Background: During ischemic stroke treatment, cerebral ischemia/reperfusion (I/R) injury results in neuronal cell death and neurological dysfunctions in brain. Previous studies indicate that basic helix-loop-helix family member e40 (BHLHE40) exerts protective effects on the pathology of neurogenic diseases. However, the protective function of BHLHE40 in I/R is unclear.
Objectives: This study aimed to explore the expression, role and potential mechanism of BHLHE40 after ischemia.
Material and methods: We established models of I/R injury in rats and of oxygen-glucose deprivation/reoxygenation (OGD/R) in primary hippocampal neurons. Nissl and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to detect neuronal injury and apoptosis. Immunofluorescence was used to detect BHLHE40 expression. Cell viability and cell damage measurements were conducted using Cell Counting Kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) assay. The regulation of BHLHE40 to pleckstrin homology-like domain family A, member 1 (PHLDA1) was assessed using the dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay.
Results: Cerebral I/R rats exhibited severe neuronal loss and apoptosis in hippocampal cornu Ammonis 1 (CA1) region, accompanied by downregulated BHLHE40 expression at both mRNA and protein levels, indicating that BHLHE40 may regulate the apoptosis of hippocampal neurons. The function of BHLHE40 in neuronal apoptosis during cerebral I/R was further explored by establishing an OGD/R model in vitro. Low expression of BHLHE40 was also observed in neurons treated with OGD/R. The OGD/R administration inhibited cell viability and enhanced cell apoptosis in hippocampal neurons, whereas BHLHE40 overexpression reversed those changes. Mechanistically, we demonstrated that BHLHE40 could repress PHLDA1 transcription by binding to PHLDA1 promoter. The PHLDA1 is a facilitator of neuronal damage in brain I/R injury and its upregulation reversed the effects caused by BHLHE40 overexpression in vitro.
Conclusions: The transcription factor BHLHE40 may protect against brain I/R injury through repressing cell damage via regulating PHLDA1 transcription. Thus, BHLHE40 may be a candidate gene for further study of molecular or therapeutic targets for I/R.
Keywords: BHLHE40; OGD/R; PHLDA1; apoptosis; cerebral I/R injury.