Network Pharmacology-Based Prediction and Verification of the Potential Mechanisms of He's Yangchao Formula against Diminished Ovarian Reserve.

He's Yangchao formula (HSYC) has been clinically proven to be effective in treating diminished ovarian reserve (DOR). However, the underlying molecular mechanisms of HSYC in DOR are unclear. This study aims to predict the underlying mechanisms of He's Yangchao formula (HSYC) against DOR through network pharmacology strategies and verify in vivo. Systematic network pharmacology was used to speculate the bioactive components, potential targets, and the underlying mechanism of HSYC in the treatment of DOR. Then, the CTX-induced DOR mouse model was established to verify the effect of HSYC against DOR and the possible molecular mechanisms as predicted in the network pharmacology approach. A total of 44 active components and 423 potential targets were obtained in HSYC. In addition, 91 targets of DOR were also screened. The identified hub genes were AKT1, ESR1, IL6, and P53. Further molecular docking showed that the four hub targets were well-bound with their corresponding compounds. In vivo experiments showed that HSYC could promote the recovery of the estrous cycle and increase the number of primordial, growing follicles and corpora lutea. Besides, The results of qRT-PCR showed HSYC could regulate the expression of AKT1, ESR1, P53, and IL6 in DOR mice. It was demonstrated that HSYC could increase ovarian reserves, and AKT1, ESR1, IL6, and P53 may play an essential role in this effect, which provided a new reference for the current lack of active interventions of DOR.

Yang L ，Zhao Y ，Xu H ，Ma Y ，Wang L ，Ma J ，Zhang Q ... -  《-》

Exploring the anti-ovarian aging mechanism of He's Yangchao formula: Insights from multi-omics analysis in naturally aged mice.

The rapid acceleration of female reproductive aging has become a major public health concern. He's Yangchao formula (HSYC), a compound comprising eight herbs, has demonstrated efficacy in enhancing ovarian function. Thus, an in-depth study of its anti-ovarian aging mechanism is required. To evaluate the anti-ovarian aging effect of HSYC in naturally aged mice and investigate the underlying mechanism by analyzing the gut microbiota (GM), metabolome, and transcriptome. Young and advanced maternal age (AMA) mice were selected for this study. Hematoxylin and eosin staining, fluorescence staining, western blotting, and qPCR analyses were used to detect the phenotypes associated with ovarian aging. Subsequently, analyses of the GM, transcriptome, and metabolome analyses were performed to explore the potential mechanisms of action of HSYC. Finally, in vivo and in vitro experiments were performed to verify potential therapeutic mechanisms. HSYC promoted follicular development in AMA mice and ameliorated age-related mitochondrial dysfunction, apoptosis, and defects in DNA damage repair. GM analysis revealed that HSYC treatment significantly increased the abundance of Akkermansia and Turicibacter. Transcriptome and metabolome analyses showed that HSYC might mitigate ovarian aging by regulating metabolic pathways, amino acid metabolism, glutathione metabolism, and the synthesis of pantothenic acid and coenzyme A. Combined transcriptomic and metabolomic analyses identified the glutathione metabolic pathway as the key pathway through which HSYC counteracts ovarian aging. Additional experimental verification confirmed that HSYC upregulated the glutathione metabolic genes GPX8, GSTA1, and GSTA4, increased glutathione-related products (GSH), and reduced ROS levels. HSYC exerts beneficial therapeutic effects on ovarian aging by regulating multiple endogenous metabolites, targets, and metabolic pathways, with an emphasis on its anti-ovarian aging effects through the glutathione metabolic pathway. These findings underscore the innovative potential of HSYC in addressing ovarian aging and offer a novel therapeutic approach that targets multiple biological pathways to improve the reproductive health of women with AMA..

Yang L ，Lai X ，Jin S ，Wang H ，Lin F ，Jin X ，Chen Y ，Wang R ，Huang Y ，Zhang Y ，Tian S ，Fang X ，Duan X ，Zhang Q ... -  《-》

He's Yangchao Recipe Ameliorates Ovarian Oxidative Stress of Aging Mice under Consecutive Superovulation Involving JNK- And P53-Related Mechanism.

To evaluate the effects of He's Yangchao Recipe (HSYC) on ameliorating ovarian oxidative stress of aging mice under consecutive superovulation. An 8-month-old C57BL/6 female mouse was chosen to establish an aging model under ovarian hyperstimulation. Mice were randomly separated into four groups: R1 as the control group, R4 as the model group, NR4 with N-acetyl-L-cysteine (NAC) administration, and TR4 with HSYC administration. Oocyte collection, in vitro fertilization, and embryo culture were performed. The serum hormone levels were measured by enzyme-linked immunosorbent assays (ELISA); the reactive oxygen species (ROS) level of oocytes, the number of growing follicles, corpus luteum, ovulated oocytes, and developing embryos at each stage, along with the proportions of fragmented oocytes and abnormal mitochondria in granulosa cells (GCs) and the apoptosis rate of GCs were calculated; the mRNA and protein levels of JNK, P53, BAX were detected by real-time PCR and the Simple Western System. HSYC enhanced estradiol, progesterone, and inhibin-B levels and increased growing follicle and corpus luteum and ovulated egg counts compared to the R4 group (P < 0.05), whereas it decreased the proportions of fragmented oocytes (P < 0.01); Meanwhile, embryos from mice subjected to four superovulation cycles with HSYC treated had a higher hatching potential. The ROS level of oocytes is downregulated by HSYC (P < 0.01) and the percentage of abnormal mitochondrial in ovaries of the TR4 group was also significantly declined compared to the R4 group (P < 0.05); the most TUNEL-positive cells proportion was detected in the R4 group; nevertheless, HSYC effectively attenuated this detrimental effect (P < 0.05). The mRNA and protein expressions of JNK and P53 in ovary tissues were reduced in the TR4 group while these genes were upregulated by repeated superovulation (P < 0.05). HSYC exerted promising effects on promoting the diminished ovarian reserve and decreased oocyte quality induced by both aging and consecutive ovarian superovulation, potentially via the ROS/JNK/p53 pathway.

Zhao Y ，Chen Y ，Miao C ，Wang R ，Yang L ，Liu J ，Chen Z ，Zhang Q ，Ma J ... -  《-》

Promising anti-ovarian aging herbal formulation He's Yangchao promotes in vitro maturation of oocytes from advanced maternal age mice.

Marveled at the discovery of artemisinin, the world's expectations for traditional Chinese medicine are rising. He's Yangchao formula (HSYC) is a traditional Chinese herbal formula with the effects of tonifying kidney and essence, and reconciling yin and yang. It has been clinically proven to have anti-ovarian aging effects. Age is the primary cause of diminished ovarian reserve and assisted reproductive failure in women, whether HSYC has the potential to improve in vitro maturation of oocytes from advanced maternal age (AMA) mice has yet to be determined. This study aims to evaluate the efficacy and possible mechanism of HSYC in promoting in vitro maturation of oocytes from AMA mice. The GV oocytes were obtained from young and aged mice. The GV oocytes from young mice were cultured in drops of M16 medium, and the GV oocytes from AMA mice were randomly divided four groups: Vehicle group (cultured in 90% M16 medium +10% blank serum), Low HSYC group (cultured in 90% M16 medium + 10% Low HSYC-medicated serum), High-HSYC group (cultured in 90% M16 medium +10% High HSYC-medicated serum), and Quercetin group (cultured in M16 medium supplemented with 10 μM quercetin). The rates of first polar body extrusion, reactive oxygen species (ROS), intracellular calcium, and mitochondrial membrane potential levels in each groups were observed. In addition, expression levels of mitochondrial function, autophagy, DNA damage, and antioxidant-related proteins were assessed. Supplementation of HSYC in vitro alleviated age-associated meiotic progression defects in maternally aged oocytes. Importantly, HSYC supplementation eliminated the age-related ROS accumulation to suppress DNA damage and autophagy during the in vitro maturation of maternally aged oocytes. Meanwhile, the mitochondrial function was improved after HSYC treatment, as manifested by higher mitochondrial membrane potential and lower Ca2+ levels. Furthermore, we found that HSYC supplementation during in vitro maturation of maternally aged oocytes upregulated the expression level of SIRT3, a crucial protein in regulating mitochondrial function. Consistently, the expression levels of the SOD2, PCG1α, and TFAM were increased, while the SOD2 acetylation level was decreased, which further proved its antioxidant function. HSYC supplementation promotes in vitro maturation of oocytes from AMA mice mainly via improving mitochondrial function and alleviating oxidative stress. The mechanism may be related to the regulation of SIRT3-dependent deacetylation of the SOD2 pathway.

Yang L ，Shang J ，Wang H ，Ma J ，Wang L ，Ma Y ，Shuo J ，Xu X ，Cheng R ，Duan X ，Zhang Q ... -  《-》

Investigation of He's Yang Chao recipe against oxidative stress-related mitophagy and pyroptosis to improve ovarian function.

Primary ovarian insufficiency (POI) is a common gynecological disease with serious ramifications including low pregnancy rate and low estrogen symptoms. Traditional Chinese medicine is regarded as an effective treatment for POI. However, the therapeutic mechanism of it is unclear. In this study, a mouse model of primary ovarian insufficiency was established by intraperitoneal injection of cyclophosphamide (CTX) and He's Yang Chao Recipe (HSYC) concentrate was used for intragastric administration. Serum hormone levels (Anti-Müllerian Hormone, Estradiol, Progesterone, Luteinizing Hormone and Follicle Stimulating Hormone) and Oxidative Stress (OS) related products, superoxide dismutase (SOD), GSH-Px, and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Pathological changes in ovarian tissue were evaluated by hematoxylin and eosin staining, and flow cytometry was used to determine reactive oxygen species content and mitochondrial membrane potential levels in granulosa cells. Mitochondrial distribution and morphology were investigated using immunofluorescence staining. The level of mitophagy was evaluated by LC3 immunofluorescence staining and autophagosome counts using electron microscopy. Western blotting and qPCR were used to detect the expression of proteins and genes related to mitophagy and the NLRP3 inflammasome. After HSYC treatment, the ovarian damage was milder than in the CTX group. Compared with the CTX group; SOD, GSH-Px, and the total antioxidant capacity were significantly increased, while MDA and ROS were decreased in the HSYC treatment groups. Furthermore, mitochondrial distribution and membrane potential levels were improved after HSYC treatment compared to the CTX group. After the HSYC treatment, the LC3 fluorescent intensity and autophagosome counts were decreased. Similarly, mitophagy related markers PINK1, Parkin, LC3, and Beclin1 were decreased, while p62 was significantly increased, compared with the CTX groups. The mRNA and protein expression of NLRP3 inflammasome, NLRP3, caspase-1, GSDMD, IL-18, and IL-1β were significantly decreased in the HSYC treatment groups. This is the first study in molecular mechanisms underlying HSYC against granulosa cell injury in POI. HSYC protects ovaries from CTX-induced ovarian damage and oxidative stress. HSYC enhanced ovarian function in mice with primary ovarian insufficiency by inhibiting PINK1-Parkin mitophagy and NLRP3 inflammasome activation.

Miao C ，Zhao Y ，Chen Y ，Wang R ，Ren N ，Chen B ，Dong P ，Zhang Q ... -  《Frontiers in Endocrinology》