SLC7A11/GPX4 Inactivation-Mediated Ferroptosis Contributes to the Pathogenesis of Triptolide-Induced Cardiotoxicity

Abstract

Triptolide exhibits promising efficacy in various cancers and immune diseases while its clinical application has been strongly restricted by its severe side effects, especially cardiotoxicity. However, the underlying mechanism of triptolide-induced cardiotoxicity (TIC) remains unclear. The RNA-seq analysis of triptolide-injured AC16 human cardiomyocyte cell line hinted that ferroptosis is involved in TIC. Further experimental validations proved that triptolide triggered ferroptosis, as evidenced by significant accumulation of lipid peroxidation (4-HNE and MDA levels) and ferrous iron, as well as depletion of intracellular GSH. Furthermore, triptolide-induced iron overload involved the upregulation of TF/TRFC/DMT1 signal axis and the degradation of ferritin, which contribute to ROS generation via Fenton reaction. In addition, inhibition of the antioxidant Nrf2/HO-1 pathway was observed in TIC, which may also lead to the overproduction of lethal lipid peroxides. Mechanistically, using streptavidin-biotin affinity pull-down assay and computational molecular docking, we unveiled that triptolide directly binds to SLC7A11 to inactivate SLC7A11/GPX4 signal axis. More importantly, employment of a ferroptosis inhibitor Ferrostatin-1 alleviated TIC by partially reversing the inhibitory effects of triptolide on SLC7A11/GPX4 signal. Altogether, our study demonstrated that SLC7A11/GPX4 inactivation-mediated ferroptosis contributed to the pathogenesis of TIC. Combating ferroptosis may be a promising therapeutic avenue to prevent TIC.

Figure 1

Triptolide induces ferroptosis in AC16 cells indicated by RNA-seq analysis. (a) Chemical structure of triptolide. (b) Cell viability of AC16 cells after treatment with triptolide for 24 h was detected using CCK-8 assay. ns: no significant; ^∗∗∗∗p < 0.0001 vs. the control group. (c) Triptolide-induced differentially enriched genes. (d) Top 20 KEGG pathway with high count. (e) Main targets of triptolide in ferroptosis. (f) DEGs identified as ferroptosis diver in FerrDb. (g) DEGs identified as ferroptosis suppressor in FerDb.

Figure 2

Triptolide provokes lipid peroxidation, excess iron accumulation, and GSH depletion. (a) MDA levels after treatment with triptolide for 24 h were detected by commercial assay kits. (b, c) The protein levels of 4-HNE were measured by Western blots. (d) Intracellular Fe²⁺ levels in AC16 cells. (e) Reduced GSH levels in AC16 cells. ns: no significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 vs. the control group.

Figure 3

Triptolide triggers iron overload through dysregulating iron absorption and storage system. (a) The protein levels of TRFC, TF, DMT1, FTH1, and LC3-II in AC16 cells were measured by Western blots. (b–f) The quantitative analysis of the indicated proteins. ns: no significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 vs. the control group.

Figure 4

Triptolide aggravated ROS accumulation via inhibiting Nrf2/HO-1 pathway. (a, b) ROS levels were assessed by flow cytometry using DCFH-DA probe. (c) The protein levels of SLC7A11 and GPX4 were measured by Western blots. (d, e) The quantitative analysis of the indicated proteins. ns: no significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 vs. the control group.

Figure 5

Triptolide downregulates SLC7A11/GPX4 axis by direct binding to SLC7A11. (a) The protein levels of SLC7A11 and GPX4 were measured by Western blots. (b, c) The quantitative analysis of the indicated proteins. (d) The synthesis procedure of biotin-triptolide. (e) Immunoblot with anti-SLC7A11 antibody of protein precipitated by streptavidin beads from AC16 cell lysates in the presence of biotin-triptolide (10 μM), biotin (10 μM) or the combination of triptolide (100 μM), and biotin-triptolide (10 μM). (f) Predicted binding sites of triptolide with SLC7A11. (g) Representations of the predicted binding mode of triptolide with SLC7A11. ns: no significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 vs. the control group.

Figure 6

Fer-1 attenuates TIC by restoring SLC7A11/GPX4 axis. (a–d) AC16 cells were pretreated with or without fer-1 (2 μM) for 1 h and then exposed to triptolide (30 nM) for another 24 h. (a) Cell viability of AC16 cells were detected using CCK8 assays. (b) MDA levels were detected by commercial assay kits. (c) Reduced GSH levels in AC16 cells. (d) The protein levels of SLC7A11 and GPX4 were measured by Western blots. (e, f) The quantitative analysis of the indicated proteins. ^∗∗∗∗p < 0.0001 vs. the control group, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. the triptolide group.

Figure 7

Schematic representation of the potential toxic mechanism involved in triptolide-induced ferroptosis.