Zinc sulfate improves insulin resistance, oxidative stress and apoptosis in liver tissues of PCOS rats through the NF-κB pathway

Abstract

Background: Polycystic ovary syndrome (PCOS) is primarily characterized by insulin resistance, which leads to increased hepatic glucose production and impaired insulin-mediated glucose disposal. Pathologically, this condition manifests as elevated liver cell apoptosis and reduced lipid transport capacity, further exacerbating insulin resistance. Liver cell apoptosis and mitochondrial dysfunction are key pathological features of PCOS-associated liver diseases, contributing significantly to the progression of PCOS. Although zinc sulfate is recognized for its antioxidant properties, its efficacy in ameliorating PCOS-related liver damage remains unclear.

Methods: Female Sprague-Dawley rats were induced with PCOS and non-alcoholic fatty liver disease (NAFLD) through a high-fat diet and letrozole administration over 28 days. Subsequently, the model rats received zinc sulfate treatment via gavage once daily for an additional 21 days. Serum hormone levels and biochemical markers were assessed using ELISA and enzymatic assays. Histological examination of ovarian and liver tissues was performed using hematoxylin and eosin (HE) staining, while hepatic lipid accumulation was evaluated by Oil Red O staining. Transmission electron microscopy was employed to examine liver cell ultrastructure, and TUNEL staining was used to assess hepatocellular apoptosis. Transcriptome sequencing was conducted on liver tissues to identify key genes and pathways, which were further validated by Western blotting and immunohistochemistry.

Results: Initial blood sampling revealed decreased serum zinc concentration in the PCOS group, alongside elevated levels of testosterone (T), luteinizing hormone (LH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), blood glucose, fasting insulin, and oral glucose tolerance test (OGTT) values. However, the levels of serum estrogen (E2) and follicle-stimulating hormone (FSH) in the PCOS group were significantly decreased. Markers of oxidative stress, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione disulfide/glutathione ratio (GSSG/GSH), glutathione peroxidase (GSH-PX), and catalase (CAT), were also increased. Zinc sulfate treatment effectively improved all these parameters. HE and Oil Red O staining confirmed that zinc sulfate mitigated high-fat diet and letrozole-induced fatty liver. Furthermore, zinc sulfate alleviated severe hepatocellular apoptosis and mitochondrial damage observed in PCOS rats. Transcriptomic analysis indicated that zinc sulfate primarily mitigated PCOS-related liver damage via the cholesterol synthesis pathway, and experimental validation demonstrated that zinc sulfate inhibited oxidative stress and apoptosis in liver cells through the NF-κB pathway.

Conclusion: Our study demonstrates that zinc sulfate ameliorates liver oxidative stress and apoptosis in PCOS by modulating the NF-κB pathway, offering a novel therapeutic approach for managing PCOS-associated liver diseases.

Keywords: hepatic lipid deposition; mitochondrial damage; oxidative stress; polycystic ovary syndrome; zinc sulfate.

Figure 1

Effects of Zinc Sulfate on Body Weight, Ovarian Pathology, and Biochemical Parameters in Polycystic Ovary Syndrome Rats. (a) Vaginal Smear Staining. (b) Changes in Body Weight and OGTT Results. (c) The changes in the emotional cycle (P, proestrus; E, estrus; M, metestrus; D, diestrus). (d) Serum Levels of Testosterone, luteinizing hormone, estrogen, follicle-stimulating hormone, Zinc Ions, Fasting Insulin, and HOMA-IR. Data are expressed as mean ± standard (n=10). (e) Histopathological Observation of Ovarian Tissue via HE Staining and Ovarian morphology score. (f) Number of follicles (PF, primary follicle; SF, secondary follicle; MF, mature follicle; AtF, atretic follicle; CL,corpus luteum). Data are expressed as mean ± standard (n=3). * means that P<0.05,** means that P<0.01, *** means that P<0.001, **** means that P<0.0001.

Figure 2

Effects of Zinc Sulfate on Serum Oxidative Stress Indicators in PCOS Rats. Zinc Sulfate Attenuates Oxidative Stress in PCOS Rats (SOD activity, CAT activity, Serum MDA levels, GSH-Px activity and GSSG/GSH ratio). Data are expressed as mean ± standard (n=10). ns means that P > 0.05, means that P<0.01, *** means that P<0.001, **** means that P<0.0001.

Figure 3

Impact of Zinc Sulfate on Liver Function in PCOS Rats. (a) Alterations in ALT, AST, TG, and TC Levels. Data are expressed as mean ± standard (n=10). ns means that P > 0.05, ** means that P<0.01, *** means that P<0.001, **** means that P<0.0001. (b) Histopathological Examination of Liver Tissue via HE Staining. (c) Detection of Lipid Droplets in Liver Tissue Using Oil Red O Staining.

Figure 4

Tunel staining was used to observe the effect of zinc sulfate on the apoptosis of liver cells. (a) Fluorescence staining (Green represents apoptotic cells and blue represents the cell nucleus). (b) The percentage of apoptotic cells. Data are expressed as mean ± standard (n=3). ns means that P > 0.05, * means that P<0.05, **** means that P<0.0001.

Figure 5

Transmission electron microscopy observation of the effect of zinc sulfate on liver cells.

Figure 6

Transcriptional Profiling of Liver Tissues. (a) Statistical distribution of gene expression levels across samples. (b) Heatmap illustrating differentially expressed genes. (c) GO enrichment analysis of differentially expressed genes. (d) String network diagram derived from GO enrichment analysis. (e) Bar chart summarizing KEGG pathway enrichment analysis for differentially expressed genes. (f) Bar chart highlighting KEGG pathway enrichment analysis of the top 20 genes. (g) Dot plot depicting the top 20 signaling pathways identified through KEGG enrichment analysis. (h) Top 10 differentially expressed genes as determined by cytoHubba module analysis. (i) Genes within cluster 1 identified via MCODE module analysis.

Figure 7

Visual representation of the docking analysis of the differentially expressed genes CYP7A1, HMGCS1, ACAT2, MSMO1, SQLE, TM7SF2, HMGCR, and IDI1 with Zn²⁺.

Figure 8

Expression of P-NFκB p65, P-IκB, NFκB p65, IκB, BCL2, Bax and Cleaved caspase3 in liver tissue by Western blotting. (a) Representative images of western blot. (b) Quantitative analysis of protein expression. On the same membrane, each internal reference is used for homogenization treatment. Data are expressed as mean ± standard (n=3). ns means that P > 0.05, ** means that P<0.01, *** means that P<0.001, **** means that P<0.0001.

Figure 9

Expression of P-NFκB p65 and P-IκB in liver tissues by immunohistochemistry. (a). Representative images of immunohistochemical staining. (b) Quantitative analysis of protein expression. On the same membrane, each internal reference is used for homogenization treatment. Data are expressed as mean ± standard (n=3). ns means that P > 0.05, **** means that P<0.0001.