Role of Ferroptosis in Paraquat-Induced Acute Lung Injury in Mice

doi:10.12116/j.issn.1004-5619.2024.440115

Abstract

Abstract:

Objective To explore the potential mechanisms by which ferroptosis contributes to paraquat-induced acute lung injury in mice and its forensic significance. Methods Sixty healthy male clean-grade Kunming mice aged 6-8 weeks were randomly divided into a control group and paraquat exposure groups （1 d, 3 d, 5 d, 7 d and 14 d）, with 10 mice per group. The exposure groups were given a single intraperitoneal injection of 20 mg/kg paraquat solution, while the control group was given an equal volume of 0.9% saline. General conditions after exposure were observed. Hematoxylin-eosin （HE） staining was used to examine pathological changes in lung tissues, and Prussian blue staining was used to detect iron content. Oxidative stress indicators, such as reduced glutathione （GSH） and malondialdehyde （MDA）, were measured by relevant testing kits. Immunohistochemistry and Western blot were employed to detect the expression levels of ferroptosis-related proteins including glutathione peroxidase 4 （GPX4）, solute carrier family 7 member 11（SLC7A11）, and heme oxygenase-1 （HO-1）. Results With the increase of exposure time, mice in the exposure groups showed progressively aggravated fibrosis and increased iron deposition in lung tissues. GSH levels gradually decreased, while MDA levels increased significantly in the 7 d and 14 d groups. The expression of GPX4 in lung tissue decreased. The expression of SLC7A11 decreased over time, except for the 1 d group. The expression of HO-1 increased with the increase of exposure time, except for the 1 d group, and the highest expression was observed in the 7 d group. The differences between the exposure groups and the control group were statistically significant （P<0.05）. Conclusion Ferroptosis is involved in acute lung injury induced by paraquat poisoning, and research on the mechanism of ferroptosis and the temporal changes of its biomarkers may provide valuable insights for the forensic identification of paraquat poisoning.

Key words: forensic pathology, forensic toxicology, paraquat, acute lung injury, ferroptosis, mice

CLC Number:

Hao-jie QIN, Chen MO, Hong-wei LI, Zhi-jiang LIU, Zhe ZHENG. Role of Ferroptosis in Paraquat-Induced Acute Lung Injury in Mice[J]. Journal of Forensic Medicine, 2025, 41(5): 494-501.

Figures/Tables 8

Fig. 1 Pathological changes of mice lung tissues at different time points after paraquat poisoning

Fig. 2 Iron staining results of mice lung tissues at different time points after paraquat poisoning

Tab. 1 The GSH and MDA contents in mice lung tissues

组别	GSH/（μg·g^-1）	MDA/（nmoL·g^-1）
对照	712.6±5.0	45.9±5.0
染毒1 d	650.8±6.0 ^1）	50.4±4.2
染毒3 d	615.9±4.8 ^1）2）	51.1±3.3
染毒5 d	560.5±13.3 ^1）2）	49.7±3.5
染毒7 d	521.1±2.7 ^1）2）	61.2±9.1 ^1）
染毒14 d	460.6±5.3 ^1）2）	76.3±11.9 ^1）

Fig. 3 Immunohistochemical staining results of GPX4 in mice lung tissues at different time points after paraquat poisoning

Fig. 4 Immunohistochemical staining results of SLC7A11 in mice lung tissues at different time points after paraquat poisoning

Fig. 5 Immunohistochemical staining results of HO-1 in mice lung tissues at different time points after paraquat poisoning

Tab. 2 Relative expression levels of GPX4， SLC7A11 and HO-1 proteins in mice lung tissues atdifferent time points after paraquat poisoning

组别	GPX4	SLC7A11	HO-1
对照	1	1	1
染毒1 d	0.58±0.09 ^1）	0.84±0.06	1.31±0.06
染毒3 d	0.46±0.07 ^1）2）	0.66±0.06 ^1）	1.40±0.11 ^1）
染毒5 d	0.34±0.08 ^1）2）	0.64±0.14 ^1）	1.55±0.06 ^1）2）
染毒7 d	0.35±0.11 ^1）2）	0.62±0.07 ^1）2）	1.79±0.10 ^1）2）
染毒14 d	0.32±0.07 ^1）2）	0.58±0.05 ^1）2）	1.52±0.06 ^1）2）

Fig. 6 Changes of GPX4， SLC7A11 and HO-1 protein expression in mice lung tissue at different time points after paraquat poisoning

References 26

[1]	LIU Z， HUANG F， ZHAO S， et al. Homicidal paraquat poisoning： Poisoned while drinking[J]. J Forensic Sci，2022，67（3）：1312-1319. doi：10.1111/1556-4029.14968 .
[2]	SHI M， ZENG M， JIAN T， et al. A mass event of paraquat poisoning via inhalation[J]. Front Public Health，2023，11：1309708. doi：10.3389/fpubh. 2023.1309708 .
[3]	ELENGA N， MERLIN C， LE GUERN R， et al. Clinical features and prognosis of paraquat poisoning in French Guiana： A review of 62 cases[J]. Medicine （Baltimore），2018，97（15）：e9621. doi：10.1097/MD.0000000000009621 .
[4]	SHADNIA S， EBADOLLAHI-NATANZI A， AHMADZADEH S， et al. Delayed death following paraquat poisoning： Three case reports and a literature review[J]. Toxicol Res （Camb），2018，7（5）：745-753. doi：10.1039/c8tx 00120k .
[5]	吉子炎. 百草枯肌肉注射后迟发性死亡1例[J].法医学杂志，2017，33（5）：552-553. doi：10.3969/j.issn.1004-5619.2017.05.025 .
	JI Z Y. Delayed death after intramuscular injection of paraquat： A case report[J]. Fayixue Zazhi，2017，33（5）：552-553.
[6]	刘勇，王方，赵波，等. 隐匿性百草枯中毒1例[J].法医学杂志，2016，32（2）：154-155. doi：10.3969/j.issn.1004-5619.2016.02.025 .
	LIU Y， WANG F， ZHAO B， et al. Occult paraquat poisoning： A case report[J]. Fayixue Zazhi，2016，32（2）：154-155.
[7]	DIXON S J， LEMBERG K M， LAMPRECHT M R， et al. Ferroptosis： An iron-dependent form of nonapoptotic cell death[J]. Cell，2012，149（5）：1060-1072. doi：10.1016/j.cell.2012.03.042 .
[8]	DIXON S J， PATEL D N， WELSCH M， et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis[J]. Elife，2014，3：e02523. doi：10.7554/eLife.02523 .
[9]	KANER R J， LADETTO J V， SINGH R， et al. Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema[J]. Am J Respir Cell Mol Biol，2000，22（6）：657-664. doi：10.1165/ajrcmb.22.6.3779 .
[10]	HENNINGSSON R， ALM P， LUNDQUIST I. Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia[J]. Am J Physiol Cell Physiol，2001，280（5）：C1242-C1254. doi：10.1152/ajpcell.2001.280.5.C1242 .
[11]	刘建辉，李欣，孙志平，等. 百草枯中毒所致大鼠急性肺损伤时肺内HO-1/CO的变化[J].中国煤炭工业医学杂志，2006，9（5）：511-512. doi：10.3969/j.issn.1007-9564.2006.05.083 .
	LIU J H， LI X， SUN Z P， et al. Changes of HO-1/CO in the lungs of levels in rats with acute lung injury induced by paraquat poisoning[J]. Zhongguo Meitan Gongye Yixue Zazhi，2006，9（5）：511-512.
[12]	CHANG L C， CHIANG S K， CHEN S E， et al. Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis[J]. Cancer Lett，2018，416：124-137. doi：10.1016/j.canlet.2017.12.025 .
[13]	TSIKAS D. Assessment of lipid peroxidation by measuring malondialdehyde （MDA） and relatives in biological samples： Analytical and biological challenges[J]. Anal Biochem，2017，524：13-30. doi：10.1016/j.ab.2016.10.021 .
[14]	王萍，李铁刚. 百草枯中毒致小鼠肺纤维化模型的建立[J].实用药物与临床，2020，23（10）：886-889. doi：10.14053/j.cnki.ppcr.202010003 .
	WANG P， LI T G. Establishment of a mouse lung fibrosis model induced by paraquat poisoning[J]. Shiyong Yaowu Yu Linchuang，2020，23（10）：886-889.
[15]	LIU Z， WANG X， LI L， et al. Hydrogen sulfide protects against paraquat-induced acute liver injury in rats by regulating oxidative stress， mitochondrial function， and inflammation[J]. Oxid Med Cell Longev，2020，2020：6325378. doi：10.1155/2020/6325378 .
[16]	SUN B， CHEN Y G. Advances in the mechanism of paraquat-induced pulmonary injury[J]. Eur Rev Med Pharmacol Sci，2016，20（8）：1597-1602.
[17]	FENG H， STOCKWELL B R. Unsolved mysteries： How does lipid peroxidation cause ferroptosis?[J]. PLoS Biol， 2018，16（5）：e2006203. doi：10.1371/journal.pbio.2006203 .
[18]	YANG W S， STOCKWELL B R. Ferroptosis： Death by lipid peroxidation[J]. Trends Cell Biol，2016，26（3）：165-176. doi：10.1016/j.tcb.2015.10.014 .
[19]	KOPPULA P， ZHANG Y， ZHUANG L， et al. Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer[J]. Cancer Commun （Lond），2018，38（1）：12. doi：10.1186/s40880-018-0288-x .
[20]	SUN L， DONG H， ZHANG W， et al. Lipid peroxidation， GSH depletion， and SLC7A11 inhibition are common causes of EMT and ferroptosis in A549 cells， but different in specific mechanisms[J]. DNA Cell Biol， 2021，40（2）：172-183. doi：10.1089/dna.2020.5730 .
[21]	CHIANG S K， CHEN S E， CHANG L C. A dual role of heme oxygenase-1 in cancer cells[J]. Int J Mol Sci，2018，20（1）：39. doi：10.3390/ijms 20010039 .
[22]	龙伟，徐丽华，成燕，等. 铁死亡参与小鼠脓毒血症相关急性肺损伤的形成[J].西南国防医药，2020，30（8）：725-728. doi：10.3969/j.issn.1004-0188.2020.08.007 .
	LONG W， XU L H， CHENG Y， et al. Ferroptosis involved in sepsis-associated acute lung injury in mice[J]. Xinan Guofang Yiyao，2020，30（8）：725-728.
[23]	DONG H， QIANG Z， CHAI D， et al. Nrf2 inhibits ferroptosis and protects against acute lung injury due to intestinal ischemia reperfusion via regulating SLC7A11 and HO-1[J]. Aging （Albany NY），2020，12（13）：12943-12959. doi：10.18632/aging.103378 .
[24]	LI X， ZHUANG X， QIAO T. Role of ferroptosis in the process of acute radiation-induced lung injury in mice[J]. Biochem Biophys Res Commun，2019，519（2）：240-245. doi：10.1016/j.bbrc.2019. 08.165 .
[25]	LI X， DUAN L， YUAN S， et al. Ferroptosis inhibitor alleviates radiation-induced lung fibrosis （RILF） via down-regulation of TGF-β1[J]. J Inflamm （Lond），2019，16：11. doi：10.1186/s12950-019-0216-0 .
[26]	GONG Y， WANG N， LIU N， et al. Lipid peroxidation and GPX4 inhibition are common causes for myofibroblast differentiation and ferroptosis[J]. DNA Cell Biol，2019，38（7）：725-733. doi：10.1089/dna.2018.4541 .