Shanyao regulates the PI3K/AKT/P21 pathway to promote oogonial stem cell proliferation and stemness restoration to alleviate premature ovarian insufficiency.

Shanyao (SY, yam, Rhizoma Dioscoreae, the dried rhizome of Dioscorea oppositifolia L.) was recorded in the Chinese pharmacopoeia and was often used in the treatment of premature ovarian insufficiency (POI). To evaluate the efficacy of shanyao in cyclophosphamide (CTX)-induced POI and explore its potential mechanism of action. We employed network pharmacology, Liquid Chromatograph Mass Spectrometer (LC-MS), and molecular docking methods to identify active compounds and core targets, and predict the mechanism of shanyao for treating POI. The mechanism was subsequently validated through a series of experiments. Female Sprague-Dawley (SD) rats were randomly divided into five groups: control (CON), model, estradiol valerate (EV), low-dose shanyao, and high-dose shanyao. An experimental rat model of POI was established using cyclophosphamide and treated with either shanyao or EV for a duration of two months. We assessed the efficacy of shanyao in vivo through methods such as weighing, Enzyme-linked Immunosorbent Assay (ELISA), and Hematoxylin and Eosin (H&E) staining. Oogonial stem cells (OSCs) were isolated, after modeling, treated them with a serum containing either shanyao or EV. Using methods such as CCK8 assay, immunofluorescence staining, flow cytometry (FCM) analysis, and Western blot analysis to verify the mechanism of shanyao in treating POI. In this study, we found that after treatment with shanyao, the general condition of POI rats was improved, body weight and the ratio of ovarian weight to body weight were increased, FSH, E2 and AMH levels were improved, primary follicles and preantral follicles were significantly increased, atretic follicles were decreased. However, the number of antral follicles and fresh corpus luteum was no statistical difference. We identified 10 active compounds of shanyao that act on 220 target genes, 176 of which are associated with POI. Denudatin B and Kadsurenone were finally identified as core components. Through topological analysis, 18 key targets were selected, and ultimately PI3K, CCND1, and CDK4 were identified as core targets. Molecular docking results showed that core components had good binding energy with core targets. The results of GO and KEGG enrichment analysis mainly focus on cell cycle regulation and PI3K/AKT signaling pathway. A series of molecular biology experiments confirmed that after shanyao treatment, the phosphorylation level of PI3K and AKT in POI rats were increased, P21 was inhibited, PI3K/AKT/P21 signaling pathway was activated, and the expression levels of CCND1 and CDK4 were increased. At the same time, the expression of Oct4, fragilis and Mvh of ovarian stem cells was up-regulated. The active compounds of shanyao can regulate the PI3K/AKT/P21 signaling pathway, promote the proliferation of oogonial stem cells, stemness restoration, and delay ovarian aging. This study provides valuable insights into shanyao treatment for POI.

Zou Y ，Li Z ，Lin Y ，Zheng Y ，Liu Z ，Li Y ，Huang L ，Chen Z ，Zhu L ... -  《-》

Zuogui pills ameliorate chemotherapy-induced ovarian aging by improving stemness, regulating cell cycle and reducing apoptosis of oogonial stem cells via the Notch1/Nrf2 pathway.

Zuogui Pills (ZGP) is a classic traditional Chinese herbal formula originating from the Ming Dynasty. It has been widely used in the treatment of kidney deficiency-related diseases, including ovarian aging. To investigate the effects and potential mechanisms of ZGP on ovarian aging induced by the chemotherapeutic agent cyclophosphamide (CTX), as well as its impact on the therapeutic target, oogonial stem cells (OSCs), involving the Notch1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. This study utilized High-Performance Liquid Chromatography (HPLC) to analyze the active components of Zuogui Pills (ZGP). In vivo experiments involved the establishment of an ovarian aging model in female rats through intraperitoneal injection of CTX, followed by an 8-week treatment with ZGP and dehydroepiandrosterone (DHEA). The Notch pathway inhibitor DAPT was administered via intraperitoneal injection, followed by ZGP intervention to validate its therapeutic effects. Transcriptomic sequencing was used to analyze the differential genes before and after ZGP treatment of CTX-induced ovarian aging, and KEGG and GO analyses were applied to assess the changes in relevant signaling pathways and biological processes. In vitro experiments included the extraction, separation, and purification of ovarian germ stem cells, followed by transfection with a Notch1 overexpression plasmid. The CTX active component 4-Hydroxycyclophosphamide (4HC) was used for model intervention, and ZGP, DHEA-containing serum, and DAPT were applied to intervene with the oogonial stem cells. The effects of CTX modeling, the therapeutic efficacy of ZGP, and the general condition of the rats were observed. H&E staining was employed to assess ovarian morphology and follicle counting at various stages. Serum hormone levels were measured using ELISA, while qPCR, Western blot, flow cytometry, immunofluorescence, and IHC were utilized to analyze the expression of the Notch1/Nrf2 pathway, cell cycle proteins, and stemness-related indicators. Flow cytometry, TUNEL fluorescence, and CCK8 assays were conducted to evaluate changes in cell cycle composition, apoptosis, and proliferation. Finally, ChIP-qPCR was employed to validate the transcriptional regulation of the target gene NFE2L2 by Notch1. ZGP improved serum sex hormones in ovarian aging rats, enhanced ovarian index, and optimized ovarian and uterine morphology, as well as follicle quantity composition. After transcriptome sequencing, KEGG analysis enriched the Notch signaling pathway and cell cycle, while GO analysis highlighted enrichment in the Notch pathway and stem cell population maintenance. Various experiments validated that ZGP significantly improved the expression of cell cycle-related proteins Cyclin D1 (CCND1), Cyclin E1 (CCNE1), cyclin-dependent kinase inhibitor 1a (CDKN1A), stemness markers Mouse Vasa Homolog (MVH), Octamer-binding Transcription Factor 4 (Oct4), Fragilis, 5-Bromo-2'-deoxyuridine (BrdU), as well as Notch1 and Nrf2 in aging ovarian tissues and OSCs. Additionally, ZGP promoted the proliferation of 4HC-damaged OSCs, optimized OSCs cell cycle composition, reduced G0/G1 phase arrest, and decreased early and late apoptosis. ZGP could reverse the detrimental effects on stemness and cell cycle of OSCs caused by blocking the Notch pathway. Furthermore, ZGP may activate the regulation of its target gene NFE2L2 by upregulating Notch1 expression in OSCs, thereby exerting therapeutic effects. ZGP protects ovarian function in CTX-induced ovarian aging rats by regulating the Notch1/Nrf2 pathway. It restores serum sex hormone levels, maintains normal follicle development, promotes the proliferation of aged OSCs, optimizes the cell cycle, reduces apoptosis, and preserves stemness, thereby alleviating ovarian aging.

Li Z ，Lin Y ，Zou Y ，Liang Y ，Zeng L ，Wang Y ，Li Y ，Zong Y ，Zhang Y ，Zheng Y ，Cui Y ，Huang L ，Chen Z ，Pan X ，Zhu L ... -  《-》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

Effect of Rhei Radix Et Rhizome on treatment of polycystic ovary syndrome by regulating PI3K/AKT pathway and targeting EGFR/ALB in rats.

Abnormal endocrine metabolism caused by polycystic ovary syndrome (PCOS) poses a serious risk to reproductive health in females. According to Traditional Chinese Medicine (TCM) theories, the leading causes of PCOS include turbid phlegm, blood stasis and stagnation of liver Qi. Rhei Radix Et Rhizome is widely used in TCM to attack stagnation, clear damp heat, relieve fire. Rhei Radix Et Rhizome is an important part of the TCM formulas for the treatment of PCOS, which has a long history of medicinal use. However, the specific effect and mechanisms of Rhei Radix Et Rhizome on PCOS have yet to be elucidated. The object of this study aimed to investigate the effect and its pharmacological mechanism of Rhei Radix Et Rhizome on the treatment of polycystic ovary syndrome. PCOS was induced in female Sprague Dawley (SD) rats by administering letrozole (1 mg/kg, per orally, p.o.) for 21 days, then treated with Rhei Radix Et Rhizome at doses of 0.6 g/kg or 1.2 g/kg. Rats weight, blood glucose and estrus period are measured, and serum hormone include free testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and ovarian lesions were observed to determine the effects of Rhei Radix Et Rhizome. Network pharmacology and molecular docking predicted the targets of Rhei Radix Et Rhizome on PCOS. Epidermal growth factor receptor (EGFR), albumin (ALB), PI3K and P-AKT/AKT protein expression levels in ovarian tissues were assessed by Western blot. Rhei Radix Et Rhizome reduce abnormal weight and fasting blood glucose induced by letrozole (n = 5, p < 0.01), and improve the disturbed estrus cycle, reduce T, LH levels and LH/FSH ratio of PCOS rats (n = 4, p < 0.01). In addition, it alleviates the polycystic changes of ovaries in PCOS rats and reduces ovarian histopathological damage (n = 4, p < 0.01). Additionally, the core active components of Rhei Radix Et Rhizome for PCOS include Sennoside D_qt, Procyanidin B-5,3'-O-gallate, and Mutatochrome, which strongly bind to core therapeutic targets ALB and EGFR. Furthermore, the treatment reduces the increase of EGFR and ALB induced by letrozole (n = 4, p < 0.01). KEGG pathway enrichment analysis highlights endocrine resistance and prolactin signaling pathway, in both of which the PI3K/AKT pathway plays a crucial role. Our results show Rhei Radix Et Rhizome rescue the abnormal expression of PI3K/AKT pathway in PCOS rats (n = 4, p < 0.01). However, no significant dose-dependent relationship was observed in the tested dose range for the above experiments. These findings suggest that Rhei Radix Et Rhizome can regulate the PI3K/AKT pathway and target EGFR and ALB to treat polycystic ovary syndrome in rats. This study provides a scientific basis for the use of Rhei Radix Et Rhizome in the treatment of PCOS and highlights its potential mechanism through modulation of the PI3K/AKT pathway.

Yang W ，Qiu J ，Zi J ，Li Y ，Li J ，Guo M ，Zhou Y ，Yang X ，Lai Y ... -  《-》

Ammopiptanthus nanus (M. Pop.) Cheng f. stem ethanolic extract ameliorates rheumatoid arthritis by inhibiting PI3K/AKT/NF-κB pathway-mediated macrophage infiltration.

Ammopiptanthus nanus (M. Pop.) Cheng f. (A. nanus), a traditional Kirgiz medicinal plant, its stem has shown potential in treating rheumatoid arthritis (RA) in China, either through oral medication or by topical application directly to the affected joints, but its underlying mechanism of action remains unexplored. The purpose of this study is to elucidate pharmacological mechanism of A. nanus in ameliorating RA using a comprehensive approach that combines network pharmacology, molecular docking and experimental evaluations. Firstly, the major constituents of A. nanus stem ethanolic extract were identified and quantified by High-Performance Liquid Chromatography (HPLC). Disease target data from Gene Cards database was then used to define RA-associated targets. A protein-protein interaction (PPI) network was created via STRING database. The DAVID database powered gene ontology (GO) function and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to gain functional insights. In vitro, RAW264.7 cells were treated with A. nanus to investigate the roles of target proteins and pathways during lipopolysaccharide (LPS) - induced inflammation. Immunofluorescence assays were performed to assess the effects of A. nanus on macrophage infiltration. The key targets and signalling pathways were validated using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), molecular docking, immunohistochemical analysis, western blotting and immunofluorescence. Finally, the therapeutic potential of A. nanus in RA was evaluated in a carrageenan-induced rat model. Network analysis identified 31 potential targets of A. nanus associated with RA, including 10 hub targets. KEGG analysis highlighted the involvement of PI3K/AKT signaling pathway. In vivo experiments demonstrated that A. nanus treatment significantly protected against carrageenan-induced inflammatory paw tissue and attenuated macrophage infiltration. Both in vivo and in vitro experiments confirmed that A. nanus significantly downregulated the protein expression of COX-2, iNOS and IL-1β, and inhibited PI3K/AKT/NFκB pathway, which are closely linked to RA. Furthermore, molecular docking and cellular thermal shift assay revealed that licoflavanone showed a strong binding affinity with key targets. In summary, this study provides the first evidence of the potent anti-inflammatory activity of A. nanus in experimental RA. The mechanism of action appears to involve inactivation of the PI3K/AKT/NF-κB pathway-mediated macrophage infiltration. These findings indicate that A. nanus has significant potential as a therapeutic potential agent for RA treatment and offer novel insights for future research and drug development in this field.

Yao Y ，Wang J ，Zhang H ，Peng T ，Sun Y ，Zhang R ，Meng X ，Lu X ，Gao Y ，Jin Y ，Zhang Y ，Chen L ... -  《-》