Research Article Notes: Kuntai Capsule and Polycystic Ovary Syndrome

Introduction to the Study

• The research article focuses on identifying bioactive compounds and potential mechanisms of Kuntai Capsule (KTC) in treating Polycystic Ovary Syndrome (PCOS).
• The study integrates network pharmacology and bioinformatics to elucidate the therapeutic effects of KTC.
• Authors: Xiushen Li, Jingxin Ma, Li Guo, Chenle Dong, Guli Zhu, Wenli Hong, Can Chen, Hao Wang, and Xueqing Wu.
• Affiliations include departments at Shenzhen University General Hospital and Shenzhen University Health Science Center.
• KTC is a proprietary Chinese medicine used for PCOS treatment.
• Objective: To understand KTC's therapeutic targets and molecular mechanisms in treating PCOS.

Materials and Methods

• Active Ingredients and Targets of KTC
  • TCMSP database (Traditional Chinese Medicine System Pharmacology Database and Analysis Platform) was used.
  • Screening criteria: drug-like properties ≥ 0.18 and bioavailability ≥ 30%.
  • Targets of active ingredients were identified via the TCMSP database and converted to gene names using the UniProt database (https://www.uniprot.org/).
  • Cytoscape 3.7.2 software was used to construct the relationship network between active ingredients and target genes.
• Therapeutic Targets of PCOS
  • Targets were obtained by searching DisGeNet, GeneCards, NCBI, OMIM, and PharmGKB using "PCOS" and "polycystic ovary syndrome" as keywords.
  • Target names were converted to gene names using the UniProt database.
• Targets Related to PCOS
  • Three PCOS-related datasets (GSE5850, GSE98421, GSE34526) from the Gene Expression Omnibus (GEO) database were merged.
  • R language "sva" and "limma" packages were used for batch correction and screening differentially expressed genes (DEGs) with |log_2(foldchange)| > 1 and p < 0.05.
• Potential Therapeutic Targets of KTC in the Treatment of PCOS
  • Therapeutic targets from CTD, DisGeNet, GeneCards, NCBI, OMIM, and PharmGKB databases.
  • Combined with DEGs from the GEO database and screened for targets appearing in at least two databases.
  • These targets were then intersected with KTC therapeutic targets to identify prospective KTC therapeutic targets for PCOS.
• Analysis of PPI network, GO, and KEGG
  • Interactions between potential therapeutic targets of KTC were obtained through the STRING database.
  • Protein-protein interaction (PPI) network was constructed using Cytoscape software.
  • Core therapeutic targets were screened according to the degree value.
  • R language was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to investigate probable molecular mechanisms.
• Molecular Docking
  • Active ingredients correlating to KTC’s core targets in PCOS treatment were identified.
  • 2D structures were retrieved via PubChem and converted into 3D structures using ChemBio3D software.
  • 3D structure of the core target was obtained through the PDB database, and water molecules and small molecule ligands were deleted using PyMOL software.
  • AutoDockTools software was used to convert protein and drug ingredient into PDBQT format files and identify active pockets.
  • Vina software was used for molecular docking.
• Identify the Core Ingredients of KTC
  • The therapeutic targets of all active ingredients in KTC were intersected with the therapeutic targets of KTC.
  • The active ingredient with the most overlapping targets was considered the core ingredient of KTC.
  • Further bioinformatics analysis of the potential therapeutic targets of the core ingredients was performed.

Results

• Active Ingredients and Targets of KTC
  • 80 active ingredients and 204 therapeutic targets of KTC were obtained through the TCMSP database.
  • The KTC regulatory network was constructed using Cytoscape software.
  • Quercetin, kaempferol, and wogonin were identified as the top three pharmaceutical ingredients in terms of degree value.
• Therapeutic Targets for PCOS
  • The number of therapeutic targets found in databases:
    • DisGeNet: 988
    • GeneCards: 2540 (relevance score ≥ 1)
    • NCBI: 477
    • OMIM: 181
    • PharmGKB: 327
• Targets Related to PCOS
  • 315 DEGs were detected using the R language "limma" package after excluding batch effects in three data datasets linked to PCOS.
• Potential Therapeutic Targets of KTC
  • 88 potential therapeutic targets of KTC for PCOS were discovered by intersecting the potential targets, DEGs, and therapeutic targets.
• Analysis Results of GO, KEGG, and PPI Networks
  • GO enrichment analysis:
    • Biological processes: reactions with metal ions, lipopolysaccharides, bacteria-derived molecules, nutritional levels, apoptosis, reactive oxygen metabolism, reproductive system, neuronal death, etc.
    • Cell components: membrane raft, membrane microdomain, membrane region, RNA polymerase II transcription factor complex, nuclear transcription factor complex, etc.
    • Molecular function: activity of steroid hormone receptors, nuclear receptors, transcription factors, oxidoreductase factors, etc.
  • KEGG enrichment analysis: IL-17 signaling pathway, TNF signaling pathway, p53 signaling pathway, Toll-like receptor signaling pathway, HIF-1 signaling pathway, etc.
  • PPI network analysis was performed, and core potential therapeutic targets were identified.
• Results of Molecular Docking
  • Five targets with the highest degree were identified: MAPK1, MAPK8, TP53, AKT1, and JUN.
  • Molecular docking data for the targets and their corresponding active ingredients were acquired.
  • The binding energies of all molecular docking results were less than -6.5.
• Core Ingredients of KTC
  • Quercetin was identified as the core ingredient of KTC.
  • Quercetin had 71 targets that overlap with the potential therapeutic targets of KTC for PCOS.
  • PPI network analysis on these 71 targets found that the 5 core targets were almost the same as those of KTC.
  • The repetition rate of the GO and KEGG enrichment analysis results of quercetin and KTC reached 75%.

Discussion

• KTC targets the pathogenesis of PCOS by invigorating the kidney and promoting blood circulation, regulating estrogen levels, and improving ovarian function.
• Network pharmacology and bioinformatics were used to explore the molecular mechanisms of KTC in the treatment of PCOS.
• 88 potential therapeutic targets of KTC for PCOS were identified, and 5 core targets (MAPK1, MAPK8, TP53, AKT1, and JUN) were screened out.
• The MAPK family participates in biological processes, with 14 members identified.
• MAPK1 plays a role in insulin resistance and ovulation dysfunction in PCOS patients.
• MAPK8 affects PCOS by regulating the autophagy of follicular cells.
• TP53 induces the apoptosis of ovarian granulosa cells and participates in PCOS.
• JUN causes fibrosis and regulates cell biological processes.
• AKT participates in the activation of primordial follicles and the differentiation of granulosa cells.
• AKT1 is involved in the proliferation of granular cells and follicle formation, and its upregulation in PCOS patients may be related to granule cell dysfunction.
• Molecular mechanisms of KTC's treatment of PCOS might be related to IL-17 signaling pathway, TNF signaling pathway, p53 signaling pathway, Toll-like receptor, etc.
• IL17A, a pro-inflammatory cytokine, is abundantly expressed in PCOS patients.
• The TNF-α signaling pathway is related to glucose uptake in tissues and may lead to a decline in female fertility.
• Increased androgen, a clinical feature of PCOS, can promote the expression of p53.
• The p53 signaling pathway may be involved in ovarian granulosa cell autophagy and death.
• The expression of Toll-like receptors in PCOS patients is significantly increased, which can lead to a decrease in the rate of available embryos.
• Molecular docking of the 5 core targets and their corresponding drug ingredients showed that wogonin-TP53, kaempferol-MAPK8, quercetin-TP53, and quercetin-MAPK1 have excellent binding efficiency.
• The core ingredient of KTC was quercetin, which had 71 targets that overlap with the potential therapeutic targets of KTC for PCOS.
• Quercetin works as an antioxidant, lowering free radical generation, preventing lipid peroxidation, and altering antioxidants.
• Studies have found that quercetin reduces body weight, cysts, and ovarian diameter and restores healthy follicle function to alleviate metabolic disorders of PCOS model rats.

Conclusion

• The study uncovered the targets and molecular mechanisms of KTC in the treatment of PCOS and confirmed that quercetin may replace KTC for the treatment of PCOS patients.
• These results may provide evidence for the clinical application of KTC in the treatment of PCOS.

Abbreviations

• KTC: Kuntai Capsule
• PCOS: Polycystic ovary syndrome
• TCMSP: Traditional Chinese Medicine System Pharmacology Database and Analysis Platform
• GEO: Gene Expression Omnibus
• TCM: Traditional Chinese medicine
• GO: Gene Ontology
• KEGG: Kyoto Encyclopedia of Genes and Genomes
• PPI: Protein-protein interaction