Knockdown of LCN2 Attenuates Brain Injury After Intracerebral Hemorrhage via Suppressing Pyroptosis

Abstract

Objective

The aims of this study are to screen novel differentially expressed genes (DEGs) for intracerebral hemorrhage (ICH) and reveal the role of Lipocalin-2 (LCN2) in ICH.

Methods

We constructed the ICH model by injection of autologous whole blood into the right basal ganglia in rats. RNA-sequencing and bioinformatics analyses were performed to identify the DEGs between ICH and sham rats, and some important ones were confirmed using quantitative real-time PCR (qRT-PCR). LCN shRNA was used to knockdown of LCN2 in ICH rats. Pathological examination was carried out using 2,3,5-triphenyltetrazolium chloride (TTC) staining and Hematoxylin-eosin (HE) staining. Immunohistochemistry detected Caspase-3, and co-staining of Terminal dUTP nick end labeling (TUNEL) and NEUN staining were performed for neuron apoptosis assessment. Western blot analysis was performed to quantify pyroptosis-related proteins. Enzyme-linked immunosorbent assay (ELISA) was used to measure inflammatory cytokine levels.

Results

ICH rats exhibited significant hematomas, higher brain water content, obvious interstitial edema, and inflammatory infiltration, as well as more apoptotic cells in brain tissues. RNA-seq analysis identified 103 upregulated and 81 downregulated DEGs. The expression of LCN2, HSPB1, CXCL10, and MEF2B were upregulated in ICH rats. ICH triggered the release of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-18, and promoted the expression of pyroptosis-related proteins Caspase-1, GSDMD, NLRP3, and ASC. LCN2 knockdown attenuated the pathological characteristics of ICH, and also reduced pyroptosis in brain tissues.

Conclusion

Inhibition of LCN2 attenuates brain injury after ICH via suppressing pyroptosis, which provide guidance for ICH management.

Keywords:

• intracerebral hemorrhage
• pyroptosis
• inflammation
• lipocalin-2

Data Sharing Statement

The datasets generated and/or analysed during the current study are available in the [NCBI] repository, [http://www.ncbi.nlm.nih.gov/bioproject/952712].

Ethical Approval and Consent to Participate

All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. The animal experiments conformed to the Guide for the Care and Use of Laboratory Animals. Animal study has been approved by the Animal Ethics Committee of The First Affiliated Hospital of Gannan Medical University. All methods were performed in accordance with Declaration of Helsinki.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no conflicts of interest to disclose.

Additional information

Funding

Jiangxi Provincial Health Committee (202130622, 20201088); Ganzhou Science and Technology Project (GZ2021ZSF060, GZ2016ZSF206).