Mechanisms of He Shi Yu Lin formula in treating premature ovarian insufficiency: insights from network pharmacology and animal experiments

Abstract

Objective: He Shi Yu Lin Formula (HSYLF) is a clinically proven prescription for treating premature ovarian insufficiency (POI), and has shown a good curative effect. However, its molecular mechanisms are unclear. This study aimed to investigate the molecular mechanisms of HSYLF and clarify how network pharmacology analysis guides the design of animal experiments, including the selection of effective treatment doses and key targets, to ensure the relevance of the experimental results.

Methods: Network pharmacology, molecular docking, and animal experiments were utilized to investigate the effects of HSYLF. Key targets were identified by intersecting herb and disease targets to construct protein-protein interaction and "active components-intersection targets-disease" networks. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using the clusterProfiler package in R. A total of 50 specific pathogen-free female mice of reproductive age were included in the animal experiments. They were divided into five groups: the positive control group, the high-dose HSYLF group, the low-dose HSYLF group, the model blank group, and the normal control group, to evaluate the serum anti-müllerian hormone levels, mitochondrial morphology in oocytes, the levels of reactive oxygen species (ROS), and mitochondrial membrane potential.

Results: Network pharmacology identified 204 active components connecting 219 key therapeutic targets for POI. Gene Ontology enrichment analysis indicated that the anti-POI targets of HSYLF mainly regulated response to xenobiotic stimulus, cellular response to chemical stress, and response to oxidative stress; and the Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested the primary pathways, including lipid and atherosclerosis, advanced glycation end product-receptor for advanced glycation end product signaling pathway in diabetic complications, bladder cancer, tumor necrosis factor signaling pathway, and interleukin-17 signaling pathway. The low-dose (33 g/kg/d) HSYLF and high-dose (66 g/kg/d) HSYLF groups exhibited a marked elevation in serum anti-müllerian hormone levels (low-dose group: 2657.63 ± 354.82 PG/ml; high-dose group: 2823.73 ± 316.04 PG/ml) and mitochondrial membrane potential compared to the model blank group (P < 0.05 or P < 0.01), along with a significant decline in fluorescence intensity of 2',7'-dichlorofluorescein for the levels of ROS in oocytes (P < 0.05 or P < 0.01). Additionally, both groups showed varying degrees of improvement in the morphology, quantity, and distribution of mitochondria.

Conclusion: This study provides definite evidence for the molecular mechanism by which HSYLF treats POI by decreasing mitochondrial ROS, increasing membrane potential, and improving mitochondrial function. The results from active components of HSYLF and their related key targets also confirmed the characteristics of its multi-component, multi-target, multi-pathway, and overall regulatory effects on POI. Further research regarding the mechanisms is required to generalize these results, and the deeper clinical value of HSYLF also needs to be investigated in the future.

Keywords: Active oxygen; He Shi Yu Lin Formula; Mitochondria; Network pharmacology; Premature ovarian insufficiency; Traditional Chinese medicine.

Fig. 1

The flow chart. HSYLF, He Shi Yu Lin Formula; POI, premature ovarian insufficiency; TCMSP, Traditional Chinese Medicine Systems Pharmacology; PPI, protein–protein interaction; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; AMH, anti-müllerian hormone; ROS, reactive oxygen species

Fig. 2

Venn diagram of intersection in HSYLF targets and POI targets

Fig. 3

Pharmacological regulatory network of TCM on key targets

Fig. 4

Bar chart for top 10 GO enrichment

Fig. 5

Bar chart of the top 20 KEGG pathways for HSYLF targets in POI treatment, highlighting lipid metabolism, AGE-RAGE signaling, and cancer-related pathways. KEGG, Kyoto Encyclopedia of Genes and Genomes; HSYLF, He Shi Yu Lin Formula; POI, premature ovarian insufficiency; AGE-RAGE, advanced glycation end product-receptor for advanced glycation end product

Fig. 6

Molecular docking results of HSYLF with high binding energy. A STAT3 and licochalcone A; B Jun and formononetin; C MAPK3 and naringenin; D TP53 and quercetin; E HSP90AA1 and 1,6-dihydroxy-5-methoxy-2-(methoxymethyl)-9,10-anthraquinone. HSYLF, He Shi Yu Lin Formula; STAT3, signal transduction and activator of transcription; Jun, Jun proto-oncogene; MAPK3: mitogen-activated protein kinase 3; TP53, tumor protein P53; HSP90AA1, heat shock protein 90 alpha family class A member 1

Fig. 7

HSYLF treatment increases serum AMH levels in mice with POI. ^▲▲P < 0.01, ^★P < 0.05, and ^★★P < 0.01 vs. normal control group (n = 8)

Fig. 8

HSYLF treatment improves the morphology and function of mitochondria in ovarian tissue of mice with POI. Magnification and scale bar (8000 × , 2 μm; 15,000 × , 1 μm), with white arrows marking the mitochondria

Fig. 9

HSYLF significantly reduces ROS levels in oocytes of mice with POI to protect ovarian function. A–B DCF fluorescence images and the expression in oocytes of mice in different groups. Magnification and scale bar: (400 × , 50 μm). ^▲▲P < 0.01 vs. normal control group; ^★P < 0.05, ^★★P < 0.01 vs. model blank group (n = 3). ROS, reactive oxygen species; DCF, 2′,7′-dichlorofluorescein; POI, premature ovarian insufficiency

Fig. 10

HSYLF treatment enhances mitochondrial membrane potential in oocytes of mice with POI. A Changes in the mitochondrial membrane potential of mice in oocytes in different groups. Magnification and scale bar: (400 × , 50 μm); B Fluorescence quantification results of mice in oocytes in different groups. ^▲▲P < 0.01 vs. normal control group; ^★P < 0.05, ^★★P < 0.01 vs. model blank group (n = 3)