Integrating Epigenetics, Proteomics, and Metabolomics to Reveal the Involvement of Wnt/β-Catenin Signaling Pathway in Oridonin-Induced Reproductive Toxicity

Abstract

Oridonin is the primary active component in the traditional Chinese medicine Rabdosia rubescens, displaying anti-inflammatory, anti-tumor, and antibacterial effects. It is widely employed in clinical therapy for acute and chronic pharyngitis, tonsillitis, as well as bronchitis. Nevertheless, the clinical application of oridonin is significantly restricted due to its reproductive toxicity, with the exact mechanism remaining unclear. The aim of this study was to investigate the mechanism of oridonin-induced damage to HTR-8/SVneo cells. Through the integration of epigenetics, proteomics, and metabolomics methodologies, the mechanisms of oridonin-induced reproductive toxicity were discovered and confirmed through fluorescence imaging, RT-qPCR, and Western blotting. Experimental findings indicated that oridonin altered m6A levels, gene and protein expression levels, along with metabolite levels within the cells. Additionally, oridonin triggered oxidative stress and mitochondrial damage, leading to a notable decrease in WNT6, β-catenin, CLDN1, CCND1, and ZO-1 protein levels. This implied that the inhibition of the Wnt/β-catenin signaling pathway and disruption of tight junction might be attributed to the cytotoxicity induced by oridonin and mitochondrial dysfunction, ultimately resulting in damage to HTR-8/SVneo cells.

Keywords: Wnt/β-catenin signaling pathway; mitochondrial dysfunction; oridonin; reproductive toxicity; tight junction.

Figure 1

(A) The chemical structure of oridonin. (B) 400 MHz ¹³C NMR spectra of oridonin. (C) 400 MHz ¹H NMR spectra of oridonin. (D) Purity analysis of oridonin by HPLC. The viability (E) and LDH release level (F) of HTR-8/SVneo cells following treatment with 0, 12.5, 25, and 37.5 μM oridonin for 24 h. * p < 0.05, *** p < 0.001, n = 3.

Figure 2

Illustration in a Venn diagram depicting the overlap of m6A-modified genes (A) and peaks (B) between oridonin and control groups. (C) Analysis of m6A motifs in the oridonin and control groups. (D) Distribution pattern of m6A modification peaks per gene. Comparing the difference in density of m6A-modified peaks in mRNA (E) and ncRNA (F). Identification of conserved m6A sites (G), m6A genes and peaks related to the disease (H) in differentially modified m6A-mRNAs.

Figure 3

Molecular diversity of HTR-8/SVneo cells post-treatment with oridonin. (A) PCA for the mRNA expression between the oridonin and control groups. (B) Venn plot illustrating the intersection between differentially modified m6A genes and DEGs. (C) Volcano plot depicting genes m6A methylation downregulated (in blue) or upregulated (in red). (D) Volcano plot depicting genes expression downregulated (in blue) or upregulated (in red). The GO (E) and KEGG (F) enrichment of 829 overlapped genes among DEGs and differentially m6A-methylated mRNAs. (G) GSEA revealed that genes in oridonin group were enriched for DNA biosynthetic process, regulation of cell cycle G2/M phase transition, and mitochondrial gene expression.

Figure 4

The associations between m6A regulators and the genes of Wnt signaling pathway (A), cellular response to DNA damage (B), regulation of cell cycle (C), and tight junction (D). (E) The impact of m6A regulators on the DEGs within Wnt signaling pathway, regulation of cell cycle, cellular response to DNA damage, and tight junction. (F) The level of m6A modification on SFRP1, CCND1, and FOXM1 mRNA transcripts visualized by IGV. The interactions of oridonin with m6A regulators of FMR1 protein (G) and HNRNPA2B1 protein (H). (I) Molecular dynamics simulation between FMR1 protein and oridonin.

Figure 5

(A) PCA for the protein expression in the oridonin and control groups. (B) The volcano plot showed the DEPs in the oridonin and control groups. (C) Heatmap of DEPs in the oridonin and control groups. Enrichment analysis for GO terms (D) and KEGG pathway (E) related to DEPs.

Figure 6

GSEA results between the oridonin and control groups. Cytoskeletal protein binding (A), positive regulation of cell differentiation (B), regulation of cytoskeleton organization (C), cadherin binding (D), DNA templated transcription initiation (E), and mRNA binding (F).

Figure 7

(A) PCA plots between the oridonin and control groups. (B) The predictive ability of the OPLS-DA model is reliable. (C) The OPLS-DA model was able to clearly separate the oridonin group from the control group. (D) OPLS-DA S-plot. Red dots indicate metabolites with VIP values greater than 1, while green dots indicate metabolites with VIP values less than or equal to 1. (E) Volcano plots showing upregulated and downregulated metabolites. (F) Correlation heatmap of DEMs.

Figure 8

(A) Bar graph of DEM multiplicity, presenting the top 20 metabolites with the highest difference in multiplicity. (B) The top 10 enriched KEGG pathways of DEMs displayed by p-value in the oridonin and control groups (p < 0.05, VIP > 1). (C) Correlation analysis among samples. The top 20 differential metabolites with the highest VIP values are included. (D) Violin plot showing the top 20 DEMs based on VIP values.

Figure 9

(A) Representative images showing the intracellular ROS production. (B) Analysis of the relative fluorescence intensity of DCF in the oridonin group and the control group. Immunofluorescence analysis (C) and quantitative analysis (D) of phosphorylated γ-H2AX induced by 0, 12.5, 25, and 37.5 μM oridonin. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 3. Scale bar = 100 μm.

Figure 10

(A) Intracellular Ca²⁺ was visualized using Fluo-4 AM in fluorescence microscopy images. (B) Statistics of intracellular Ca²⁺ level. Oridonin induced mitochondrial damage in HTR-8/SVneo cells. JC-10 (C) and Calcein AM (E) probes were used to detect the MMP and mPTP in oridonin-treated HTR-8/SVneo cells, respectively. Quantitative results of the JC-10 probe (D) and Calcein AM probe (F) in oridonin-treated HTR-8/SVneo cells. ** p < 0.01, *** p < 0.001, n = 3. Scale bar = 100 μm.

Figure 11

(A) The mRNA levels were measured through RT-qPCR analysis. (B) Expression levels of WNT6, β-catenin, TCF7L1, and GSK-3β proteins in 0, 12.5, 25, and 37.5 μM oridonin-treated HTR-8/SVneo cells. (C) Protein expression levels of CLDIN1, ZO-1, and CCND1 in 0, 12.5, 25, and 37.5 μM oridonin-treated HTR-8/SVneo cells. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 3.