A series of estratriene-based hydroxamic acid derivatives were rationally designed as histone deacetylase (HDAC) inhibitors, utilizing estrone and estradiol scaffolds with hydroxamic acid groups attached at the 3-positio...A series of estratriene-based hydroxamic acid derivatives were rationally designed as histone deacetylase (HDAC) inhibitors, utilizing estrone and estradiol scaffolds with hydroxamic acid groups attached at the 3-position via alkoxy linkers of varying chain lengths. Structure-activity relationship studies indicated that compounds with n = 4 exhibited optimal activity. The lead compounds CFT-2b and CEC-2b showed potent antiproliferative effects against HeLa and SKOV-3 cells (IC, 6.09-8.36 μM) and favorable selectivity indices (8.5 to >13.1 versus 293 T cells). Notably, several compounds showed superior HDAC inhibitory activity compared to SAHA. Mechanistic studies showed that CFT-2b and CEC-2b induced dose-dependent apoptosis, caused G1-phase cell-cycle arrest, and significantly increased acetylated histone H3 levels in HeLa cells, consistent with intracellular HDAC inhibition. Molecular docking supported favorable binding within the HDAC2 and HDAC6 active sites via zinc chelation and proper cap-group positioning. These findings establish estratriene-based hydroxamic acids as promising HDAC inhibitor scaffolds for cancer therapy development.
Human cytochrome P450 enzyme CYP7A1 catalyzes the rate-limiting 7α-hydroxylation of cholesterol in bile acid biosynthesis but is difficult to express functionally in microbial hosts. Here, we report a whole-cell Escheric...Human cytochrome P450 enzyme CYP7A1 catalyzes the rate-limiting 7α-hydroxylation of cholesterol in bile acid biosynthesis but is difficult to express functionally in microbial hosts. Here, we report a whole-cell Escherichia coli biocatalytic platform co-expressing truncated CYP7A1 and its redox partner cytochrome P450 reductase (CPR) using bicistronic or dual-promoter vector systems. Protein expression was improved via GroES-GroEL co-expression, and Rosetta(DE3)-pET-tCYP7A1-tCPR was identified as the optimal strain. With the aid of hydroxypropyl-β-cyclodextrin and polymyxin B, 7α-hydroxy-4-cholesten-3-one (7α-HCO) was produced at 118.3 mg·L⁻¹ ·d⁻¹ with > 98 % purity. The product was verified by NMR and high-resolution mass spectrometry. This work presents the first microbial system for 7α-HCO biosynthesis via human CYP7A1, offering a scalable strategy for steroid production and potential applications in drug metabolism and inhibitor screening.
7-Ketocholesterol (7-KC), a major oxysterol formed through cholesterol autoxidation, is increasingly recognized as a pathogenic mediator in ageing and chronic disease. Detected in atherosclerotic plaques, Alzheimer's cor...7-Ketocholesterol (7-KC), a major oxysterol formed through cholesterol autoxidation, is increasingly recognized as a pathogenic mediator in ageing and chronic disease. Detected in atherosclerotic plaques, Alzheimer's cortex, aged retina, and lysosomal storage disorders, 7-KC actively drives oxidative stress, chronic inflammation, organelle dysfunction, and oxiapoptophagy. These mechanisms underpin its role in cardiovascular, neurodegenerative, and metabolic pathologies. Recent advances highlight nutritional antioxidants, pharmacological agents, microbial bioremediation, and nanotechnology as promising therapeutic avenues. Recognizing 7-KC as both a biomarker and therapeutic target offers opportunities for innovation in diagnostics and treatment of age-related and inflammatory disorders.
17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) can catalyze the reduction of the less active estrone (E) to the more active estradiol (E). It has a significant impact on the reproduction of female animals, follicular deve...17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) can catalyze the reduction of the less active estrone (E) to the more active estradiol (E). It has a significant impact on the reproduction of female animals, follicular development, the development of the breasts and reproductive organs in reproductive-age women, as well as the physical health, bones and cardiovascular system of postmenopausal women. This review summarizes the research progress on the expression, biological function, and regulatory mechanisms of 17β-HSD1 in estrogen-dependent diseases, including cancer. It also discusses the role of 17β-HSD1 in female reproduction processes, such as follicle development, and the regulation of its enzyme activity by activin A and insulin-like growth factor 1 (IGF-1). Furthermore, the review explores how phosphorylation at key sites influences its enzyme's activity, aiming to enhance the understanding of its regulatory mechanisms and improve the clarity of related research findings. This review systematically summarizes the research progress of 17β-HSD1 expression and enzyme activity regulation, which can provide theoretical reference for the development of animal breeding technology and the treatment of estrogen dependent diseases.
7-Ketocholesterol (7-KC), a cytotoxic oxysterol generated through cholesterol oxidation, plays a central role in the progression of atherosclerosis, neurodegeneration, and metabolic syndromes through mitochondrial dysfun...7-Ketocholesterol (7-KC), a cytotoxic oxysterol generated through cholesterol oxidation, plays a central role in the progression of atherosclerosis, neurodegeneration, and metabolic syndromes through mitochondrial dysfunction, ROS overproduction, and NLRP3 inflammasome activation. This study presents the first integrative systems pharmacology analysis exploring the molecular mechanisms by which β-sitosterol (BS), a phytosterol with antioxidant and anti-inflammatory properties, mitigates 7KC-induced toxicity. Shared targets between BS and 7KC were identified through target prediction databases and subjected to protein-protein interaction (PPI) network analysis using Cytoscape with bottleneck centrality. Top hub genes were functionally enriched using Gene Ontology and KEGG pathway tools, revealing BS's modulation of nuclear receptor activity, redox homeostasis, and OXPHOS pathways. BS targets were localized across cytosol, nucleus, and membrane compartments, supporting its multi-compartmental regulatory role. This mechanistic framework highlights BS as a potential nutraceutical intervention for 7KC-driven chronic diseases, including atherosclerosis, NAFLD, and Alzheimer's disease, warranting further biological validation.
We aim to explore the key metabolic components and underlying mechanisms of the Bushen Huoxue Formula (BH) in treating Osteoarthritis (OA). The mouse knee OA model was constructed using the destabilization of the medial...We aim to explore the key metabolic components and underlying mechanisms of the Bushen Huoxue Formula (BH) in treating Osteoarthritis (OA). The mouse knee OA model was constructed using the destabilization of the medial meniscus method. OA mice were orally administered the BH. Mouse cartilage damage was assessed. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS), network pharmacology analysis and molecular docking were employed to analyze the serum metabolite components and target protein of BH. After lipopolysaccharide (LPS) treatment, different concentrations of β-Estradiol 3-acetate were added to primary chondrocytes. Flow cytometry was utilized for detecting cell apoptosis. The Ubiquitin-Specific Protease 13 (USP13)/Toll-like Receptor 4 (TLR4)/Myeloid Differentiation Primary Response Protein 88 (MYD88)/NF-κB pathway and the TLR4 ubiquitination levels were assessed using immunological quantification and biochemical methods. Relative to normal mice, OA mice exhibited decreased knee joint cartilage thickness and increased inflammatory damage. BH treatment reversed these effects. Furthermore, BH enhanced TLR4 ubiquitination. Estradiol acetate was identified as the metabolic component of BH that alleviates OA. Estradiol acetate and its subtype molecule β-Estradiol 3-acetate could bind to the USP13 protein. The β-Estradiol 3-acetate concentration-dependently decreased the elevated levels of USP13, TLR4, MYD88, p-p65/p65 in chondrocytes induced by LPS, while increasing the TLR4 ubiquitination. β-Estradiol 3-acetate reversed LPS-induced chondrocyte apoptosis and elevation of inflammatory factors. Moreover, USP13 overexpression abolished the protective effects of BH and β-Estradiol 3-acetate against LPS-induced chondrocytes. In Conclusion, the BH metabolite β-Estradiol 3-acetate promotes TLR4 ubiquitination to relieve inflammation and apoptosis in OA chondrocytes by inhibiting USP13.
Estrogen modulates sensory neuron excitability via metabolic pathways, regulating women's pain perception. pro-opiomelanocortin (POMC), an endogenous polypeptide precursor, regulates pain response and is highly expressed...Estrogen modulates sensory neuron excitability via metabolic pathways, regulating women's pain perception. pro-opiomelanocortin (POMC), an endogenous polypeptide precursor, regulates pain response and is highly expressed in the trigeminal ganglion (TG). In this study, we used ovariectomized female rats to study how trigeminal ganglion POMC links to orofacial allodynia in hypoestrogenic state, and verified at both the gene and protein levels that the expression of POMC in the trigeminal ganglion decreased under the hypoestrogenic state. Subsequently, overexpressing the POMC gene in the TG reversed the pain hyperalgesia in ovariectomized rats. To further explore the regulatory mechanism of estrogen on POMC, we injected a selective estrogen receptor agonist at the trigeminal ganglion. Estradiol (E2) in the TG regulates the expression of POMC through estrogen receptor α (ERα). Subsequently, the Chromatin Cleavage and Tagging technology (CUT&Tag) and the dual-luciferase assay revealed that estrogen receptor α in the trigeminal ganglion has a positive regulatory effect on the promoter of POMC. In conclusion, this study has found that in the trigeminal ganglion, estrogen receptor α may reduce the expression of the POMC gene by inhibiting the activity of the POMC promoter. Meanwhile, this study has also found that in the TG, ERα may further regulate the biological activity of the POMC protein by binding to it. This dual regulation at both the transcriptional level and the protein level collectively mediates a decrease in the orofacial mechanical pain threshold and triggers an orofacial allodynia response.
Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder characterized by ovarian dysfunction, with limited effective treatments. This study investigates the therapeutic effects and mechanisms of white kidney...Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder characterized by ovarian dysfunction, with limited effective treatments. This study investigates the therapeutic effects and mechanisms of white kidney bean extract (WKBE) in a PCOS rat model. A PCOS model was established using letrozole, followed by intervention with varying doses of WKBE. Serum sex hormone levels, insulin resistance, and metabolic markers were measured. Ovarian histopathology, fibrosis, and apoptosis were assessed. Transcriptomic sequencing was performed on ovarian tissues from control, PCOS, and high-dose WKBE groups. High-dose WKBE significantly ameliorated endocrine-metabolic disturbances in PCOS rats, including reduced testosterone, LH/FSH ratio, insulin resistance, and lipid abnormalities, outperforming low/medium doses. It decreased body weight, ovarian index, and organ fat deposition, repaired ovarian histopathological damage, and reduced fibrosis and apoptosis. Transcriptomic analysis revealed that high-dose WKBE altered the expression of Wnt signaling pathway-related genes, suggesting its therapeutic role may involve modulation of this pathway. High-dose WKBE alleviates endocrine-metabolic dysregulation and ovarian dysfunction in PCOS rats by regulating the Wnt signaling pathway, offering a potential novel therapeutic strategy.
Vitamin D has been proposed to attenuate chemotherapy-induced gastrointestinal mucositis (GM). In the intestine, local catabolism of active vitamin D [1,25-dihydroxyvitamin D₃] is mediated by the enzyme Cyp24a1. This stu...Vitamin D has been proposed to attenuate chemotherapy-induced gastrointestinal mucositis (GM). In the intestine, local catabolism of active vitamin D [1,25-dihydroxyvitamin D₃] is mediated by the enzyme Cyp24a1. This study assessed whether deletion of Cyp24a1 specifically in intestinal epithelial cells can protect against 5-fluorouracil (5-FU)-induced intestinal injury and microbiome disruption in mice. Using the Cre-loxP system, Cyp24a1 was selectively ablated in the intestinal epithelium (IEC-KO mice). Male IEC-KO and Cyp24a1^fl/fl^ littermate control mice received a single intraperitoneal injection of 5-FU (450 mg/kg) or saline and were euthanised 48 h later. In control mice, 5-FU markedly reduced duodenal villous height and crypt area (p < 0.01), whereas IEC-KO mice retained intestinal architecture. Proliferation, measured by Ki-67 immunostaining, was preserved in both the small and large intestine of IEC-KO mice following 5-FU treatment (p < 0.05). Notably, colonic Tlr4 mRNA was significantly upregulated in IEC-KO mice (p < 0.001), with no corresponding increase in inflammatory cytokines. 16S rRNA sequencing revealed no change in overall microbial diversity; however, there were notable differences in the relative abundance of key taxa, such as Bifidobacteriaceae and Alistipes. These findings suggest that intestinal Cyp24a1 contributes to susceptibility to chemotherapy-induced intestinal injury and microbial dysbiosis, and that its deletion enhances epithelial regeneration, potentially via innate immune pathways.
7-Ketocholesterol (7-KC) is a biologically active oxysterol formed through the oxidation of cholesterol, predominantly under conditions of oxidative stress. It is generated both enzymatically in specific tissues such as...7-Ketocholesterol (7-KC) is a biologically active oxysterol formed through the oxidation of cholesterol, predominantly under conditions of oxidative stress. It is generated both enzymatically in specific tissues such as the brain and liver, and non-enzymatically via reactive oxygen species (ROS), especially in aging tissues and heat-processed animal-derived foods. 7-KC exerts multifaceted effects on human health, extending beyond lipid metabolism to disrupt glucose and amino acid utilization, impair mitochondrial function, and provoke endoplasmic reticulum (ER) stress. These disturbances contribute to chronic inflammation and oxidative damage, playing pivotal roles in the development of various diseases, including atherosclerosis, neurodegenerative disorders, diabetes, cancer, hepatic steatosis, and ocular and gastrointestinal pathologies. Additionally, 7-KC is a marker of cholesterol oxidation in the food industry, where it signals product degradation and potential toxicity in long-stored or thermally processed animal-based foods. This review explores the biosynthesis, metabolic fate, and pathophysiological role of 7-KC, highlighting its critical role in intermediary metabolism, disease progression, and food safety. Furthermore, it outlines mitigation strategies to reduce 7-KC exposure through dietary modifications, antioxidant interventions, and advanced food processing technologies aimed at enhancing public health.
7-Ketocholesterol (7-KC), a cytotoxic cholesterol oxidation product, drives oxidative stress, inflammation, and apoptosis in cardiovascular, neurodegenerative, and metabolic disorders. Current lipid-lowering agents, such...7-Ketocholesterol (7-KC), a cytotoxic cholesterol oxidation product, drives oxidative stress, inflammation, and apoptosis in cardiovascular, neurodegenerative, and metabolic disorders. Current lipid-lowering agents, such as statins, do not eliminate pre-formed oxysterols, highlighting an unmet therapeutic need. Guggulsterone(GGS), a steroidal phytoconstituent from Commiphora mukul, exhibits dual-function potential by reducing 7-KC formation through antioxidant, anti-inflammatory, and Nrf2-activating effects, while enhancing cholesterol efflux via LDLR and ABC transporters. Additionally, it improves endothelial and neuronal resilience to oxysterol-induced apoptosis. Guggulsterone's amphiphilic nature supports its integration into nanocarriers, enabling co-delivery with therapeutics for synergistic effects. Advanced formulations such as SEDDS, nanoparticle co-encapsulation, and solid lipid nanoparticles enhance its bioavailability, stability, and tissue targeting, including brain delivery. These properties position guggulsterone as both a therapeutic agent and bioenhancer, offering a promising strategy to mitigate oxysterol burden and improve clinical outcomes in 7-KC-related disorders.
7-Ketocholesterol (7-KC), a prominent oxysterol found in oxidized LDL, plays a central role in atherosclerosis through mechanisms involving oxidative stress, NF-κB activation, and mitochondrial dysfunction. Guggulsterone...7-Ketocholesterol (7-KC), a prominent oxysterol found in oxidized LDL, plays a central role in atherosclerosis through mechanisms involving oxidative stress, NF-κB activation, and mitochondrial dysfunction. Guggulsterone (GGS), a bioactive steroid from Commiphora mukul, exhibits antioxidant, anti-inflammatory, and FXR-antagonistic properties. This work highlights guggulsterone's ability to counteract 7-KC-induced endothelial injury by inhibiting NF-κB translocation, reducing reactive oxygen species (ROS), and modulating apoptosis. These multimodal effects suggest guggulsterone as a promising natural agent for vascular protection. A systems-based pharmacological approach may further define its therapeutic utility in oxysterol-driven cardiovascular diseases.
Elucidating the mechanisms of action of natural metabolites may be promising in the emergence of alternative candidate therapeutics. In the present study, the combined approaches of in silico molecular docking (MD) and i...Elucidating the mechanisms of action of natural metabolites may be promising in the emergence of alternative candidate therapeutics. In the present study, the combined approaches of in silico molecular docking (MD) and in vitro analyses were conducted to investigate the interacting partners of 24-epibrassinolide (EBR) as a steroid-derived phytohormone in cancer cells and evaluate the cell death mechanisms associated with these partners. EBR scoring functions were initially calculated against the selected 35 functional target proteins, which may interact with steroids, for tumor biology using AutoDock Tools-1.5.7 receptor-ligand MD software. Molecular analyses were carried out in breast, pancreatic, and hepatocellular carcinoma cell lines. Our results showed that the retinoic acid nuclear receptor γ (RARγ) was the most stable interacting partner with a binding energy (BE). Furthermore, the secondary simulation analyses obtained the lowest BE score for EBR among RARγ selective agonistic compounds. According to our data, EBR was significantly inhibited the cell viability of MDA-MB-231, MIA-PaCa-2, and Hep-G2 cells, and diminished the colony formation potential. We showed that RARγ was inhibited after increasing concentration of EBR, by affecting the downstream target's expressions including p21, p16, p27, p57 and cyclin D1 detected by qRT-PCR. We also investigated the effect of EBR treatment on the expression levels of the proteins linked to nuclear hormone receptor (NHR) expressions, apoptosis, endoplasmic reticulum stress, and Hippo-Yes-associated protein (YAP)/ Transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways. Our findings indicated that EBR is a strong ER stress modulator, apoptosis inducer in a caspase-dependent manner, and effector for the modulation of Hippo-YAP-TAZ signaling pathways through the interaction with retinoic acid receptor.
Research on the effects of the mineralocorticoid receptor (MR) suggested a role in innate and adaptive immune responses. The inflammatory profile is directly linked to MR activation in several pathologies such as cardiov...Research on the effects of the mineralocorticoid receptor (MR) suggested a role in innate and adaptive immune responses. The inflammatory profile is directly linked to MR activation in several pathologies such as cardiovascular diseases, autoimmunity, chronic renal disease and obesity. MR is a high-affinity receptor binding both mineralocorticoids and glucocorticoids. In this study, we explored the pharmacological modulation of MR with the mineralocorticoid agonist deoxycorticosterone (DOCA) and the antagonist spironolactone (SPIRO) on corticosterone levels in plasma, neuroinflammation, myelin status and neurodegeneration in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE) model of Multiple Sclerosis. Animals were treated from day 1 until sacrificed on day 17 post-induction, and experimental groups were divided into: EAE+DOCA (0.75 mg/kg s.c every 3 days), EAE+DOCA+SPIRO (Spironolactone: 25 mg/kg i.p daily), vehicle-treated EAE (EAE+VEH) and Control (CTRL). Administration of DOCA or vehicle to EAE conducted to similar neuropathological alterations. The MR antagonist (a) significantly decreased inflammatory parameters TLR4, IL-1β and microglial CD11b mRNAs and showed a tendency to reduced osteopontin, b) reduced the % of infiltrated cellular and demyelinated area, as well as the reactive gliosis (GFAP+ area and number of IBA1 + cells) vs EAE+DOCA (c) increased the area of the neuronal marker NeuN vs EAE+DOCA and EAE+VEH groups (d) improved functional performance in the rotarod test and clinical signs vs EAE+DOCA. Interestingly, plasma corticosterone was increased in EAE+VEH and EAE+DOCA vs CTRL, while SPIRO administration raised even more corticosterone levels. This hypercorticosteronemia had functional consequences, because the glucocorticoid receptor (GR) and the target gene serum glucocorticoid regulated kinase 1 (SGK1) mRNAs expression were also increased vs DOCA alone. We hypothesized that MR blockage with SPIRO downregulated inflammation-related spinal cord pathology whereas excess glucocorticoids circulating in the EAE+DOCA+SPIRO group may contribute to anti-inflammatory effects.
Alzheimer's disease (AD) is a progressive neurodegenerative condition characterized by persistent cognitive decli ne, tau-containing intracellular neurofibrillary tangles, and β-amyloid (Aβ)-containing extracellular plaq...Alzheimer's disease (AD) is a progressive neurodegenerative condition characterized by persistent cognitive decli ne, tau-containing intracellular neurofibrillary tangles, and β-amyloid (Aβ)-containing extracellular plaques. Early symptoms include patchy memory loss and some behavioural abnormalities. There is a plethora of studies that have reported sexual dimorphism and a higher prevalence of Alzheimer's disease in women. However, the molecular mechanisms responsible for these differences remain an enigma. The increasing aging population, as well as the decline in estrogen levels, have been attributed to increased risk in the development of AD in women. Hormone replacement therapy (HRT) has been proposed as an approach for tackling the increased AD susceptibility in women; increased AD vulnerability in men is also linked to testosterone levels. In addition to the hormonal influence as one of the causative factors for increased risk of AD, there is the involvement of genetic factors, with APOE ε4 gene documented as a risk gene leading to tau pathological changes in the brain of female AD patients. Here, we aim to systematically examine literature on the factors and molecular mechanisms responsible for sexual dimorphism in increased vulnerability and pathological features of AD, with the hope that it may provide information on the diagnosis and therapeutic interventions in AD.
The skin is a vital organ that protects the body from external insults. Ceramides, the major lipid components of the skin, are synthesized through three main pathways: de novo synthesis, sphingomyelin hydrolysis, and the...The skin is a vital organ that protects the body from external insults. Ceramides, the major lipid components of the skin, are synthesized through three main pathways: de novo synthesis, sphingomyelin hydrolysis, and the salvage pathway. Ceramides are crucial for maintaining the skin barrier and hydration, and their deficiencies are associated with various skin diseases such as atopic dermatitis, psoriasis, and Netherton's syndrome. In the cosmetic industry, ceramides are used for skin barrier repair and moisturization. However, their poor water solubility necessitates the development of effective delivery systems. Alternatively, exogenous substances can be utilized to promote ceramide synthesis in skin. Therefore, elucidating the mechanisms by which ceramides influence the skin barrier and hydration, and developing ceramide-containing cosmetic products based on these mechanisms, represent promising research directions for improving skin health.
Polycystic Ovary Syndrome (PCOS) is a severe and heterogeneous endocrine disorder affecting 6-20 % of women of reproductive age globally. Despite its high prevalence, the underlying etiology and pathophysiology remain un...Polycystic Ovary Syndrome (PCOS) is a severe and heterogeneous endocrine disorder affecting 6-20 % of women of reproductive age globally. Despite its high prevalence, the underlying etiology and pathophysiology remain unclear, necessitating the use of animal models to study disease mechanisms and therapeutic targets. This review critically evaluates various induction agents used in PCOS animal models and their ability to mimic the clinical, metabolic, and reproductive manifestations of the human condition. Induction agents explored include androgens [Testosterone, Dihydrotestosterone (DHT), Dehydroepiandrosterone (DHEA)], estrogen (estradiol valerate), aromatase inhibitors (letrozole), endocrine disruptors (bisphenol A), and dietary modifications (high-fat or high-sugar diets). These agents, administered in species such as rats, mice, zebrafish, reproduce hallmark PCOS features, including hyperandrogenism, anovulation, polycystic ovaries, and insulin resistance. The review highlights the mechanisms, symptom profiles, and translational relevance of each model. Comparative analysis is provided to assess the strengths and limitations associated with each agent, considering factors such as hormonal balance, metabolic function, and reproductive outcomes. Animal models serve as essential tools for understanding PCOS and testing therapeutic interventions. Each inducing agent offers unique insights into specific aspects of the disorder, although none fully replicates the human syndrome. The selection of the agent and animal species based on research goals is vital for clinical relevance. Future work should focus on integrating models that reflect both reproductive and metabolic features of PCOS to improve translational value.
Polycystic Ovary Syndrome (PCOS) is a prevalent endocrine disorder characterized by metabolic dysfunction. This study investigated whether Mogroside V (MV) ameliorates hyperandrogenism-induced glycolytic dysfunction in t...Polycystic Ovary Syndrome (PCOS) is a prevalent endocrine disorder characterized by metabolic dysfunction. This study investigated whether Mogroside V (MV) ameliorates hyperandrogenism-induced glycolytic dysfunction in testosterone (TES)-treated KGN cells. KGN cells treated with 150 µM TES exhibited significantly reduced viability, decreased lactate production, and increased pyruvate levels, which were reversed by 60 µM MV. Transcriptomic analysis revealed that TES dysregulated gene expression associated with alternative splicing (AS) and glycolytic pathways, while MV normalized glycolysis-related genes (LDHA, PKM) without affecting AS events. Although TES upregulated splicing factors HNRNPH3 and SRSF1, MV restored the expression of HNRNPH3 and SRSF1 without inducing aberrant splicing. Mechanistically, MV significantly reduced TES-induced hypermethylation of the LDHA promoter, thereby restoring LDHA mRNA and protein expression. MV mitigates PCOS-associated metabolic dysfunction primarily through LDHA promoter demethylation, independent of alternative splicing regulation. This study highlights MV as a natural compound with epigenetic regulatory potential for PCOS therapy.