ATOX1 alleviates radiation‐induced cardiac injury by modulating AMPK/NRF2 to inhibit myocardial oxidative stress and mitochondrial dysfunction

Figures

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  FIGURE 1. ATOX1 encourages the survival of cardiomyocytes after radiation exposure. (A) WB analysis of ATOX1 protein expression changes in H9C2 cells following radiation; (B, C) RT‐PCR and WB assessed ATOX1 overexpression efficiency; (D) MTT assay of H9C2 cell proliferation; (E, G) IF analysis of KI‐67 expression levels in H9C2 cells; (F, H) TUNEL assay of apoptosis in H9C2 cells. *p < 0.05. WB, western blot.

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  FIGURE 2. ATOX1 attenuates radiation‐induced oxidative stress and mitochondrial dysfunction in cardiomyocytes. (A) Assay kit analysis of mitochondrial reactive oxygen species levels in H9C2 cells; (B) JC‐1 staining to evaluate changes in mitochondrial membrane potential in H9C2 cells; (C) Assay kit detection of mitochondrial permeability transition pore opening in H9C2 cells. *p < 0.05.

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  FIGURE 3. ATOX1 promotes the AMPK/NRF2 signaling. (A) WB analysis of AMPK protein expression and phosphorylation levels following radiation; (B) IF detection of AMPK phosphorylation levels in different cell groups; (C) WB analysis of the activation of the AMPK/NRF2 signaling pathway. *p < 0.05. WB, western blot.

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  FIGURE 4. ATOX1 alleviates radiation‐induced oxidative stress and mitochondrial dysfunction by activating the AMPK signaling pathway. (A) WB analysis of the activation status of the AMPK/NRF2 signaling pathway; (B, E) Assay kit measured and quantified mitochondrial ROS levels in H9C2 cells; (C, F) JC‐1 staining evaluated and quantified mitochondrial membrane potential changes in H9C2 cells; (D, G) Assay kit detected and quantified mPTP opening in H9C2 cells. (H) WB assessed the activation of the AMPK/NRF2 signaling pathway. (I, L) Assay kit measured and quantified mitochondrial ROS levels in M‐VCM cells; (J, M) JC‐1 staining evaluated and quantified changes in mitochondrial membrane potential in M‐VCM cells. (K, N) Test kit detected and quantified mPTP opening in M‐VCM cells; *p < 0.05. mPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; WB, western blot.

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  FIGURE 5. ATOX1 reduces myocardial injury and fibrosis in a murine thoracic irradiation model. (A) Post‐irradiation body weight changes in mice; (B) Heart‐to‐body weight ratio at the end of the experiment; (C) IF detection of KI‐67 in myocardial tissue; (D, E) IHC和 WB analysis of ATOX1 expression in myocardial tissue; (F) ELISA measurement of cTnT, NT‐proBNP, and CKMB in mouse serum; (G) Masson's trichrome staining to assess fibrosis in myocardial tissue; (H) RT‐PCR analysis of Tgfβ, Collagen‐I, and Collagen‐III mRNA levels in tissue. *p < 0.05.