Baicalin mitigates polycystic ovary syndrome‑associated non‑alcoholic fatty liver disease by inhibiting the AR/SREBP1 axis

Figures

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  Figure 1. Validation of the PCOS mouse model. (A) Chemical structure of BA. (B) The experimental workflow for animal studies. (C) Representative images of vaginal exfoliated cells in control mice at different estrous cycle stages. Proestrus, predominantly nucleated epithelial cells (single or clustered) with few leukocytes; estrus, large, anucleated, keratinized squamous cells with irregular edges and minimal leukocyte presence; metestrus, reduced keratinized epithelial cells accompanied by numerous leukocytes and nucleated epithelial cells; diestrus, dominated by leukocytes. (D) Representative estrous cycle patterns in control and PCOS mice. (E) Representative ovarian sections stained with H&E. Corpus luteum is indicated by & and cystic follicles are indicated by #. Quantitative comparison of (F) corpora lutea and (G) cystic follicles in control and PCOS groups. Serum levels of (H) FSH, (I) LH, (J) DHT and (K) LH/FSH ratio in control and PCOS groups. Data are presented as the mean ± standard deviation (n=6). **P<0.01. Statistical significance was determined using an unpaired twotailed Student's ttest. PCOS, polycystic ovary syndrome; BA, baicalin; H&E, hematoxylin and eosin; FSH,…

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  Figure 2. BA treatment alleviates NAFLD-associated features in PCOS mice. (A) Body weight, (B) visceral fat-to-body weight ratio, serum levels of (C) TG, (D) TC and (E) NEFA in each group. (F) Representative liver histology in each group. (G) Quantitative analysis of Oil Red O-positive areas in liver sections. Hepatic levels of (H) TG, (I) TC and (J) NEFA in each group. Data are presented as the mean ± standard deviation (n=6-8). **P<0.01. Statistical significance was assessed using oneway ANOVA followed by Tukey's post hoc test. BA, baicalin; NAFLD, non-alcoholic fatty liver disease; PCOS, polycystic ovary syndrome; TG, triglycerides; TC, total cholesterol; NEFA, non-esterified fatty acids.

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  Figure 3. BA modulates lipid metabolism-associated transcriptomic profiles in the liver of PCOS mice. (A) PCA revealed distinct clustering of samples from control, PCOS and BA treatment groups. Volcano plots depicted DEGs between (B) PCOS vs. control and (C) BA-treated vs. PCOS groups. (D) Venn diagram illustrated overlapping DEGs in each group. DEGs were defined by DESeq2 with |log2(fold change)|>1.5 and BH-adjusted q<0.05. (E) GO enrichment analysis of overlapping DEGs highlighted the top 15 enriched biological processes associated with lipid metabolism. (F) KEGG pathway enrichment analysis of overlapping DEGs identified key pathways modulated by BA. GO and KEGG enrichment was assessed using the default hypergeometric test in clusterProfiler, followed by BH-adjusted q<0.05. BA, baicalin; PCOS, polycystic ovary syndrome; PCA, principal component analysis; DEGs, differentially expressed genes; BH, Benjamini-Hochberg; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

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  Figure 4. BA regulates de novo lipogenesis in PCOS mice through modulating the SREBP1 signaling pathway. (A) Transcriptomic analysis of Srebp1 gene expression levels in liver tissues (n=4). (B) Relative mRNA expression of Srebp1 determined using qualitative PCR (n=8). (C) Western blot analysis of SREBP1, ACC and FASN levels in the liver tissue. Quantitative analysis of (D) SREBP1, (E) ACC and (F) FASN protein expression levels. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01. Statistical significance was assessed using oneway ANOVA followed by Tukey's post hoc test. BA, baicalin; PCOS, polycystic ovary syndrome; Srebp1, sterol regulatory element-binding protein 1; ACC, acetyl-CoA carboxylase; FASN, fatty acid synthase.

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  Figure 5. Identification and analysis of therapeutic targets of BA for PCOS-associated NAFLD. (A) BATMAN-TCM identified potential drug targets of BA. GeneCards, OMIM and DrugBank identified disease-associated targets for (B) PCOS and (C) NAFLD. (D) Venn diagram illustrated the overlapping targets among PCOS, NAFLD and BA. (E) PPI network of 25 common genes identified in the Venn diagram. (F) CytoHubba analysis identified the top 10 core genes. BA, baicalin; PCOS, polycystic ovary syndrome; NAFLD, non-alcoholic fatty liver disease; PPI, protein-protein interaction.

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  Figure 6. Molecular docking, protein purification and ITC analysis reveal binding between BA and AR. Molecular docking analysis illustrated (A) 2D and (B) 3D binding interactions between BA and AR. (C) Structural diagram of the pGEX4T1-AR plasmid, with the sequence from the N-terminal to C-terminal consisting of a GST tag, restriction sites and the truncated AR protein. (D) GST-AR protein purification and Coomassie Brilliant Blue staining for identification. The order of the samples is indicated at the top of the image. The last lane contains the purified GST-AR protein. ITC analysis of BA binding to a truncated AR protein in (E) sample cells containing 1 μM AR protein (GST-tagged) and in (F) sample cells containing 1 μM GST protein (control). Upper panels, heat flow vs. time; lower panels, normalized heat effects vs. ligand-to-protein molar ratio. ITC, isothermal titration calorimetry; BA, baicalin; AR, androgen receptor; BSA, bovine serum albumin; GST, glutathione S-transferase.

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  Figure 7. BA inhibits AR transcriptional activity in MDA-kb2 cells. Results of the luciferase reporter assay revealed (A) AR activation through increasing DHT concentrations and (B) BA-mediated AR inhibition in the presence of 1 nM DHT in MDA-kb2 cells. BA, baicalin; AR, androgen receptor; DHT, dihydrotestosterone.

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  Figure 8. Proposed mechanism by which BA ameliorates PCOS-associated NAFLD via the AR/SREBP1 signaling axis. BA binds to AR and inhibits its transcriptional activity, thereby downregulating the AR-mediated activation of SREBP1. This suppression leads to decreased expression of key lipogenic enzymes, including FASN and ACC, resulting in reduced hepatic lipogenesis. Through this pathway, BA alleviates hepatic steatosis and metabolic dysfunction in a DHT-induced mouse model of PCOS with NAFLD. BA, baicalin; PCOS, polycystic ovary syndrome; NAFLD, non-alcoholic fatty liver disease; AR, androgen receptor; SREBP1, sterol regulatory element-binding protein 1; FASN, fatty acid synthase; ACC, acetyl-CoA carboxylase; DHT, dihydrotestosterone.