Study on the Mechanism of Tumor Necrosis Factor α Promoting Ferroptosis of H9c2 Cardiomyocytes through Inhibiting the Nrf2/GPX4 Signaling Pathway

ZHANG Zongli^1,2,3, XI Shibing^1,2,3, YAO Li⁴, WANG Xiaoli⁵, XUE Lisha², LI Dan³, QUAN Jia^1,2, LI Tao^1,2,3*

(¹Institute of Pediatric Disease, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China; ²Pediatric Department, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China; ³Institute of Pediatric Diseases, Hubei University of Medicine, Shiyan 442000, China; ⁴Department of Pathology, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China; ⁵Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Shiyan 442000, China)

Abstract:

This study aims to investigate the role of TNFα (tumor necrosis factor α) in inducing ferropto sis in H9c2 cardiomyocytes and the regulatory role of the Nrf2/GPX4 signaling pathway in this process. A dam age model of H9c2 cardiomyocytes was established by TNFα stimulation. The expression of related proteins was detected by Western blot and immunofluorescence. The accumulation of ferrous ions, production of ROS (reactive oxygen species), and changes in mitochondrial membrane potential were observed under a microscope. qRT-PCR was performed to examine the effects of the ferroptosis inhibitor on the mRNA levels of Nrf2, GPX4, and PTGS2 induced by TNFα. The regulatory effects of Nrf2 gene silencing on the transcription levels of GPX4, PTGS2, and ACSL4 mRNA, as well as the effects on cellular ROS and ferrous ion content, were analyzed using siRNA technolo gy. TNFα treatment significantly downregulated the expression of GPX4 protein in H9c2 cells (P<0.05), which was accompanied by elevated ROS levels, significantly increased positive staining rate of ferrous ions, and decreased mitochondrial membrane potential. Mechanistic studies showed that TNFα significantly inhibited GPX4 expression in H9c2 cells; meanwhile, it suppressed the Nrf2-driven antioxidant proteins HO-1 and NQO1, and concomitantly up-regulated ACSL4 (P<0.05 or P<0.01). TNFα significantly upregulated the mRNA levels of Nrf2, GPX4, and PTGS2, and Fer-1 effectively inhibited the TNFα-induced upregulation of these genes. Silencing Nrf2 inhibited the transcription of GPX4 mRNA and significantly upregulated the mRNA levels of ACSL4 and PTGS2 (P<0.001 or P<0.000 1), which exacerbated the accumulation of ROS and ferrous iron overload in cells, ultimately promoting the ferroptosis process. TNFα induces ferroptosis in H9c2 cardiomyocytes by inhibiting the Nrf2/GPX4 signaling pathway, disrupting the balance between antioxidant defense and lipid peroxidation.

CSTR: 32200.14.cjcb.2025.10.0006