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| Cell | Gene | NCBI ID | Species | Deliverable | Order |
|---|---|---|---|---|---|
| {[ item.cell_name ]} | {[item.gene_symbol]} | {[item.gene_id]} | {[item.taxon]} | {[item.deliverable]} |
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Di-(2-ethylhexyl) phthalate (DEHP) exposure leads to ferroptosis via the HIF-1α/HO-1 signaling pathway in mouse testes.
Research Plan:
1. Validate that DEHP stimulation can cause testicular damage in vivo and in vitro.
2. Omics analysis and experimental validation indicate abnormal signaling pathways such as
HIF-1α.
3. Explore the mechanism of action of the HIF-1α signaling pathway.
4. Further validate the role of HIF-1α by knocking out HIF-1α in vitro.
Conducting rigorous research requires a complete experimental workflow that validates findings at each step of the process. Generally, omics analysis is used to scientifically screen genes, and multiple experimental results must be cross-validated. Gene knockout (KO) is still one of the conventional strategies for gene function studies. A successful gene KO cell line is crucial for the accuracy of the validation results.
In this study, the authors used phenotype analysis, RNA-Seq analysis, and ChIP-qPCR analysis after MEHP stimulation to identify the regulatory gene associated with testicular dysfunction: HIF-1α. They also found the preliminary regulation mechanism: MEHP (the main biological metabolite of DEHP in vivo) can inhibit PHDs, affect HIF-1α accumulation and stability, cause HIF-1α to translocate to the nucleus, and induce the binding of HIF-1/Hmox1 in Leydig and Sertoli cells, leading to an upregulation in HO-1 expression. Overexpression of HO-1 leads to ferrous overload (hypoferraemia), ROS accumulation, and ultimately causes ferroptosis, damaging the viability of Leydig and Sertoli cells.
To further confirm these results, the researchers used Leydig and Sertoli cells with HIF-1α gene KO (HIF-1α gene KO Leydig and Sertoli cells provided by Cyagen) for subsequent experiments. Sanger sequencing and Western blotting were used to confirm the successful knocking out of HIF-1α in the KO cell lines. Compared with wild-type cells, the HIF-1α-KO cell lines showed significant improvement in the degree of cell viability damage under MEHP stimulation.
Meanwhile, lipid peroxidation (Figure 1B, I) and ferrous overload (Figure 1C, J) were inhibited. Expressions of Hmox1 and HO-1 were evaluated using qPCR and Western blotting respectively, demonstrating that knocking out Hif-1α can reverse the up-regulation of Hmox1 (Figure 1D, K) and HO-1 (Figure 1G, N) expression levels under MEHP stimulation.
Furthermore, the ROS burst (Figure 1E, L) and cell death (Figure 1F, M) levels after MEHP stimulation were also reduced after knockdown HIF-1α.
Western blotting showed that knockdown HIF-1α restored the expression of ACSL4, FTH1, and SLC7A11, and suppressed the expression of GPX4 (Figure 1G, N). Overall, these findings suggest that MEHP stimulation leads to ferroptosis in testicular Leydig and Sertoli cells in a HIF-1α-dependent manner.
