KLF2 up-regulates IRF4/HDAC7 to protect neonatal rats from hypoxic-ischemic brain damage

Fan Wu,Chunlin Li

doi:10.1038/s41420-022-00813-z

Abstract

Neonatal brain hypoxic ischemic injury is a devastating event causing permanent brain damage. The current study set out to explore the role of Kruppel-like factor 2 (KLF2) and its downstream molecular mechanism on hypoxic-ischemic brain damage (HIBD) in neonatal rats. First, we adopted a modified Rice method to develop a HIBD model in postnatal day seven Sprague Dawley (SD) rat pups. Next, neuronal damage, morphological changes, and neuronal apoptosis were documented in the vulnerable hippocampal CA1 region and evaluated using Nissl staining, H&E staining, and TUNEL assay, respectively. Meanwhile, a hypoxic-ischemic model using the oxygen-glucose deprivation (OGD) method was established in cortical neurons isolated from day one SD rat pups, followed by MTT and flow cytometry detections of the cell survival rate and apoptotic ability. Experimental findings revealed that KLF2 was poorly-expressed in the brain tissues of HIBD rats and in the OGD-induced neurons. We found that KLF2 overexpression inhibited neuron apoptosis in vitro and in vivo, which was also observed to inhibit brain injury in the HIBD rats and alleviate neuronal damage of OGD-treated neurons. Besides, as dual luciferase reporter gene assay and chromatin immunoprecipitation established that KLF2 bound to the interferon regulatory factor 4 (IRF4) promoter, which promoted the binding of IRF4 in the promoter of histone deacetylase 7 (HDAC7) to augment its expression, thereby inhibiting neuronal apoptosis and brain damage. In conclusion, our findings indicated that KLF2 could increase the expression of IRF4 to up-regulate the expression of HDAC7, which protects against HIBD in neonatal rats.

Highlights

Hypoxic-ischemic brain injury can affect all age-groups of the population, and has an incidence in human neonates of ~3–5 per 1000 births [1]
To further explore the mechanism of Kruppel-like factor 2 (KLF2) involvement in hypoxic-ischemic brain damage (HIBD), RESULTS KLF2is poorly-expressed in HIBD rats First, we found that the severity of neurological deficit increased the cortical neurons of neonatal Sprague Dawley (SD) rats were isolated and cultured
Taken A dual luciferase assay was performed to explore the binding together, these findings suggested that the HIBD model was relationship between KLF2 and interferon regulatory factor 4 (IRF4), which revealed that established successfully and that KLF2 was poorly-expressed in luciferase activity in the IRF4 promoter-WT was increased by hippocampus of HIBD rats

Summary

INTRODUCTION

Hypoxic-ischemic brain injury can affect all age-groups of the population, and has an incidence in human neonates of ~3–5 per 1000 births [1]. After KLF2 over-expression treatment, the Nissl In addition, we observed that HDAC7 mRNA and protein body staining was deepened and the number of cells was expressions were significantly reduced in the brain tissues of increased, while the neuronal damage in CA1 area was HIBD rats (Fig. S5A, B). Expressions of KLF2, IRF4, and HDAC7 (Fig. 6A, B), reduced mRNA and protein levels of Cleaved-caspase-3 and Bax and increased that of Bcl-2 (Fig. 6A, B), decreased neurological deficit score (Fig. 6C), decreased infarct volume (Fig. 6D, Fig. S7A), improved hippocampal tissue structure (Fig. S7B), increased the number of Nissl-positive cells (Fig. 6E, Fig. S7C), and decreased neuronal apoptosis (Fig. 6F, Fig. S7D). COX IV was only expressed in mitochondrial components and β-actin was only expressed in cytoplasmic components (Fig. S8)

DISCUSSION

MATERIALS AND METHODS

Findings

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