Transcriptomic and proteomic investigations identify PI3K-akt pathway targets for hyperthyroidism management in rats via polar iridoids from radix Scrophularia

Abstract

High-polarity iridoids from Radix Scrophulariae (R. Scrophulariae) offer a range of benefits, including anti-inflammatory, antioxidant, antitumour, antibacterial, antiviral, and antiallergic effects. Although previous studies have indicated the potential of R. Scrophulariae for hyperthyroidism prevention and treatment, the specific active compounds involved and their mechanisms of action are not fully understood. This study explored the effects of high-polarity iridoid glycosides from R. Scrophulariae on hyperthyroidism induced in rats by levothyroxine sodium. The experimental design included a control group, a hyperthyroidism model group, and a group treated with iridoid glycosides. Serum triiodothyronine (T3) and thyroxine (T4) levels were quantified using an enzyme-linked immunosorbent assay (ELISA). Transcriptomic and proteomic analyses were applied to liver samples to identify differentially expressed genes and proteins. These analyses were complemented by trend analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The effectiveness of key factors was further examined through molecular biology techniques. ELISA results indicated a notable increase in T3 and T4 in the hyperthyroid rats, which was significantly mitigated by treatment with iridoid glycosides. Transcriptomic analysis revealed 6 upregulated and 6 downregulated genes in the model group, showing marked improvement following treatment. Proteomic analysis revealed changes in 30 upregulated and 50 downregulated proteins, with improvements observed upon treatment. The PI3K-Akt signalling pathway was investigated through KEGG enrichment analysis. Molecular biology methods verified the upregulation of Spp1, Thbs1, PI3K, and Akt in the model group, which was reversed in the treatment group. This study revealed that highly polar iridoids from R. Scrophulariae can modulate the Spp1 gene and Thbs1 protein via the PI3K-Akt signalling pathway, suggesting a therapeutic benefit for hyperthyroidism and providing a basis for drug development targeting this condition.

Keywords: Highly polar iridoids from radix scrophulariae; Hyperthyroidism; PI3K-Akt signaling pathway; Proteomics; Transcriptomics.

Fig. 1

Effects of high-polarity iridoid glycosides from R. Scrophulariae on T3 and T4 levels in hyperthyroidism rats ($\bar{x}$ ±s, n = 10). *P < 0.05, **P < 0.01 compared to the blank group; ^#P < 0.05, ^##P < 0.01 compared to the model group.

Fig. 2

Volcano plot and MA plot. (A) Volcano plot of levothyroxine sodium tablet (LST)-induced hyperthyroidism rats. The x-axis represents the log-fold change in expression, and the y-axis represents the negative log of the significance of differential expression. The vertical line represents a 2-fold difference threshold, and the horizontal line represents a P < 0.05 threshold. Genes that met the differential expression criteria are represented by blue dots, while genes that did not meet the criteria are represented by orange dots. (B) MA plot of the rats with LST-induced hyperthyroidism. The MA plot reflects the trend of expression differences between samples regardless of expression level. (C) Volcano plot of the effects of highly polar iridoids from R. Scrophulariae on hyperthyroidism model rats. (D) MA plot of the effects of highly polar iridoids from R. Scrophulariae on hyperthyroidism model rats.

Fig. 3

Heatmap of commonly differentially expressed genes (DEGs) in the control, model, and treatment groups. A heatmap was generated by clustering the RPKM values of DEGs using the median expression value as the median and the Pearson distance method (absolute value) as the clustering method. The red color in the heatmap represents highly expressed genes, while the blue color represents genes with low expression. K represents the control group, M represents the model group, and L represents the group treated with high-polarity iridoid glycosides from R. Scrophulariae.

Fig. 4

GO enrichment analysis and pathway classification enrichment. (A) Bar chart showing the enrichment analysis of differentially expressed genes (DEGs) in hyperthyroidism-induced rats treated with highly polar iridoids from R. Scrophulariae. The x-axis represents gene GO functional categories, and the y-axis represents the significance of enrichment calculated using the hypergeometric distribution (P value). The red line in the graph represents a P value = 0.05, indicating the GO functional categories that may be enriched in DEGs compared to the entire gene expression background. (B) KEGG pathway enrichment analysis of DEGs in hyperthyroidism-induced rats treated with highly polar iridoids from R. Scrophulariae. The x-axis represents KEGG pathways, and the y-axis represents the significance of enrichment calculated using the hypergeometric distribution (P value). The red line in the graph represents a P value = 0.05, indicating the KEGG pathways that may be enriched in DEGs compared to the entire gene expression background.

Fig. 5

GO enrichment analysis, KEGG pathway analysis, and protein‒protein interaction network diagram. (A) Biological process (BP), (B) cellular component (CC), and (C) molecular function (MF) terms. (D) The node size indicates the number of connections (degree), and the node color indicates the betweenness centrality (blue: low betweenness centrality, red: high betweenness centrality). The key proteins with the highest betweenness centrality values shown in the table are marked by the red nodes in the subnetwork. (E) Protein‒protein interaction network diagram of significantly differentially expressed proteins (DEPs) at the protein level.

Fig. 6

Validation of the microarray data by qRT‒PCR analysis (n = 3). The results are expressed as the relative quantification normalized to β-actin mRNA expression. **P < 0.01 vs. the control group. ^##P < 0.01 vs. model group.