Molecular mechanism of toxicity of Triptolide on hamster ovary cells based on transcriptomics research
Triptolide belongs to the class of epoxy diterpenoid lactones and is the main active ingredient in the traditional Chinese medicine Tripterygium wilfordii. Modern pharmacological research has shown that Triptolide can induce cell apoptosis, regulate autophagy, inhibit cell cycle, inhibit angiogenesis, and suppress the expression of pro-inflammatory cytokines, thus having various pharmacological effects such as anti-tumor, anti-inflammatory, immune regulation, and neuroprotection. Triptolide related preparations have also been widely recognized in clinical applications. However, in contrast to its excellent pharmacological effects, Triptolide has also shown significant toxic side effects in clinical applications. Multiple in vitro and in vivo studies have shown that Triptolide can cause damage to multiple organs such as the liver, kidneys, and ovaries, greatly limiting its clinical application and development. Therefore, a deep understanding of the molecular mechanism of the toxic side effects of Triptolide is of great significance for optimizing clinical medication, designing new derivatives, reducing toxicity, and improving safety.

The reproductive toxicity of Tripterygium wilfordii Hook. f. has always been a concern for researchers, but most studies have focused on the pathological changes and related physiological and biochemical abnormalities in the ovaries and other organs caused by Tripterygium wilfordii Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. f. Hook. Due to the ability of Triptolide to regulate multiple pathways to achieve its pharmacological effects, the study of its toxicity mechanism should also be considered as a whole. This study used Chinese hamster ovary cells as a model and utilized transcriptome sequencing technology to investigate the overall changes in gene expression in ovarian cells before and after administration of Triptolide, in order to provide reference for the molecular mechanism of Triptolide induced ovarian injury.

Triptolide has various pharmacological effects such as anti-inflammatory, anti-tumor, and immunosuppressive effects, and is commonly used in the treatment of various autoimmune diseases such as rheumatoid arthritis in clinical practice, achieving good application results. However, it can also damage ovarian function, induce ovarian cell apoptosis, cause various reproductive system problems such as menstrual disorders, amenorrhea, and premature ovarian failure, but the molecular mechanism of its ovarian toxicity is still unclear. This study used hamster ovary cell line as a model to detect and analyze the effect of Triptolide on gene expression in ovarian cells. Based on the comprehensive analysis of GO biological functions and KEGG metabolism and pathways, it was found that Triptolide promotes the IL-17 signaling pathway (cge04657) and TNF signaling pathway (cge04668), while exhibiting inhibitory effects on the PI3K Akt signaling pathway (cge04151).

There are currently six members of the interleukin-17 (IL-17) family, numbered IL-17A to IL-17F, which interact with receptors to activate downstream pathways including NF – κ B, MAPKs, and C/EBPs, inducing the expression of antimicrobial peptides, cytokines, and chemokines, thereby playing an important role in protecting hosts from invasion by extracellular pathogens. However, numerous studies have shown that abnormal IL-17 signaling pathway is a key trigger for many inflammatory diseases. The expression level of IL-17 is closely related to psoriasis, multiple sclerosis, ankylosing spondylitis, and cancer cell invasion. In the research of ovarian related diseases, the relationship between IL-17 and ovarian cancer is the most common. Studies have shown that IL-17 can promote the deterioration of ovarian cancer and serve as a potential biomarker for poor prognosis. Endometriosis, polycystic ovary syndrome, and premature ovarian failure have also been shown to be related to abnormal expression of IL-17. At the same time, Tripterygium wilfordii Hook. f. polysaccharide tablets have also been used to establish a rat model of premature ovarian failure disease. Through transcriptome sequencing and enrichment analysis, it was found that Tripterygium wilfordii Hook. f. can increase the expression levels of genes such as il17c, traf6, fos, cxcl1, cxcl3, and ptgs2, thereby promoting the IL-17 signaling pathway and downstream gene expression in ovarian cells (see Table 3 and Figure 6). This may be the main factor in the toxicity of Tripterygium wilfordii Hook. f. to ovarian cells and even inducing premature ovarian failure.

Tumor necrosis factor (TNF) is a multifunctional cytokine that includes TNF – α and TNF – β, both of which have the same receptor and can induce a series of intracellular signaling pathways. It plays an important role in anti-tumor response, inflammation control, and immune system homeostasis balance. Research has found that TNF receptors are present in almost all types of cells. After TNF binds to its receptor, it recruits connecting proteins such as TRADD and TRAF2 to further induce downstream gene activation and generate responses. The downstream effects of TRADD mainly include apoptosis and anti-inflammatory effects, while the downstream effects of TRAF2 mainly include anti apoptosis and inflammatory signals. As an important pro-inflammatory cytokine, abnormal expression of tumor necrosis factor can lead to various diseases such as systemic lupus erythematosus, ulcerative colitis, Crohn’s disease, etc. The existing research results confirm that serum TNF – α levels are significantly elevated in patients with ovarian endometriosis, polycystic ovary syndrome, and premature ovarian failure, indicating its important role in the process of ovarian injury. Through transcriptome sequencing and enrichment analysis, it was found that Triptolide can increase the expression levels of genes such as traf1, traf5, fos, cxcl1, csf2, and vcam1, promote the TNF signaling pathway and downstream gene expression (see Table 3 and Figure 7), which may be an important trigger for Triptolide induced ovarian cell pathology.

The phosphatidylinositol 3-kinase Akt signaling pathway (PI3K Akt signaling pathway) can be activated by various cellular stimuli or toxic damage, and plays a role in transcription, translation, cell proliferation, growth, and other processes. This pathway plays an important role in the activation, growth, and ovulation process of mammalian follicles. The transcriptome analysis results showed that Triptolide can inhibit the PI3K Akt signaling pathway and affect ovarian cell morphogenesis (GO: 0009653), which is consistent with reported research results and indirectly confirms the accuracy of our study.

In addition, enrichment analysis results showed that Triptolide can promote the biosynthesis of cholesterol in ovarian cells (cge00100 cge00900、GO:0008299、GO:0006720) 。 Research has shown that estradiol deficiency can affect lipid metabolism and cause an increase in cholesterol levels. Therefore, cholesterol levels in the body are negatively correlated with estradiol levels, and estradiol deficiency is an important pathological indicator of premature ovarian failure. Meanwhile, studies have shown that Triptolide can induce dyslipidemia in rats, leading to fatty liver toxicity. The results of this study suggest that Triptolide’s impact on lipid metabolism in ovarian cells may promote its ovarian toxicity.

In addition to the above pathways, other significantly enriched effects are mainly related to the pharmacological mechanism of Triptolide, such as the MAPK signaling pathway, p53 signaling pathway, ECM receptor interaction signaling pathway, as well as disease-related pathways such as rheumatoid arthritis and cancer, which are consistent with existing research results.

In summary, Triptolide can disrupt ovarian cell signaling and metabolic processes by affecting various pathways such as IL-17 signaling pathway, TNF signaling pathway, PI3K Akt signaling pathway, cholesterol metabolism, etc., leading to ovarian cell damage and ovarian toxicity. The research results provide useful references for further clarifying the toxic mechanism of Triptolide.