). Specifically, we analyzed the differential expression of IL6R in PCOS samples and normal samples based on the GEO database, and then verified IL6R expression in KGN cells (Human granulosa-like tumor cell line) using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blot. MiRNA targeting IL6R was predicted by bioinformatics analysis and verified by luciferase reporter assay, qRT-PCR and western blot. KGN cells were transfected with miR-520h inhibitor and si-IL6R, and then the cell viability and apoptosis were detected by cell counting kit-8 (CCK-8) and flow cytometry assays. Additionally, western blot was applied to examine the expressions of cell cycle-, apoptosis-, and JAK / STAT pathway-related proteins. IL6R was highly expressed in PCOS and KGN cells, and IL6R silencing inhibited the viability, while promoting the apoptosis of KGN cells. Importantly, miR-520h directly targeted IL6R and inhibited IL6R expression. Moreover, downregulation of miR-520h enhanced the cell viability, impeded the cell apoptosis, upregulated the expressions of CDK2, CCNB1, Bcl-2, activated JAK/STAT pathway and downregulated Bax expression in KGN cells. Of note, knockdown of IL6R can reverse the biological functions of miR-520h in KGN cells. Collectively, miR-520h hindered the proliferation and promoted the apoptosis of KGN cells via targeting IL6R to inhibit the development of PCOS, and these effects were possibly realized by JAK/STAT pathway. However, the effect of miR-520h in the progression of PCOS need to further study in the GCs.

CircASPH promotes KGN cells proliferation through miR-375/MAP2K6 axis in Polycystic Ovary Syndrome.

Polycystic Ovary Syndrome (PCOS) is a kind of endocrine disorder which is prevalent in adult women, so exploring more biomarkers for PCOS is imperative. Recently, circular RNA and microRNA are confirmed to be related with PCOS development. Whether circular RNA ASPH (circASPH) is involved in PCOS need to be studied further. We utilized RT-qPCR to measure the expression levels of circASPH, miR-375 and MAP2K6 in PCOS patients and normal group. The effects of circASPH and miR-375 on KGN cells proliferation and apoptosis were observed by CCK-8 assay, EdU incorporation assay and apoptosis assay, separately. Then Dual-luciferase reporter assay was carried out to verify the circASPH/miR375 axis and miR375/MAP2K6 axis. The interaction between circASPH and MAP2K6 were detected with the support of RT-qPCR and Western blot. We found circASPH and MAP2K6 were both over-expressed in PCOS patients, while miR-375 was in the opposite direction. Moreover, miR-375 was negatively regulated by circASPH, while MAP2K6 was positively regulated by circASPH. In addition, circASPH directly targeted miR-375, which targeted MAP2K6. More than that, the knockdown of circASPH repressed KGN cells proliferation and enhanced apoptosis, while the silence of miR-375 reversed the above effects. In conclusion, circASPH promotes KGN cells proliferation through miR-375/MAP2K6 axis in PCOS, and they are thought-provoking biomarkers for PCOS diagnosis and therapy.

miR-613 inhibits the proliferation of human ovarian granulosa cells by arresting cell cycle progression via the targeting of IGF-1.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder, and microRNA (miRNA) molecules have been implicated in the pathological process of PCOS. The aim of the present study was to elucidate the regulatory effects of miR-613 and insulin-like growth factor-1 (IGF-1) on the pathological process of polycystic ovary syndrome (PCOS). The targeting of IGF-1 by miR-613 was investigated by dual-luciferase reporter assay. The regulatory effect of miR-613 on the mRNA and protein levels of IGF1 was determined by reverse transcription-quantitative PCR and western blot analysis. The regulatory effects of miR-613 and IGF-1 on the proliferation and cell cycle progression of KGN cells were evaluated by colony formation assay and flow cytometric analysis. The results revealed that miR-613 targeted IGF-1 and reduced its translational level. In KGN cells, miR-613 arrested cell cycle progression in the G2/M phase and downregulated the expression of cyclin D1 and CDK1. The overexpression of IGF-1 attenuated the inhibitory effects of miR-613 on cell cycle arrest, cyclin D1 and CDK1 expression, and the proliferation of KGN cells. In conclusion, the present study demonstrated that miR-613 targets IGF-1 and thus suppresses its translation. It arrests cell cycle progression and attenuates the proliferation of KGN cells via the targeting of IGF-1. Therefore, it is suggested that miR-613 and IGF-1 could potentially be diagnostic biomarkers and therapeutic targets for PCOS.

CircPSMC3 alleviates the symptoms of PCOS by sponging miR-296-3p and regulating PTEN expression.

Polycystic ovary syndrome (PCOS), the most common female endocrine disease that causes anovulatory infertility, still lacks promising strategy for the accurate diagnosis and effective therapeutics of PCOS attributed to its unclear aetiology. In this study, we determined the abnormal reduction in circPSMC3 expression by comparing the ovarian tissue samples of PCOS patients and normal individuals. The symptom relief caused by up-regulation of circPSMC3 in PCOS model mice suggested the potential for further study. In vitro functional experiments confirmed that circPSMC3 can inhibit cell proliferation and promote apoptosis by blocking the cell cycle in human-like granular tumour cell lines. Mechanism study revealed that circPSMC3 may play its role through sponging miR-296-3p to regulate PTEN expression. Collectively, we preliminarily characterized the role and possible insights of circPSMC3/miR-296-3p/PTEN axis in the proliferation and apoptosis of KGN cells. We hope that this work provides some original and valuable information for the research of circRNAs in PCOS, not only to better understand the pathogenesis but also to help provide new clues for seeking for the future therapeutic target of PCOS.

miR-155 is high-expressed in polycystic ovarian syndrome and promotes cell proliferation and migration through targeting PDCD4 in KGN cells.

Polycystic ovarian syndrome (PCOS) is a typical disease of female endocrine and metabolic abnormalities. miR-155, famous as a multifunctional miRNA, promotes the proliferation, migration and invasion of human cancer cells. Therefore, we aimed to explore its regulation mechanism in PCOS. BrdU incorporation and apoptosis assay were used to test KGN cell survival. Luciferase activity experiment was employed to test targeting link between miR-155 and programmed cell death 4 (PDCD4). Migration and invasion assay were operated to examine the influence of miR-155 and PDCD4 in migration and invasion of KGN cells. In addition, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay and western blot analysis were utilized to measure expression of miR-155 and other relative factors. We found that expression of miR-155 was high in PCOS patients' tissues and it promoted proliferation, migration and invasion in KGN cells. Further studies found that PDCD4 was down-regulated by miR-155 and was a target of miR-155. Overexpression of PDCD4 promoted cell apoptosis to mitigate PCOS. Besides, up-regulation of PDCD4 suppressed PI3K/AKT and JNK signal pathways. To sum up, miR-155 promoted proliferation, migration, invasion and the activation of PI3K/AKT and JNK pathways in KGN cells through negatively regulating PDCD4.

Excessive levels of diverse phytoestrogens can modulate steroidogenesis and cell migration of KGN human granulosa-derived tumor cells.

Phytoestrogens are plant-derived estrogen-like compounds that are increasingly used for their suggested health promoting properties, even by healthy, young women. However, scientific concerns exist regarding potential adverse effects on female reproduction. In this study, naringenin (NAR), 8-prenylnaringenin (8-PN), genistein (GEN), coumestrol (COU), quercetin (QUE) and resveratrol (RSV) up-regulated steroidogenic acute regulatory protein (StaR) mRNA levels in KGN human granulosa-like tumor cells. Most of the phytoestrogens tested also increased CYP19A1 (aromatase) mRNA levels via activation of ovary-specific I.3 and II promoters. Yet, only NAR (3 and 10 µM), COU (10 and 30 µM) and QUE (10 µM) also statistically significantly induced aromatase activity in KGN cells after 24 h. 8-PN, aromatase inhibitor letrozole and estrogen receptor antagonist ICI 182,780 concentration-dependently inhibited aromatase activity with IC50 values of 8 nM, 10 nM and 72 nM, respectively. Co-exposure with ICI 182,780 (0.1 µM) statistically significantly attenuated the induction of aromatase activity by QUE and COU, but not NAR. Cell cycle status and proliferation of KGN cells were not affected by any of the phytoestrogens tested. Nonetheless, the migration of KGN cells was significantly reduced with approximately 30% by COU, RSV and QUE and 46% by GEN at 10 µM, but not NAR and 8-PN. Our results indicate that phytoestrogens can affect various pathways in granulosa-like cells in vitro at concentrations that can be found in plasma upon supplement intake. This implies that phytoestrogens may interfere with ovarian function and caution is in place regarding the use of supplements with high contents of phytoestrogens.

Effects of an inhibitor of the γ-secretase complex on proliferation and apoptotic parameters in a FOXL2-mutated granulosa tumor cell line (KGN).

Ovarian granulosa cell tumors (GCTs) represent 3%-5% of all ovarian malignancies. Treatments have limited proven efficacy and biologically targeted treatment is lacking. The aim of this study was to investigate the role of Notch signaling in the proliferation, steroidogenesis, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/AKT pathway in a FOXL2-mutated granulosa tumor cell line (KGN) representative of the adult form of GCTs. When Notch signaling is initiated, the receptors expose a cleavage site in the extracellular domain to the metalloproteinase TACE and, following this cleavage, Notch undergoes another cleavage mediated by the presenilin-gamma-secretase complex. To achieve our goal, DAPT, an inhibitor of the gamma-secretase complex, was used to investigate the role of the Notch system in parameters associated with cell growth and death, using a human granulosa cell tumor line (KGN) as an experimental model. We observed that JAGGED1, DLL4, NOTCH1, and NOTCH4 were highly expressed in KGN cells as compared to granulosa-lutein cells obtained from assisted reproductive techniques patients. The proliferation and viability of KGN cells, as well as progesterone and estradiol production, decreased in the presence of 20 µM DAPT. Apoptotic parameters like PARP and caspase 8 cleavages, BAX, and BCLXs increased in KGN cells cultured with DAPT, whereas others such as BCL2, BCLXl, FAS, and FAS ligand did not change. AKT phosphorylation decreased and PTEN protein increased when Notch signaling was inhibited in KGN cells. We conclude that the Notch system acts as a survival pathway in KGN cells, and might be interacting with the PI3K/AKT pathway.