Identification of Lipocalin 2 as a Ferroptosis-Related Key Gene Associated with Hypoxic-Ischemic Brain Damage via STAT3/NF-κB Signaling Pathway

Lianxiang Luo; Liyan Deng; Yongtong Chen; Rui Ding; Xiaoling Li

doi:10.3390/antiox12010186

Identification of Lipocalin 2 as a Ferroptosis-Related Key Gene Associated with Hypoxic-Ischemic Brain Damage via STAT3/NF-κB Signaling Pathway

Antioxidants (Basel). 2023 Jan 12;12(1):186. doi: 10.3390/antiox12010186.

Authors

Lianxiang Luo^{1

2}, Liyan Deng³, Yongtong Chen³, Rui Ding¹, Xiaoling Li⁴

Affiliations

¹ The Marine Biomedical Research Institute, Guangdong Medical University, Zhanjiang 524023, China.
² The Marine Biomedical Research Institute of Guangdong Zhanjiang, Zhanjiang 524023, China.
³ The First Clinical College, Guangdong Medical University, Zhanjiang 524023, China.
⁴ Animal Experiment Center, Guangdong Medical University, Zhanjiang 524023, China.

Abstract

Hypoxic-ischemic brain damage (HIBD) is a common cause of death or mental retardation in newborns. Ferroptosis is a novel form of iron-dependent cell death driven by lipid peroxidation, and recent studies have confirmed that ferroptosis plays an important role in the development of HIBD. However, HIBD ferroptosis-related biomarkers remain to be discovered. An artificial neural network (ANN) was established base on differentially expressed genes (DEGs) related to HIBD and ferroptosis and validated by external dataset. The protein-protein interaction (PPI) network, support vector machine-recursive feature elimination (SVM-RFE) algorithms, and random forest (RF) algorithm were utilized to identify core genes of HIBD. An in vitro model of glutamate-stimulated HT22 cell HIBD was constructed, and glutamate-induced ferroptosis and mitochondrial structure and function in HT22 cells were examined by propidium iodide (PI) staining, flow cytometry, Fe²⁺ assay, Western blot, JC-1 kit, and transmission electron microscopy (TEM). In addition, Western blot and immunofluorescence assays were used to detect the NF-κB/STAT3 pathway. An HIBD classification model was constructed and presented excellent performance. The PPI network and two machine learning algorithms indicated two hub genes in HIBD. Lipocalin 2 (LCN2) was the core gene correlated with the risk of HIBD according to the results of differential expression analysis and logistic regression diagnostics. Subsequently, we verified in an in vitro model that LCN2 is highly expressed in glutamate-induced ferroptosis in HT22 cells. More importantly, LCN2 silencing significantly inhibited glutamate-stimulated ferroptosis in HT22 cells. We also found that glutamate-stimulated HT22 cells produced mitochondrial dysfunction. Furthermore, in vitro experiments confirmed that NF-κB and STAT3 were activated and that silencing LCN2 could have the effect of inhibiting their activation. In short, our findings reveal a molecular mechanism by which LCN2 may promote ferroptosis in HIBD through activation of the NF-κB/STAT3 pathway, providing new and unique insights into LCN2 as a biomarker for HIBD and suggesting new preventive and therapeutic strategies for HIBD.

Keywords: NF-κB; PPI; STAT3; WGCNA; ferroptosis; hypoxic-ischemic brain damage; lipocalin 2; machine learning; mitochondrial.

Grants and funding

2XK17040/Guangdong medical university