Effect and mechanism of ionizing radiation on ferroptosis in mouse hepatocytes
Zhou Yali1, Xu Jie2, Li Ming3, He Yang1
1Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou 215006, China; 2Department of Blood Transfusion, the First Affiliated Hospital of Soochow University, Suzhou 215000, China; 3School of Radiation Medicine and Protection of Soochow University, Suzhou 215123, China
Abstract：Objective To investigate the effect and mechanism of ionizing radiation on ferroptosis in mouse hepatocytes. Methods Twenty-four C57BL/6J mice were divided into two groups by random number table method: healthy control group (control group, n=6) and irradiation group (whole liver was irradiated with a single dose of 30 Gy X-ray, n=18). Mice were sacrificed at 6, 24 and 72 h (6 mice per time point) after irradiation to obtain liver tissue and plasma samples. The contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma were measured by automatic biochemical analyzer. The prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured by automatic biochemical analyzer. Pathological changes of liver tissues were observed by hematoxylin-eosin (HE) staining. The iron deposition in liver tissues was detected by Prussian blue staining. The expression levels of 4-Hydroxynonenal (4HNE) and hepcidin in the liver were determined by immunohistochemical staining, and quantitative analysis was performed. Plasma malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC) and glutathione (GSH) content were determined by microplate reader analysis according to the kit instructions. The expression levels of transferrin receptor 1 (TfR1), p53, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in the liver were measured by Western blot. Results Compared with the control group, the plasma contents of ALT (t=5.15, 5.47, both P<0.001) , AST at 6 and 24 h after irradiation were increased (t=8.42, 2.50, both P<0.001), the plasma PT was prolonged (t=3.12, P=0.011) and the APTT was shortened (t=3.26, P=0.009) at 72 h after radiation in the irradiation group. Histopathological results showed that evident liver edema was observed at 6, 24 and 72 h after irradiation (t=9.58, 10.09, 18.70, all P<0.001). Different degrees of iron deposition were observed (t=8.57, 15.31, 32.11, all P<0.001). The infiltration of hepcidin positive cells was significantly increased after irradiation (t=5.36, 13.17, 17.11, all P<0.001). The number of 4HNE positive cells was significantly increased (t=18.86, 22.67, 9.12, all P<0.001). At the same time, ionizing radiation induced a significant increase in plasma MDA content (t=4.36, 7.47, 8.22, all P<0.001), and a decrease in SOD (t=4.52, 5.80, 7.60, all P<0.001), T-AOC (t=13.24, 20.49, 24.96, all P<0.001) and GSH (t=2.78, 6.07, 11.25, P=0.020, <0.001, <0.001), respectively. The expression level of TfR1 protein was significantly up-regulated (t=3.46, 5.40, P=0.026, 0.006), whereas that of GPX4 protein was significantly down-regulated (t=11.88, 30.63, both P<0.001) at 24 and 72 h after irradiation. At 6, 24 and 72 h after irradiation, the expression level of p53 protein was significantly up-regulated and maintained at a high level (t=7.84, 4.25, 8.22, P=0.001, 0.013, 0.001), while that of SLC7A11 protein was significantly down-regulated (t=9.29, 19.96, 9.09, all P<0.001). Conclusion Ionizing radiation induces the ferroptosis in hepatocytes, and its mechanism may be related to the activation of p53-SLC7A11-GPX4 pathway.
Zhou Yali,Xu Jie,Li Ming et al. Effect and mechanism of ionizing radiation on ferroptosis in mouse hepatocytes[J]. Chinese Journal of Radiation Oncology, 2023, 32(7): 626-632.
[1] Liao JB, Jin HL, Li SQ, et al.Apatinib potentiates irradiation effect via suppressing PI3K/AKT signaling pathway in hepatocellular carcinoma[J]. J Exp Clin Cancer Res, 2019,38(1):454. DOI: 10.1186/s13046-019-1419-1. [2] Shojaie L, Iorga A, Dara L.Cell death in liver diseases: a review[J]. Int J Mol Sci, 2020,21(24):9682. DOI: 10.3390/ijms21249682. [3] 康传杰, 张相彤, 马威. 细胞铁死亡发生与调控机制的研究进展[J].中国病理生理杂志,2017,33(3):567-571. DOI: 10.3969/j.issn.1000-4718.2017.03.032. Kang CJ, Zhang XT, Ma W.Progress in occurrence and development of ferroptosis[J].Chin J Pathophysiol,2017,33(3):567-571. DOI: 10.3969/j.issn.1000-4718.2017. 03.032. [4] Chen X, Li JB, Kang R, et al.Ferroptosis: machinery and regulation[J]. Autophagy, 2021,17(9):2054-2081. DOI: 10.1080/15548627.2020.1810918. [5] Ding YH, Chen XP, Liu C, et al.Identification of a small molecule as inducer of ferroptosis and apoptosis through ubiquitination of GPX4 in triple negative breast cancer cells[J]. J Hematol Oncol, 2021,14(1):19. DOI: 10.1186/s13045-020-01016-8. [6] Stockwell BR, Jiang XJ, Gu W.Emerging mechanisms and disease relevance of ferroptosis[J]. Trends Cell Biol, 2020,30(6):478-490. DOI: 10.1016/j.tcb.2020.02.009. [7] Lei G, Zhang YL, Koppula P, et al.The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression[J]. Cell Res, 2020,30(2):146-162. DOI: 10. 1038/s41422-019-0263-3. [8] Lei G, Mao C, Yan YL, et al.Ferroptosis, radiotherapy, and combination therapeutic strategies[J]. Protein Cell, 2021,12(11):836-857. DOI: 10.1007/s13238-021-00841-y. [9] Zhang XH, Li X, Zheng CY, et al.Ferroptosis, a new form of cell death defined after radiation exposure[J]. Int J Radiat Biol, 2022,98(7):1201-1209. DOI: 10.1080/09553002. 2022.2020358. [10] Zhou H, Zhou YL, Mao JA, et al.NCOA4-mediated ferritinophagy is involved in ionizing radiation-induced ferroptosis of intestinal epithelial cells[J]. Redox Biol, 2022,55:102413. DOI: 10.1016/j.redox.2022.102413. [11] Chen JY, Li XP, Ge CD, et al.The multifaceted role of ferroptosis in liver disease[J]. Cell Death Differ, 2022, 29(3): 467-480. DOI: 10.1038/s41418-022-00941-0. [12] Du SS, Chen GW, Yuan BY, et al.DNA sensing and associated type 1 interferon signaling contributes to progression of radiation-induced liver injury[J]. Cell Mol Immunol, 2021,18(7):1718-1728. DOI: 10.1038/s41423- 020-0395-x. [13] Das U, Manna K, Sinha M, et al.Role of ferulic acid in the amelioration of ionizing radiation induced inflammation: a murine model[J]. PLoS One, 2014,9(5):e97599. DOI: 10.1371/journal.pone.0097599. [14] Sempoux C, Horsmans Y, Geubel A, et al.Severe radiation-induced liver disease following localized radiation therapy for biliopancreatic carcinoma: activation of hepatic stellate cells as an early event[J]. Hepatology, 1997,26(1):128-134. DOI: 10.1002/hep.510 260117. [15] Pan CC, Kavanagh BD, Dawson LA, et al.Radiation- associated liver injury[J]. Int J Radiat Oncol Biol Phys, 2010,76(3 Suppl):S94-100. DOI: 10.1016/j.ijrobp.2009.06.092. [16] Ayala A, Muñoz MF, Argüelles S.Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal[J]. Oxid Med Cell Longev, 2014,2014:360438. DOI: 10.1155/2014/360438. [17] Zhang HX, Ostrowski R, Jiang DZ, et al.Hepcidin promoted ferroptosis through iron metabolism which is associated with DMT1 signaling activation in early brain injury following subarachnoid hemorrhage[J]. Oxid Med Cell Longev, 2021,2021:9800794. DOI: 10.1155/2021/980 0794. [18] Feng HZ, Schorpp K, Jin J, et al. Transferrin receptor is a specific ferroptosis marker[J]. Cell Rep, 2020,30(10):3411-3423.e7. DOI: 10.1016/j.celrep.2020.02.049. [19] Huang S, Wang YH, Xie SW, et al.Hepatic TGFβr1 deficiency attenuates lipopolysaccharide / D- galactosamine- induced acute liver failure through inhibiting GSK3β-Nrf2-mediated hepatocyte apoptosis and ferroptosis[J]. Cell Mol Gastroenterol Hepatol, 2022,13(6):1649-1672. DOI: 10.1016/j.jcmgh.2022. 02.009. [20] Rodier F, Campisi J, Bhaumik D.Two faces of p53: aging and tumor suppression[J]. Nucleic Acids Res, 2007,35(22):7475-7484. DOI: 10.1093/nar/gkm744. [21] Li YC, Cao YM, Xiao J, et al.Inhibitor of apoptosis- stimulating protein of p53 inhibits ferroptosis and alleviates intestinal ischemia/reperfusion-induced acute lung injury[J]. Cell Death Differ, 2020,27(9):2635-2650. DOI: 10.1038/s41418-020-0528-x. [22] Jiang L, Kon N, Li TY, et al.Ferroptosis as a p53-mediated activity during tumour suppression[J]. Nature, 2015,520(7545):57-62. DOI: 10.1038/nature14344. [23] Chen X, Yu CH, Kang R, et al.Cellular degradation systems in ferroptosis[J]. Cell Death Differ, 2021,28(4):1135-1148. DOI: 10.1038/s41418-020-00728-1. [24] Lei G, Zhang YL, Hong T, et al.Ferroptosis as a mechanism to mediate p53 function in tumor radiosensitivity[J]. Oncogene, 2021,40(20):3533-3547. DOI: 10.1038/s41388-021-01790-w. [25] Xie L, Song XR, Yu JM, et al.Solute carrier protein family may involve in radiation-induced radioresistance of non-small cell lung cancer[J]. J Cancer Res Clin Oncol, 2011, 137(12): 1739-1747. DOI:10.1007/s00432-011- 1050-9.
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