Ferroptosis of alveolar epithelial cells induced by HAdV-7 infection

Zhongying YANG; Jianhua WEI; Luo REN; Shiyi CHEN; Enmei LIU; Na ZANG

doi:10.16016/j.2097-0927.202204131

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Ferroptosis of alveolar epithelial cells induced by HAdV-7 infection

Basic Medicine | 更新时间：2026-01-09

- Ferroptosis of alveolar epithelial cells induced by HAdV-7 infection
- Journal of Army Medical University Vol. 44, Issue 21, Pages: 2146-2156(2022)
- 作者机构：
  
  400014 重庆，重庆医科大学附属儿童医院呼吸科，国家儿童健康与疾病临床医学研究中心，儿童发育与疾病教育部重点实验室，儿科学重庆市重点实验室, 儿童感染免疫重庆市重点实验室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(32071123);the Project of Natural Science Foundation of Chongqing(cstc2020jcyj-msxmX0239);the Natural Science Foundation of Chongqing Education Committee(KJQN201900445)
- DOI：10.16016/j.2097-0927.202204131
  CLC： R322.35;R329.25;R563.1
- Received：17 April 2022，
  
  Revised：2022-07-12，
  
  Published：15 November 2022
- 稿件说明：
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Zhongying YANG, Jianhua WEI, Luo REN, et al. Ferroptosis of alveolar epithelial cells induced by HAdV-7 infection[J]. Journal of Army Medical University, 2022, 44(21): 2146-2156.
DOI：

Zhongying YANG, Jianhua WEI, Luo REN, et al. Ferroptosis of alveolar epithelial cells induced by HAdV-7 infection[J]. Journal of Army Medical University, 2022, 44(21): 2146-2156. DOI： 10.16016/j.2097-0927.202204131.

摘要

目的

探究人7型腺病毒(human adenovirus type 7

HAdV-7)感染对肺泡上皮细胞发生铁死亡的诱导作用。

方法

体外培养肿瘤性肺泡上皮细胞(A549)和正常人肺泡Ⅱ型上皮细胞(HPAEpiC)，感染HAdV-7(24、48、72 h)后，CCK-8检测细胞活性变化，电子透射显微镜观察胞内线粒体形态，活性氧(ROS) 探针、脂质过氧化物(MDA)检测试剂盒分别检测细胞ROS和MDA水平，胞内亚铁离子荧光探针观察胞内Fe

水平，实时荧光定量PCR(RT-qPCR)和Western blot检测铁死亡通路中关键分子的表达水平。HAdV-7滴鼻感染CD46人源化小鼠后，普鲁士蓝染色和Western blot分别检测肺组织的铁沉积和铁死亡相关蛋白的表达水平。体内外予铁死亡抑制剂后，CCK-8检测HAdV-7感染肺泡上皮细胞后细胞活性变化，HE染色观察HAdV-7感染CD46人源化小鼠肺组织病理情况改变。

结果

HAdV-7感染导致A549、HPAEpiC细胞活性降低，细胞线粒体出现体积缩小，线粒体嵴减少等铁死亡特征性改变，HAdV-7组感染后48、72 h ROS水平显著高于对照组(

0.01)；感染后48 h的MDA水平也明显升高(

0.05)。HPAEpiC细胞感染后胞内铁离子显著高于对照组(

0.01)。RT-q PCR结果显示，HAdV-7感染降低了两种细胞胱氨酸/谷氨酸逆向转运蛋白溶质载体家族7成员11(SLC7A11

也称为xCT)(

0.01)、谷胱甘肽过氧化物酶4(GPX4)(

0.05)、膜铁转运蛋白(SLC40

A1)(

0.05)、铁蛋白重链1(FTH1)(A549: 72 h

0.01; HPAEpiC: 48、72 h

0.01)等基因的表达，而上调了铁调素(HAMP)基因的表达(A549: 72 h

0.01; HPAEpiC:

0.05)。HPAEpiC细胞感染后xCT和GPX4的蛋白表达水平降低。HAdV-7感染CD46人源化小鼠后，肺组织铁染色加深，而xCT(

0.05)和Gpx4(

0.01)表达显著下调。予以铁死亡抑制剂，DFO能够抑制HAdV-7诱导的肺泡上皮细胞死亡(HPAEpiC:

0.01)

Fer-1抑制细胞死亡的作用更明显(A549:

0.05; HPAEpiC:

0.01)。此外，Fer-1能够明显改善HAdV-7感染CD46人源化小鼠肺损伤状态。

结论

HAdV-7感染致使肺泡上皮细胞铁离子累积、脂质过氧化物增加，从而诱导细胞发生铁死亡，肺泡上皮细胞的铁死亡可能参与了HAdV-7感染所致的肺损伤。

Abstract

Objective

To investigate the inductive effect of human adenovirus type 7 (HAdV-7) infection on ferroptosis in alveolar epithelial cells.

Methods

Human alveolar epithelial carcinoma cells A549 and human pulmonary type Ⅱ alveolar epithelial cells HPAEpiC were infected with HAdV-7

respectively

and in 24

48 and 72 h after infection

cell viability was measured using CCK-8 assay

mitochondrial morphology was observed by transmission electron microscopy (TEM)

productions of reactive oxygen species (ROS) and malondialdehyde (MDA) were measured with ROS probe and Lipid Peroxidation

respectively

and intracellular Fe

level were observed by Fe

fluorescence probe. RT-qPCR and Western blotting were applied to determine the expression changes of key genes in ferroptosis. After CD46 humanized mice were infected with HAdV-7 by nose dropping

Perls staining and Western blotting were applied to detect iron deposition and the expression of ferroptosis-related proteins in the lung tissue. After administration of ferroptosis inhibitor

deferoxamine mesylate (DFO)

in the mice with HAdV-7 infection

CCK-8 assay and HE staining were performed to observe the alveolar epithelial cell death a

nd lung pathology.

Results

After HAdV-7 infection

cell viability were reduced in the A549 and HPAEpiC cells

and smaller mitochondria and decreased even disappeared mitochondrial crista were observed in the ferroptotic cells. ROS level (A549 and HPAEpiC cells: 48 h and 72 h

0.01) and MDA level (both A549 and HPAEpiC cells:

0.05) were significantly higher in the HAdV-7 group than the control group. The intracellular iron concentration was obviously higher in the infected HPAEpiC cells than the control cells (

0.01). RT-qPCR results showed that the mRNA levels of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11

also known as xCT) (

0.01)

glutathione peroxidase 4 (GPX4) (

0.05)

ferroportin (SLC40A1) (

0.05) and ferritin heavy chain (FTH1) (A549 cells: 72 h

0.01; HPAEpiC cells: 48

72 h

0.01) were down-regulated after HAdV-7 infection

whereas that of HAMP was up-regulated (A549 cells: 72 h

0.01; HPAEpiC cells:

0.05). The protein levels of xCT and GPX4 were also decreased in the infected HPAEpiC cells than the control group. HAdV-7 infection in the CD46 humanized mice resulted in deepened iron stains in the lung tissue

and decreased protein levels of xCT (

0.05) and Gpx4 (

0.01). Ferroptosis inhibitor DFO could partially prevent HAdV-7-induced cell death (HPAEpiC:

0.01)

and obviously enhance Fer-1 inhibited cell death (A549 cells:

0.05; HPAEpiC cells:

0.01). In addition

Fer-1 treatment could effective ameliorate pathological damages in the CD46 humanized mice after HAdV-7 infection.

Conclusion

HAdV-7 infection increases iron accumulation and lipid peroxides

and thus induces ferroptosis in alveolar epithelial cells

which may be involved in lung injury caused by HAdV-7 infection.

关键词

Keywords

references

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